Plant paper digest

Rice blast, caused by Magnaporthe oryzae, is one of the most destructive diseases threatening global rice production. Race-independent broad-spectrum resistance (BSR) holds the ideal strategy for durable rice blast management, yet its molecular mechanisms remain largely obscure. Here, we identified and characterized elicitor-triggered cell death 1 (etd1), a rice lesion mimic mutant that displays spontaneous cell death and hypersensitive response (HR)-like necrosis upon challenge with M. oryzae elicitors. Genetic analysis reveals that etd1 encodes a hypermorphic haplotype of rice Cyclic Nucleotide-Gated Channel 13 (OsCNGC13) protein, harboring a single glycine-to-glutamic acid substitution (G483E) in the highly conserved gating domain of the CNGC family. This mutation transforms OsCNGC13 into a hyperactive Ca2+-permeable channel (etd1), driving ectopic Ca2+ influx under both developmental and pathogenic conditions that lead to cell death. Although detrimental to agronomic traits, etd1 confers robust BSR against all 108 tested M. oryzae isolates by effectively blocking fungal colonization through ectopic Ca2+ influx-induced cell death. Our data demonstrate that hyperactivation of a Ca2+-permeable channel can confer rice BSR through Ca2+-mediated cell death. The discovery of etd1 provides a valuable clue for breeding rice with broad-spectrum blast resistance based on Ca2+-permeable channels.

Flufenoxuron is extensively applied in agricultural pest control; however, its effects on plant physiological processes remain insufficiently understood. Hence, this study evaluated the effects of flufenoxuron on photosynthetic processes and stomatal regulation in barley. The findings indicated that flufenoxuron significantly reduced photosynthetic efficiency and gas-exchange capacity. Furthermore, flufenoxuron exposure markedly increased abscisic acid (ABA) in leaves but not in roots. Nonetheless, reactive oxygen species (ROS) generation and membrane lipid peroxidation were triggered by flufenoxuron. Concomitantly, defense responses were activated, including the upregulation of peroxidase activity and the accumulation of soluble sugars to mitigate osmotic imbalance. Correlation analysis revealed that stomatal conductance (Gs) showed significant negative correlations with ABA, H2O2 (hydrogen peroxide), and O2·- (superoxide anion) levels. Furthermore, flufenoxuron exposure altered the expression of genes associated with ABA biosynthesis and catabolism, accompanied by changes in the expression of genes related to ABA- and ROS-associated signaling. These findings suggest that ABA- and ROS-associated processes may be involved in stomatal regulation under flufenoxuron exposure. Overall, this study provides a conceptual and mechanistic basis for understanding the phytotoxicity of benzoylurea compounds.

Arabidopsis thaliana Ethylene Response 1 (ETR1) has long been viewed as a canonical hormone receptor initiating ethylene signaling at the endoplasmic reticulum (ER). Recent studies demonstrate that ETR1 also acts as an ER redox sensor, with disulfide-dependent dimerization controlling receptor conformation and activity. In parallel, subfamily I ethylene receptors, including ETR1, function as Ca2+-permeable channels, directly linking ethylene perception to rapid cytosolic Ca2+ elevation. Here, we summarize emerging evidence that redefines ETR1 as a multifunctional signaling hub connecting ethylene perception, redox regulation, and Ca2+ signaling. We present this integration as a working model and highlight unresolved questions concerning the mechanistic relationships among these functions. This framework provides a new perspective on how plants integrate ER homeostasis with hormone signaling to coordinate development and stress adaptation.

Plant cells are turgid and exert pressure against a stiff extracellular matrix, the plant cell wall, which resists turgor pressure and prevents cell bursting due to osmotic pressure. During growth, the cell wall yields under turgor pressure and deforms to guide the expansion of the cytoplasm. Together, these suggest that the mechanical properties of the cell wall are tightly regulated during development and that a tight coordination between turgor pressure and cell wall plasticity is required. In this review we explore the anisotropic mechanical properties of the cell wall and the mechanisms involved in its regulation. We discuss some of the theories supporting passive regulation of the cell wall such as the multinet theory and the scaffolding theory. We then discuss the role of CrRLK1Ls such as FERONIA and mechanosensing ion channels like MSL8 in the perception of mechanical stress and the role of Ca2+ as a central second messenger of mechanical stress response. Finally, we highlight some of the transcriptional responses induced by mechanosensing via small signal peptides such as SCOOP and Pep1 and cell wall remodelling enzymes such as TCH4.

Aflatoxin B1 (AFB1), a pervasive food contaminant affecting approximately 25% of global staple crops, represents an underrecognized environmental risk factor for nonalcoholic fatty liver disease (NAFLD). This study investigates the protective role of peroxiredoxin II (Prx II), a mitochondrial antioxidant enzyme, against AFB1-induced hepatic steatosis and elucidates the underlying molecular mechanisms. Bioinformatic analysis of the publicly available AFB1-exposed liver transcriptome dataset (GSE26838) combined with gain- and loss-of-function studies in AML12 hepatocytes revealed that AFB1 induces lipid accumulation, mitochondrial reactive oxygen species (ROS) overproduction, and membrane potential depolarization, while simultaneously suppressing PPARα expression and upregulating PPARγ-a metabolic shift favoring lipid storage. Prx II knockdown exacerbated these pathological changes, whereas overexpression attenuated lipid accumulation (~ 60%) and mitochondrial ROS production (~ 58%), restoring PPARα activity and attenuating ROS-associated p38 MAPK activation and PPARγ upregulation. Pharmacological ROS scavenging with N-acetylcysteine (NAC) recapitulated the protective effects of Prx II overexpression, supporting ROS as an upstream contributor to AFB1-induced steatosis. These findings define a Prx II-ROS-PPARα/γ regulatory axis that mitigates AFB1-induced hepatic steatosis by scavenging mitochondrial ROS, preserving PPARα-dependent fatty acid oxidation, and dampening lipogenic signaling. Prx II emerges as a promising candidate biomarker and therapeutic target for aflatoxin-associated metabolic liver disease, with significant implications for food-safety risk assessment and intervention strategies in AFB1-endemic regions.

Drought stress severely constrains global maize productivity. Although ethylene (ETH)-responsive transcription factors play key roles in plant stress adaptation, the functional mechanism of ZmEIL2 in drought tolerance remains poorly understood. Here, transgenic maize lines with ZmEIL2 overexpressing (OE) and ZmEIL2 knockout (KO) were generated and subjected to progressive drought stress followed by rehydration. Phenotypic analysis indicated that ZmEIL2 OE lines exhibited improved plant survival and reduced leaf water loss, while KO lines exhibited displayed enhanced drought sensitivity. Physiological assessments demonstrated that ZmEIL2 preserves leaf relative water content by regulating stomatal movement and mitigates drought-induced membrane damage and lipid peroxidation, which correlated with increased proline (Pro) accumulation. Furthermore, ZmEIL2 modulated endogenous ETH and abscisic acid (ABA) levels under drought stress and enhanced the antioxidant defense system by increasing the activities of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX), thereby diminishing reactive oxygen species (ROS) accumulation, as evidenced by 3,3'-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining. We performed RNA sequencing (RNA-seq)on wild-type (WT), OE, and KO plants sampled at 0, 10, and 13 days of drought treatment and identified 120 core drought-responsive genes involved in stress perception, oxidoreductase activity, and metabolic reprogramming. Notably, 11 candidate genes, including ZmSOD16, ZmPAL7, ZmHSP101, ZmHSP70, ZmGA2ox3, and ZmSMT1, exhibited strictly opposite expression trends between OE and KO under normal conditions, suggesting their roles in antioxidant defense, protein protection, and hormone and carbohydrate metabolism. Collectively, these results demonstrate that ZmEIL2 as a positive regulator of drought tolerance in maize via a multi-layered regulatory mechanism encompassing stomatal regulation, hormonal balance, membrane integrity, osmotic adjustment, and ROS scavenging. This work provides valuable candidate genes for the breeding of drought-resilient maize varieties.

BackgroundPotato (Solanum tuberosum L.) is the world's fourth most important staple crop, but its production is increasingly threatened by soil salinization, particularly alkaline-salt stress caused by excessive NaHCO3. Although exogenous spermidine (Spd) has been reported to alleviate abiotic stresses in various plants, its physiological and molecular mechanisms in conferring alkaline-salt tolerance in potato remain largely unknown.ResultsThe results demonstrated that exogenous Spd enhances alkaline-salt tolerance in potato by increasing antioxidant enzyme activities and maintaining osmotic balance. RNA sequencing (RNA-seq) and weighted gene co-expression network analysis (WGCNA) revealed obvious tissue-specific transcriptional reprogramming in potato under Spd-combined alkaline-salt stress, with 5,968 differentially expressed genes (DEGs) identified in leaves and only 187 in roots. KEGG pathway analysis indicated that Spd mainly regulates plant hormone signal transduction, carbon metabolism and photosynthesis pathways in leaves, as well as ribosome and energy metabolism-related pathways in roots. Three candidate hub genes, StHK4, StbZIP27 and StERF106, were screened from key Spd-responsive modules. Among them, StHK4 exhibited the highest upregulation ( Log2FC = 3.40) and positively mediates the cytokinin-ABA signaling pathway, StbZIP27 functions in bZIP-dependent regulatory pathways to promote osmotic adjustment and antioxidant defense, and StERF106 integrates ethylene signaling and reactive oxygen species (ROS) scavenging pathways. These pathways jointly improve membrane stability, maintain osmotic balance and enhance ROS-scavenging capacity, ultimately improving potato tolerance to alkaline-salt stress.ConclusionsThis study demonstrates that exogenous Spd enhances alkaline-salt tolerance in potato by modulating multiple physiological processes and transcriptional networks. The screened candidate hub genes (StHK4, StbZIP27, and StERF106) serve as potential pivotal regulators responsible for Spd-induced stress tolerance, providing valuable gene resources and novel mechanistic insights for understanding polyamine-mediated stress adaptation in potato.

BackgroundDrought tolerance in wheat is a complex trait controlled by multiple regulatory networks, among which calcium-dependent protein kinases (CDPKs) act as important components linking stress perception with downstream cellular responses. However, the functional contribution of individual CDPK members to drought adaptation in wheat remains largely unclear. This study aimed to identify and functionally characterize drought-responsive CDPK genes associated with differential drought responses in wheat.MethodsTwo wheat lines derived from the same breeding background exhibiting contrasting drought adaption, 23B1 and 23B39, were subjected to PEG6000-induced osmotic stress. Growth traits, osmotic adjustment-related metabolites, membrane damage indicators, and antioxidant enzyme activity were evaluated. Transcriptomic analysis was performed at early drought-response stages, followed by differential expression analysis, functional enrichment, CDPK family screening, and qRT-PCR validation. The role of TaCDPK22-5A was further investigated using barley stripe mosaic virus (BSMV)-mediated virus-induced gene silencing (VIGS).ResultsThe drought-responsive line 23B1 maintained stronger growth, accumulated higher levels of proline and soluble sugars, exhibited enhanced peroxidase activity, and showed reduced membrane lipid peroxidation compared with 23B39. Transcriptome analysis revealed extensive transcriptional reprogramming under drought stress, with differentially expressed genes mainly associated with metabolic adjustment, transport regulation, secondary metabolism, and stress-responsive pathways. Among the identified CDPK members, TaCDPK22-5A showed a strong drought-responsive expression pattern in the line exhibiting stronger drought tolerance (23B1). Virus-induced gene silencing of TaCDPK22-5A significantly impaired drought tolerance, resulting in reduced growth, biomass accumulation, and chlorophyll retention under drought conditions.ConclusionsThese findings demonstrate that TaCDPK22-5A contributes positively to drought adaptation in wheat and highlight CDPK-mediated calcium signaling as an important regulatory component of drought responses. The identified gene provides a potential target for improving drought resilience in wheat breeding.

Calmodulin-binding protein 60b (CBP60b) is a pivotal transcription factor in the transcriptional reprogramming of immune responses. Although its downstream regulatory genes and associated genetic pathways have been reported, its post-translational regulation and modifications remain unexplored. Here, we report that calcium-dependent protein kinases CPK4/5/6/11 physically interact with CBP60b and phosphorylate its serine 568 residue, thereby enhancing its transcriptional activity. The phosphodeficient variant CBP60bS568A compromises immune responses, whereas the phosphomimetic variant CBP60bS568D enhances resistance against pathogens, demonstrating that ser568 is essential for the function of CBP60b. Moreover, we observed that the expression of CPK4/5/6/11 is induced by pathogens, while this pathogen-induced expression of CPKs is reduced in the cbp60b mutant. Through experiments including dual luciferase (LUC) reporter assays and chromatin immunoprecipitation (ChIP) assays, we further demonstrated that CPK4, CPK5, and CPK11 are direct transcriptional targets of CBP60b. In conclusion, our results demonstrate for the first time that CBP60b is post-translationally regulated by CPKs via phosphorylation, while the pathogen-induced expression of CPKs is dependent on CBP60b. This mutual regulation establishes a positive feedback loop, which enables plants to mount a robust immune response against pathogens.

The escalating frequency and intensity of recent droughts threaten global crop production and food security. Although drought priming is crucial for plant drought resilience, the mechanisms imparting drought stress memory remain poorly understood, especially in crops such as tomato. This study elucidates the mechanisms of drought stress memory in tomato by integrating phenotypic screening with transcriptomic and proteomic analyses. Initial screening of 30 genotypes identified varieties with contrasting basal drought tolerance as indicated by gas exchange and drought damage index. Subsequent priming experiments revealed that acquired drought tolerance is independent of basal resistance, with both sensitive and tolerant genotypes exhibiting enhanced adaptation. Priming improved photosynthetic capacity and water use efficiency via optimised stomatal regulation. Moreover, elevated reactive oxygen species (ROS), particularly H2O2, was a key signalling molecule that initiated the drought memory establishment. This acquired drought tolerance was stress-specific, enhancing tolerance of tomato plants to subsequent drought but not heat stress. Integrative multi-omics analysis identified 518 memory-associated transcripts and pinpointed key candidate genes, including HSP90 (heat shock protein 90), WRKY26 and WRKY31 (WRKY transcription factor 26/31), which were linked to central pathways such as MAPK (mitogen-activated protein kinase) signalling and phenylpropanoid biosynthesis. The VIGS (Virus-Induced Gene Silencing) assay further demonstrated that WRKY26 functions as a negative regulator of acquired drought tolerance. Our work established a mechanistic framework for drought stress memory, highlighting ROS signalling and memory-specific gene networks. These insights, along with the generated datasets, provide valuable resources for the strategic breeding of climate-resilient tomato varieties.

The typical nucleotide-binding leucine-rich repeat (NLR) proteins, such as TIR-NBS-LRR (TNL) and CC-NBS-LRR (CNL), are known to be engaged in effector-triggered immunity (ETI). However, several atypical resistance (R) proteins with truncated NLR domains or with nonclassical domains are emerging to be key regulators in plant immunity. SMV resistance cluster 4 (SRC4) is an atypical NLR protein in soybean, distinguished by an N-terminal Domain (NTD)-TIR-shortened NBS oligomerization region (SNOR)-EF-hand (EFh) domain architecture. SRC4 lacks both NBS and LRR domains but retains basal antiviral activity through its TIR-SNOR region, with SNOR contributing to TIR-dependent antiviral activity and SRC4 self-association. The EFh domain recognizes coat protein (CP) of soybean mosaic virus (SMV) and serves as a Ca2+-responsive module to distinctly regulate TIR-SNOR-mediated immune response in soybean. SRC4-overexpression (Ox-SRC4) soybean plants show SMV resistance without obvious growth penalty. Furthermore, several SRC4 high-expression soybean varieties showed stronger SMV resistance and Ca2+ responsiveness than the SRC4 low-expression soybean varieties. Our study highlights the essential roles of atypical resistance proteins in plant immunity and growth-defense balance.

Cadmium (Cd) is a toxic heavy metal that severely disrupts plant growth, induces oxidative stress and disruption of physiological processes in plants. In this study, we evaluated the potential of two biostimulants, 28‐homobrassinolide (28‐HBL), a brassinosteroid, and the root endophytic fungus Piriformospora indica, to alleviate Cd‐induced stress in Brassica juncea. seedlings. Exposure to Cd significantly impaired seedling morphology and elevated reactive oxygen species (ROS) levels, while biopriming with 28‐HBL and root inoculation with P. indica markedly improved morphology and stress resilience. Enzymatic antioxidants (SOD, CAT, APOX, DHAR, MDHAR) and non‐enzymatic antioxidants (GSH, phenolics, flavonoids) effectively attenuated ROS levels, with the combined treatment showing the strongest impact. These biochemical enhancements were supported by transcriptional upregulation of antioxidant defense genes. In addition, osmolyte accumulation (proline, glycine betaine [GB]) supported cellular homeostasis under stress. Atomic absorption spectroscopy confirmed reduced Cd uptake in treated seedlings. Notably, P. indica colonization upregulated key brassinosteroid signaling genes (BRI1, BAK1, BES1, and BZR1), suggesting a hormone‐mediated mechanism of stress mitigation. The findings highlight a promising synergy between 28‐HBL and P. indica, offering an eco‐friendly strategy to enhance plant tolerance to Cd stress.

Cadmium (Cd) accumulation in crops seriously threatens agricultural productivity and food safety, highlighting the need for effective strategies to mitigate Cd toxicity. Uniformly dispersed zinc-doped carbon quantum dots (Zn-CQDs; 2.67 ± 0.36 nm) were synthesized in this study. The synthesized dots demonstrated enzyme-mimetic activities in vitro and potent radical-scavenging capacity. Foliar application of Zn-CQDs markedly alleviated Cd-induced growth inhibition in lettuce (Lactuca sativa L.), increasing shoot fresh and dry biomass by 30.8% and 23.9%, respectively. Moreover, Zn-CQDs treatment reduced the Cd concentration in lettuce shoots by 44.7% while elevating the Zn concentration by 167.9%. Photosynthetic performance was also substantially restored, as evidenced by a 61.0% increase in total chlorophyll content. Concurrently, Zn-CQDs mitigated Cd-triggered oxidative damage, reducing H2O2 and MDA levels by 31.4% and 38.9%, respectively, and lowering the activities of the antioxidant enzymes POD, SOD, and CAT by 23.3%, 71.5%, and 44.3%, respectively. Transcriptomic profiling revealed that Zn-CQDs modulate stress-responsive signaling networks involving MAPK cascades, Ca2 +, and phytohormone signaling, as well as their associated transcription factors, thereby regulating downstream genes involved in ROS detoxification and metal transport. Metabolomic analysis further demonstrated that Zn-CQDs extensively reprogram Cd-responsive metabolic pathways, particularly those related to phenylpropanoid, lipid, amino acid, and phytohormone metabolism, accompanied by increased accumulation of jasmonic acid, flavonoids, and organic acid derivatives. The above-mentioned findings offer mechanistic insights into Zn-CQD-mediated alleviation of Cd stress and lay a theoretical basis for functional carbon nanomaterials' application to reduce heavy metal risks in agricultural systems.

NAC transcription factors are key regulators of plant growth and stress responses, but their role in low-temperature tolerance of octoploid strawberry (Fragaria × ananassa) remains largely unexplored. In particular, little is known about the evolutionary characteristics of the NAC gene family in this species or the molecular mechanisms by which these genes mediate low-temperature stress responses. Here, we identified 371 FaNAC genes unevenly distributed across 28 chromosomes, with the Fve subgenome being the most abundant. Members of the same subgroup share highly conserved gene structures and motifs. Promoter analysis revealed cis-elements responsive to plant hormones (MeJA, ABA) and abiotic stresses including low-temperature, drought, and anaerobic. Most FaNAC‑Fve genes responded transcriptionally to low-temperature. One representative, FaNAC7-Fve6, localized to the nucleus and exhibited transcriptional self-activation activity. RT‑qPCR revealed that FaNAC7‑Fve6 was markedly induced by low-temperature, salt, heat, drought and ABA treatments. Overexpression of FaNAC7-Fve6 in 'Asia' strawberry enhanced the expression of antioxidant enzyme genes and reduced reactive oxygen species (ROS) accumulation. These findings reveal the evolutionary expansion of the FaNAC gene family in octoploid strawberry and establish a molecular mechanism whereby FaNAC7-Fve6 enhances low-temperature tolerance via the ROS scavenging pathway, providing valuable genetic targets for improving strawberry low-temperature stress resistance.

The geomagnetic field (GMF) is a ubiquitous yet poorly understood environmental factor that affects plant growth and development. It has been suggested that the blue-light photoreceptor cryptochrome (CRY) functions as a critical magnetic-field sensor in birds and insects through the radical-pair mechanism (RPM). However, whether CRY acts as a bona fide transducer of GMF signals and what are the downstream cascades remain largely elusive in plants. To address these key issues, we generated hypomagnetic field (HMF) via Helmholtz coils and passive magnetic shielding to investigate the GMF deprivation effects in Arabidopsis thaliana . HMF significantly delays the germination speed without altering the final germination rate, suggesting that HMF specifically modulates germination kinetics rather than seed viability. Time-resolved transcriptomic profiling revealed a coordinated transcriptional shift characterized by downregulation of growth-promoting genes and concurrent upregulation of defense-related genes under HMF. This biphasic transcriptional reprogramming coincides with a significant increase of reactive oxygen species (ROS), linking redox perturbation to the germination delay. Consistently, supplementation with ROS-scavenging antioxidants (e.g., reduced glutathione and ascorbic acid) rescues both germination speed and the misregulated expression of a subset of HMF-responsive genes, suggesting the central role of ROS in mediating HMF bioeffects. Genetic analysis using cry1 cry2 double mutants further indicated that HMF-induced germination delay operates through both CRY-dependent and -independent signaling pathways, suggesting the involvement of additional magnetosensitive modules beyond the canonical CRY-based RPM. These findings suggest that the GMF acts as a positive environmental cue that fine tunes the growth-defense trade-off through a redox-dependent signaling.

Abstract AbstractBackgroundSoil salinization is a major abiotic constraint limiting rice growth and productivity worldwide. Rice (Oryza sativa L.) is moderately salt-sensitive, and salt stress significantly inhibits tillering, a key determinant of panicle number and final yield. Prohexadione calcium (Pro-Ca) is a novel plant growth regulator that inhibits gibberellin biosynthesis and has been shown to alleviate salt stress in rice seedlings. However, the molecular mechanisms by which Pro-Ca regulates salt tolerance in rice tillers remain poorly understood. In this study, we investigated the regulatory effects of exogenous Pro-Ca on salt-stressed tillers of the indica rice variety 9311 using integrated physiological, transcriptomic, and metabolomic approaches.ResultsExogenous Pro-Ca treatment significantly alleviated salt-induced inhibition of tillering, increasing tiller number by 64.7%–137.5% compared with salt stress alone, while its effect on stem basal width was relatively modest. Pro-Ca enhanced antioxidant enzyme activities (SOD, APX, POD, CAT) by 4.9%–30.6%, reduced MDA accumulation by 15.2%–26.0%, and partially restored soluble sugar and protein levels. Ion analysis revealed that Pro-Ca decreased Na⁺ content by 50.7%, increased K⁺ content by 13.7%, and elevated the K⁺/Na⁺ ratio by approximately 1.3-fold, while IAA and SA contents increased by 40.0% and 22.5%, respectively. Transcriptomic analysis showed that Pro-Ca shifted the transcriptional response from passive damage repair (photosynthesis inhibition, cell wall degradation) toward active defense signaling, with significant enrichment of salicylic acid response, ethylene-activated signaling, and defense-related pathways. Metabolomic profiling revealed that Pro-Ca redirected metabolic flux from amino acid and energy metabolism toward alpha-linolenic acid metabolism, while Z-score analysis identified N-Carbamoylputrescine as a consistently upregulated key metabolite and 4-hydroxy-2-nonenal as a downregulated oxidative stress marker. Integrated multi-omics analysis pinpointed N-Carbamoylputrescine and Vanillin as central hubs linking gene expression changes with metabolic adjustments, and pathway integration revealed that the TCA cycle functions as a carbon–nitrogen metabolic hub, with fumarate significantly elevated across three interconnected pathways. Furthermore, Pro-Ca activated cutin, suberine, and wax biosynthesis pathways, suggesting physical barrier reinforcement.ConclusionOur findings demonstrate that exogenous Pro-Ca enhances salt tolerance in rice tillers through a multi-layered synergistic strategy: reconstructing ion homeostasis by reducing Na⁺ accumulation while preserving K⁺ levels and coordinately elevating IAA (growth promotion) and SA (defense activation) contents; reprogramming the transcriptome from passive repair to active defense signaling, coupled with metabolic reprogramming of polyamine biosynthesis, lipid peroxidation alleviation, and TCA cycle maintenance; and reinforcing physical barriers through cutin, suberine, and wax biosynthesis. This study provides novel insights into the molecular mechanisms by which Pro-Ca regulates salt tolerance in rice tillers and offers a theoretical basis for the application of plant growth regulators in saline-alkaline rice production. Keywords: metabolome; rice; salt stress; tillering; transcriptome

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), remains a major threat to global wheat production, which necessitates a better understanding of the molecular mechanisms underlying resistance. WRKY transcription factors are pivotal regulators of plant immunity; however, their specific functions in the wheat-Pst pathosystem remain poorly characterized. In this study, we identified and functionally characterized a WRKY transcription factor gene, TaWRKY24, in wheat. Expression profiling revealed that TaWRKY24 was continuously induced upon Pst inoculation. Subcellular localization assays demonstrated that the TaWRKY24 protein is predominantly localized to the nucleus. Functional analyses indicated that transient overexpression of TaWRKY24 significantly enhanced wheat resistance to Pst, whereas virus-induced gene silencing (VIGS) compromised wheat resistance to Pst. Histological observations revealed that TaWRKY24 promotes reactive oxygen species (ROS) accumulation at early infection sites and restricts fungal hyphal expansion. To further explore the underlying mechanism, combined transcriptomic and qRT-PCR analyses showed that silencing TaWRKY24 resulted in significant downregulation of key genes involved in MAPK signaling (MKK4/5, WRKY33), glutathione metabolism (GSS), and phenylpropanoid biosynthesis (PAL). In addition, yeast one-hybrid and dual-luciferase reporter assays demonstrated that TaWRKY24 directly binds to and activates the promoters of PAL- and MAPK-related genes. Collectively, our findings suggest that TaWRKY24 positively regulates wheat resistance to Pst, likely through the modulation of MAPK-mediated immune signaling, ROS homeostasis, and defense-related metabolic pathways. This study provides novel insights into the regulatory functions of WRKY transcription factors in wheat innate immunity.

Abstract NAC transcription factors (TFs) constitute a major plant-specific regulatory family that coordinates diverse biological processes, including organ growth, developmental progression, and responses to unfavorable environmental conditions. In this study, we isolated a NAC gene from Suaeda liaotungensis whose expression was induced by salt treatment and named it SlNAC6 . The coding region of SlNAC6 is 1,059 bp in length and encodes a protein comprising 353 amino acids. Subcellular localization analysis based on fluorescent signals revealed that Sl NAC6 is distributed in both the nucleus and cytoplasm. Yeast one-hybrid assays further confirmed that Sl NAC6 functions as a transcriptional activator, with its activation capacity primarily attributable to the C-terminal region. Expression profiling further showed that Sl NAC6 is constitutively transcribed in the roots, stems, and leaves of S. liaotungensis , although its transcript level is highest in the leaves. In addition to salt, drought, cold, and abscisic acid treatment markedly enhanced Sl NAC6 expression. To characterize the biological function of SlNAC6 , two independent Arabidopsis lines constitutively expressing this gene were established. When exposed to salt or drought stress, the SlNAC6 -overexpressing lines displayed greater stress resistance and survival rate than wild-type (WT) plants and pBI121-GUS transgenic controls (VT). These phenotypic improvements were accompanied by elevated proline (Pro) levels, higher maximum quantum efficiency of photosystem II photochemistry (Fv/Fm), and increased activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT). By contrast, relative electrical conductivity and malondialdehyde (MDA) accumulation were significantly reduced. Ectopic expression of SlNAC6 also enhanced the transcription of several stress-associated genes. These observations demonstrate that SlNAC6 expression improves Arabidopsis tolerance to salinity and drought, potentially by promoting reactive oxygen species (ROS) detoxificationand limiting membrane lipid peroxidation. Thus, SlNAC6 may serve as a valuable genetic resource for improving plant resilience to adverse environmental conditions.

Reactive oxygen species (ROS) regulate plant growth and stress responses. Catalases play a central role in detoxifying hydrogen peroxide, predominantly within peroxisomes, yet key aspects of catalase regulation remain incompletely understood. Using affinity purification of the UV-B photoreceptor UVR8 coupled with mass spectrometry, we identified CATALASE-INTERACTING RCC1-LIKE 1 (CAIR1), which interacts with all three Arabidopsis catalases and their chaperone NO CATALASE ACTIVITY 1. Loss of CAIR1 reduces catalase activity and causes oxidative stress sensitivity, impaired root growth, and alkaline sensitivity, resembling cat2 and nca1 mutants. CAIR1 promotes peroxisomal import and proper localization of CAT2, preventing CAT2 aggregation and maintaining its activity. CAIR1 undergoes reversible redox-dependent oligomerization that enhances catalase binding, whereas mutation of Cys-356 and Cys-545 compromises this interaction and fails to rescue the oxidative stress sensitivity of cair1 mutants. UV-B weakens CAIR1-catalase interactions and suppresses catalase activity, linking light signalling with redox homeostasis. These findings identify CAIR1 as a redox-responsive regulator of catalase localization and activity.

Background/Objectives: Soil salinization threatens global potato production, yet the molecular mechanisms underlying salt tolerance remain poorly understood. This study aimed to elucidate dynamic transcriptomic responses to salt stress, identify key regulatory genes, and characterize the corresponding gene family and its stress-responsive roles. Methods: Potato cv. Atlantic plantlets were subjected to 150 mM NaCl stress, with samples collected at 0, 3, 12, 24, and 48 h. Physiological and transcriptomic analyses were performed. WGCNA was employed to identify salt-responsive modules. Genome-wide identification, phylogenetic analysis, gene structure characterization, conserved motif analysis, and promoter cis-element prediction of the annexin gene family were conducted. Results: Transcriptome sequencing identified 1394 differentially expressed genes (DEGs) that were consistently responsive to salt stress, with functional enrichment highlighting stress signaling, hormone transduction, and phenylpropanoid biosynthesis pathways. Weighted gene co-expression network analysis (WGCNA) revealed the MEblack module as the key salt-responsive regulatory module, in which annexin genes exhibited high intramodular connectivity. Genome-wide identification uncovered nine annexin genes (StANN1-StANN9), characterized by uneven chromosomal distribution, conserved exon-intron structures and annexin repeat domains, and expansion through tandem, proximal and segmental duplication under purifying selection. Promoter analysis revealed abundant stress-responsive cis-elements, including ABRE and DRE/CRT motifs, in salt-inducible annexins. Expression profiling demonstrated that StANN5 and StANN6 were continuously upregulated under prolonged salt stress, whereas StANN2 and StANN3 showed transient early induction. Conclusions: These findings establish the central role of annexins in calcium signaling, redox homeostasis, and hormone-mediated stress adaptation, providing candidate targets for molecular breeding of salt-tolerant potato varieties.

Photosynthetic machinery reprogramming is essential for Cold Stress Tolerance (CST) in plants. STAY-GREEN 1 (SlSGR1) plays vital roles in chlorophyll breakdown and leaf senescence, but its function in tomato cold acclimation has not been clarified. Here, we constructed two inbred tomato lines (T048, T069) carrying SlSGR1 homozygous knockout mutants via CRISPR/Cas9 editing, and conducted integrated physiological, biochemical, metabolomic, and transcriptome analyses to dissect the role of SlSGR1 in regulating cold tolerance. Loss of function of SlSGR1 triggered extensive physiological, metabolic and transcriptional readjustments that improved tomato cold tolerance. sgr1 mutant seedlings maintained higher water retention and proline levels, along with reduced MDA and reactive oxygen species (ROS) accumulation, alongside elevated SOD, POD and CAT antioxidant enzyme activities. Multi-omics data revealed prominent activation of jasmonic acid (JA) biosynthesis and signaling in cold-stressed sgr1 lines, accompanied by elevated JA-Ile and strong upregulation of JA synthetic and signaling genes including multiple JAZ family members. Activated JA cascades further induced the master cold regulators ICE1, CBF1 and CBF2, driving downstream shifts in carbohydrate and amino acid metabolism as well as widespread ethylene-responsive transcription factor expression. Our multi-line genetic and multi-omics evidence collectively confirms that SlSGR1 acts as a negative regulator of tomato cold adaptation via restraining JA-ICE-CBF signaling. This study expands the biological functions of SlSGR1 beyond chlorophyll breakdown and leaf senescence, and provides valuable resources for molecular breeding.

Agricultural productivity is increasingly constrained by interacting abiotic stresses, yet translation of nano-enabled seed treatments from controlled experiments to field agriculture remains limited. Seed nanopriming can initiate physiological and molecular priming before radicle emergence, but outcomes depend strongly on nanomaterial identity, particle size and morphology, surface charge, colloidal stability, cargo chemistry, exposure regime, and crop genotype. This review critically examines mesoporous silica nanoparticles, chitosan-based systems, metal-organic frameworks, hybrid/core-shell platforms, and catalytic nanozymes, distinguishing true cargo-delivery carriers from catalytic functional nanomaterials. We integrate interfacial physics with seed-coat and cell-wall barriers, clarifying how zeta potential, surface charge density, hydrodynamic size, porosity, functionalization, and material transformation influence adhesion, uptake, release, and translocation. At the molecular level, we connect nanoparticle-surface interactions with ROS and Ca2+ signaling, membrane-potential changes, MAPK/CDPK cascades, phytohormone cross-talk, transcription-factor activation, antioxidant defenses, and stress-memory processes. We also distinguish transient physiological memory from mitotically persistent and experimentally demonstrated transgenerational inheritance, and critically examine bio-nano combinations in soil, including dissolution, redox transformation, heteroaggregation, and eco-corona formation. Particular emphasis is placed on dose- and material-dependent phytotoxicity, multi-stress cross-tolerance, regulatory status of transgene-free editing, and practical barriers involving reproducibility, cost, scalability, field stability, and environmental safety. Finally, we identify realistic research priorities, including standardized nanoformulation reporting, field-validated dose windows, multi-omics and mechanistic imaging, Nano-QSAR/graph-learning workflows, and safer-by-design criteria for precision seed treatment.

Abstract Bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv. oryzae , is a major constraint to rice production, especially when infection occurs at the seedling stage. The executor resistance gene Xa7 confers durable and broad‑spectrum resistance to BLB; however, the underlying protein‑level mechanisms governing Xa7‑mediated resistance during early development remain largely unclear. This study aims to perform comparative proteomic analysis between near‑isogenic lines of Xa7 locus in the background of KD18 variety. Rice seedlings were inoculated with Xoo , and total proteins were analyzed by two-dimensional gel electrophoresis followed by MALDI‑TOF/TOF mass spectrometry. Eight protein spots showing reproducible expression level changes (≥ 1.5-fold) between resistant and susceptible genotypes were identified, including six upregulated and two downregulated proteins in Xa7-NIL seedlings. Peroxiredoxin‑2C showed the strongest induction (+ 3.15-fold), together with increased levels of a glycine‑rich RNA‑binding protein (+ 1.87-fold) and lactoylglutathione lyase (+ 2.14-fold), indicating enhanced regulation of reactive oxygen species, post‑transcriptional processes, and methylglyoxal detoxification. In contrast, the cytochrome b6/f complex iron-sulfur subunit was markedly downregulated (-3.84-fold), suggesting reprogramming of photosynthetic electron transport during defense activation. These results provided direct proteomic evidence linking an executor resistance gene to specific defense‑associated protein responses during early rice development.

Tandem kinases are emerging regulators of plant immunity, but the downstream defense programs they activate remain poorly understood. Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a major constraint on global wheat production. The Yr15 resistance gene, derived from wild emmer wheat, encodes the tandem kinase protein WTK1. To define the temporal basis of this resistance, we analysed resistant and susceptible near-isogenic lines across a 7-day infection time course using histopathology, ROS phenotyping, RNA sequencing, co-expression network analysis, and qRT-PCR validation. WTK1-mediated resistance limited fungal colonization, enhanced ROS accumulation, and triggered extensive transcriptional reprogramming. We identified 2509 pathogen-responsive differentially expressed genes, including 1530 genes upregulated in the resistant line, revealing an early phase enriched for ROS and Ca2+ signaling, autophagy-related processes, and metacaspases consistent with hypersensitive cell death observed in pathogen-invaded cells. This was followed by later defense reinforcement and metabolic reprogramming in later stages of infection, including pathogenesis-related proteins, cell-wall reinforcement, and phytoalexin biosynthesis, consistent with the confinement of Pst to localized necrotic lesions. Our results show that WTK1 triggers coordinated waves of transcriptional reprogramming and co-expression network responses during infection, providing insight into tandem kinase-mediated immunity and a framework for improving durable stripe rust resistance in wheat.

Ca2+-ATPase (CAP) is a key Ca2+ efflux protein in plants. Our previous research suggests that CAPs may play a crucial role in the adaptation of rhododendrons to high calcium environments. However, the evolution, variation, characteristic expression, and subfunctionalization of this gene family in Rhododendron remain unknown. Through the analysis of pan-genomes and pan-transcriptomes, we elucidated the systematic evolution of CAPs in plants, as well as their characteristic expression patterns in Rhododendron. During the evolutionary process from lower to higher plants, CAPs can be divided into six clades and exhibit structural conservation. CAPs have emerged and differentiated in lower plants such as algae, and they have undergone significant amplification in Eudicots plants like rhododendrons. Three Rhododendron species (Rhododendron bailiense, R. delavayi, and R. irroratum) located in the karst province of Guizhou in Southwest China exhibit the highest copies of CAPs, suggesting a strong association between CAP copy number variation and habitat, particularly in high calcium environments. Through multiple transcriptome analyses, we revealed that CAPs are induced under various environmental/developmental conditions (e.g. karst environments, high altitude, early flower development, hormones, etc.). Co-expression network analysis highlighted key members of calcineurin B-like protein (CBL) and CBL-interacting protein kinases (CIPK) that are associated with the high expression of CAPs. Experimental validation demonstrated that CAPb1 and CAPd1 significantly alleviate high calcium stress, and the CAPb1-CIPK1-CBL1 and CAPd1-CIPK2-CBL1 modules can further enhance the alleviation. These findings provide new insights into the evolution, characteristic expression, and function of CAPs, as well as new perspectives on the high calcium adaptability of rhododendrons.

Key messageStERF87 directly activates StPR1a via GCC-box binding and integrates SA/ET signaling to enhance bacterial wilt resistance. Potato (Solanum tuberosum) is an important food crop worldwide, yet its yield is severely constrained by bacterial wilt caused by Ralstonia solanacearum. We used RNA-Seq to study gene expression in 'Z1076-1' at 0, 1, and 2 days post-inoculation (dpi) with R. solanacearum (10⁶ CFU mL⁻1). We identified 6663 differentially expressed genes at 1 dpi and 7390 at 2 dpi. Calcium signaling and MAPK cascade genes were upregulated at 1 dpi. PR protein and ROS-related genes showed stronger induction at 2 dpi. The ethylene-responsive transcription factor StERF87 was continuously upregulated. Its expression increased 4.7-fold at 2 dpi, with FPKM values over 100. We selected this gene for functional analysis. Transgenic potato plants overexpressing StERF87 showed lower disease severity and reduced bacterial growth in both whole plants and tuber slices. StERF87 is a transcriptional activator that directly binds the GCC-box in the StPR1a promoter to activate its transcription. After R. solanacearum inoculation, StERF87 overexpression also increased PR1b1 expression, elevated salicylic acid and ethylene levels, reduced jasmonic acid accumulation, and altered the activities of ROS-scavenging enzymes including SOD, POD, and CAT. These results show that StERF87 regulates potato defense against R. solanacearum and may be useful for breeding bacterial wilt-resistant varieties.

Stomatal regulation is governed by interconnected developmental, hormonal, metabolic, and environmental signaling networks that enable plants to balance carbon acquisition with water conservation. This review highlights lipid metabolism and lipid-mediated signaling as an emerging regulatory layer integrating these processes during stomatal development and movement. We synthesize evidence that diverse lipid classes, including fatty acids, sterols, phosphoinositides, phosphatidic acid (PA), diacylglycerol (DAG), sphingolipids, and oxylipins, function not only as membrane components but also as dynamic metabolic and signaling regulators in guard cells. A central conclusion is that lipid flux, rather than lipid abundance alone, provides a flexible regulatory interface linking environmental and hormonal cues to guard-cell signaling and stomatal behavior. In particular, PA-DAG metabolism integrates ABA, ROS, Ca²⁺, and ion-transport pathways during stress-induced stomatal closure, whereas triacylglycerol turnover supplies metabolic substrates that support blue-light-induced stomatal opening. Lipid metabolism also intersects with the SPCH-MUTE-FAMA-dependent developmental program, linking lipid homeostasis to stomatal differentiation and environmental plasticity. Key gaps remain in understanding the spatial and temporal dynamics of lipid signaling, the specificity of individual lipid species, and their crosstalk with hormonal, ROS, Ca²⁺, and ion-transport networks. Clarifying these mechanisms will advance our understanding of how lipid metabolism coordinates stomatal responses and may provide new opportunities to improve plant stress resilience.

Abstract Reproductive-stage drought disrupts cellular redox homeostasis in camelina ( Camelina sativa L.) through uncontrolled reactive oxygen species (ROS) accumulation, causing membrane lipid peroxidation and photosynthetic impairment. While arbuscular mycorrhizal fungi (AMF) and melatonin independently confer drought tolerance, their synergistic coordination of antioxidant enzyme induction, osmolyte-mediated osmotic adjustment, and phytohormone signaling remains unresolved. Over two field seasons under arid soils, the triple combination of 50% chemical fertilizer, Funneliformis mosseae inoculation, and foliar melatonin (100 µM) activated a hierarchical physiological defense program under reproductive-stage drought. Root colonization reached 68.4%, serving as the upstream trigger for coordinated upregulation of catalase and peroxidase the primary enzymatic ROS-scavenging module. This antioxidant priming was mechanistically linked to reduced malondialdehyde accumulation and elevated membrane stability index, indicating preserved thylakoid and plasma membrane integrity. Concurrent proline and soluble sugar accumulation reinforced osmotic adjustment, lowering cellular osmotic potential and sustaining turgor-dependent water relations and photosynthetic pigment retention. Structural equation modeling resolved the causal architecture: AMF colonization → antioxidant enzyme induction (β = 0.52) → membrane stabilization (β = 0.47) → photosynthetic maintenance (β = 0.58) → yield formation (β = 0.63). Melatonin contributed through hormone-mediated antioxidant priming and direct membrane stabilization, acting synergistically with AMF-induced osmotic adjustment. The resultant 106% increase in oil yield over unfertilized controls represents a downstream agronomic consequence of enhanced physiological resilience AMF-mediated antioxidant priming and melatonin-supported membrane and osmotic stabilization rather than direct nutritional effects. These findings establish that reduced chemical fertilization can be achieved through mechanistic exploitation of symbiotic and hormonal defense pathways.

AGL (AGAMOUS-LIKE) transcription factors are key regulators of plant growth and development, but their function in plant defense against pathogenic fungi is less studied. Apple (Malus × domestica) ring rot, caused by Botryosphaeria dothidea, is an important disease affecting apple production. In this study, we systematically analyzed the function and regulatory mechanism of the transcription factor MdAGL15 in apple resistance to B. dothidea. The results showed that overexpression of MdAGL15 in apple calli and fruits significantly enhanced susceptibility to B. dothidea, as evidenced by the enlargement of lesion area and the intensification of pathogen infection. Further mechanistic studies showed that the expression of key genes in the jasmonic acid (JA) biosynthesis and signal transduction pathways was significantly suppressed in MdAGL15-overexpressing material after inoculation with B. dothidea. Meanwhile, MdAGL15 also affected the basal immune response of plants by regulating reactive oxygen species (ROS) accumulation. This study reveals the molecular mechanism by which AGL transcription factors impair resistance to B. dothidea by negatively regulating the JA signaling pathway and ROS accumulation in apple. These findings not only expand the functional knowledge of AGL transcription factors in plant immunity but also provide a potential theoretical basis and gene targets for disease resistance breeding in apple.

Bacterial wilt caused by Ralstonia solanacearum is a devastating soil‐borne disease threatening tomato production with limited control strategies. NAC transcription factors play vital roles in plant stress responses, but their function in tomato resistance to bacterial wilt remains unclear. Our previous investigation revealed that SlNAP2 expression is markedly induced following R. solanacearum infection. Here, subcellular localization confirmed SlNAP2 is localized in the nucleus and possesses transcriptional activation activity. Functional analysis revealed that SlNAP2 overexpression enhanced resistance to bacterial wilt, whereas a CRISPR/Cas9 knockout increased susceptibility. Physiological and biochemical assays demonstrated that SlNAP2 alleviates pathogen‐induced reactive oxygen species (ROS) accumulation by enhancing the activities of superoxide dismutase (SOD), peroxidase (POD), phenylalanine ammonia‐lyase (PAL), and polyphenol oxidase (PPO), while reducing malondialdehyde (MDA) content. Hormonal profiling indicated SlNAP2 negatively regulates the jasmonic acid (JA) pathway but positively modulates the salicylic acid (SA) pathway: overexpression lines displayed decreased JA levels along with downregulated expression of JA signaling‐related genes (SlMYC2, SlCOI1, SlJAZ, and SlAOS), and increased SA levels accompanied by upregulated expression of SA pathway genes (SlNPR1, SlTGA, SlPR1, and SlICS1). Molecular interaction assays confirmed that SlNAP2 physically interacts with SlMYC2 in vivo and in vitro and specifically binds to the SlMYC2 promoter. However, dual‐luciferase reporter assays indicated SlNAP2 does not directly activate SlMYC2 transcription. In summary, SlNAP2 positively regulates tomato resistance to bacterial wilt by enhancing antioxidant defense, coordinately modulating JA and SA signaling pathways, and maintaining ROS homeostasis. This study provides new insights into the functional mechanisms of NAC transcription factors in plant immunity.

Abstract Plant Rho/ROP GTPases act as essential molecular switches in disease resistance. However, how their protein stability is regulated and integrated with lignin metabolism remains poorly understood. Here, we demonstrate that the cotton small GTPase GhARAC3 serves as a positive regulator of resistance against the vascular pathogen Verticillium dahliae (V. dahliae). Genetic analyses reveal that overexpression of GhARAC3 enhances defense responses in cotton and Arabidopsis, whereas its silencing significantly compromises plant immunity. Under normal physiological conditions, the E3 ubiquitin ligase GhKEG acts as a key regulator that targets GhARAC3 for proteasomal degradation by ubiquitinating multiple lysine residues. Notably, the lysine 161 (K161) residue within the conserved G5 motif is identified as an in vivo ubiquitination site. Mutation of this residue (K161R) impairs GTPase activity and compromises disease resistance, without altering its subcellular localization at the plasma membrane and nucleus. Pathogen infection attenuates this ubiquitination to promote the stabilization of GhARAC3. Genetic epistasis analysis indicates that GhARAC3 requires GhLAC14, a transmembrane laccase, to confer effective immunity.Rather than altering GhLAC14 protein stability, GhARAC3 physically interacts with and stimulates the enzymatic activity of the laccase. Additionally, GhARAC3 promotes reactive oxygen species (ROS) production. This dual activation of laccase activity and the ROS burst drives the rapid deposition of guaiacyl lignin to fortify the plant cell wall. Furthermore, transcriptomic profiling reveals that GhARAC3 broadly upregulates downstream defense-related pathways. Collectively, our findings uncover a regulatory module linking ubiquitin-mediated GTPase turnover to laccase activation, providing a mechanistic framework for improving cotton disease resistance.

Plant viruses have evolved sophisticated virulence strategies to counteract host antiviral defenses. Blue light, as an important environmental signal, can regulate plant growth and tolerance to biological stress. Nevertheless, how blue light regulates plant responses to antiviral immunity remains largely obscure. Here, we demonstrate that blue light suppresses rice black-streaked dwarf virus (RBSDV) replication. The blue light receptor ZmPHR2 functions as a key immune component, positively regulating maize resistance to RBSDV. Mechanistically, ZmPHR2 interacts with ZmISP in chloroplasts to trigger a defensive reactive oxygen species (ROS) burst, thereby conferring resistance. The RBSDV effector P6 enhances the ZmPHR2-ZmISP interaction, alters their subcellular distribution, and suppresses ROS accumulation. A functionally analogous P6-OsPHR2-OsISP module similarly mediates RBSDV resistance in rice. Our findings reveal that blue light perception directly activates antiviral defense pathways, uncovering a molecular basis for RBSDV resistance. This study provides a theoretical basis for artificially modifying the blue light receptor protein to enhance the resistance of cereal crops.

Nitrate (NO3-) is an essential nitrogen source and an important signaling molecule that regulates plant growth and development in response to nitrogen availability. Although several CBL-interacting protein kinases (CIPKs) have been implicated in nitrate signaling, the role of CIPK11 (PKS5) in nitrate availability responses remains unknown. Here, we investigated the function of CIPK11 using Arabidopsis mutants under nitrate-deficient and nitrate-resupply conditions by analyzing growth phenotypes, nitrate and protein accumulation, nitrate-responsive gene expression, extracellular H+ dynamics, and responses to the plasma membrane (PM) H+-ATPase inhibitor protonstatin-1 (PS-1). CIPK11 expression was downregulated under nitrate deficiency and rapidly induced after nitrate resupply. The loss-of-function mutant pks5-1 exhibited enhanced growth, increased nitrate and protein accumulation, and stronger induction of nitrate-responsive genes (NRT2.1, NRT2.4, NIA1, and NIA2), whereas the gain-of-function mutants pks5-3 and pks5-4 showed opposite phenotypes. Altered extracellular H+ dynamics and PS-1 sensitivity further suggested that PM H+-ATPase activity contributes to CIPK11-mediated nitrate responses. Collectively, our findings identify CIPK11 as a negative regulator of plant adaptation to nitrate availability and suggest that CIPK11 modulates nitrate acquisition and assimilation, at least partly through regulation of PM H+-ATPase activity.

Cadmium (Cd) toxicity requires plants to coordinate growth restraint with cellular protection, but the upstream regulatory mechanism remains unclear. Here, we identify the gibberellin (GA)-DELLA module as a positive regulator of Cd tolerance in Arabidopsis thaliana. Cd exposure reduced endogenous GA levels, shifted GA metabolic gene expression toward a low-GA state, and increased DELLA protein abundance; exogenous GA₃ attenuated this increase. Genetic and pharmacological analyses showed that elevated DELLA activity improved growth under Cd stress, whereas loss of DELLA function increased sensitivity. Mechanistically, DELLA activity strengthened redox protection and restricted root-to-shoot Cd translocation, as indicated by lower reactive oxygen species and lipid peroxidation, higher antioxidant capacity, better maintenance of photosynthetic performance, and enhanced expression of genes associated with Cd chelation and vacuolar sequestration. These results support a model in which a Cd-induced low-GA state promotes DELLA-dependent protection by coordinating oxidative defense with Cd retention in roots.

Flooding stress (FS) at the maize seedling stage disrupts energy metabolism and redox homeostasis, impairing photosynthesis and causing cellular damage. This study evaluated whether exogenous melatonin (MT) alleviates FS injury in maize and characterized physiological and molecular responses associated with this protection. Maize seedlings were subjected to a 2 × 2 design (CK, CK-MT, FS, and FS-MT), while transcriptomic, metabolomic, and hormone profiling focused on CK, FS, and FS-MT. FS reduced net photosynthetic rate and stomatal conductance by 61.93% and 77.98%, respectively, increased H2O2 by 194.15%, and decreased leaf area. Relative to FS, MT increased leaf area by 25.46%, net photosynthetic rate by 79.98%, stomatal conductance by 155.58%, Fv/Fm by 3.35%, and relative water content by 10.22%, while decreasing H2O2, NO, and ethanol levels by 23.27%, 12.67%, and 13.86%, respectively. MT restored SOD and CAT activities toward CK levels and further enhanced POD and APX activities. Multi-omics analyses revealed treatment-associated changes in RBOH-associated ROS responses, NADPH-related metabolic pathways, antioxidant-related genes, ZmPIP expression, osmotic and fermentative metabolism, and hormone profiles. These responses were accompanied by improved chloroplast ultrastructure, reduced qualitative TUNEL-positive signals, and an 18.24% increase in projected grain yield relative to FS. Collectively, the key novel finding is that MT-associated improvement under flooding coincided with coordinated redox, osmotic, fermentative, and chloroplast responses across physiological and multi-omics layers. These treatment-associated patterns support an integrated stress-alleviation response but do not establish a causal role for NADPH regeneration or a flooding-specific MT molecular mechanism.

Chromium (Cr) toxicity poses a significant threat to crop growth and agricultural productivity. Fulvic acid (FA) and hydrogen sulfide (HS) have emerged as promising agents for mitigating metal-induced toxicity; however, their combined effects on physiological and biochemical responses under Cr stress remain largely unexplored. This study investigated the synergistic effects of FA and HS on Cr accumulation, seedling growth, physio-biochemical attributes, leaf ultrastructure, and the expression of photosynthesis-related genes in tobacco seedlings subjected to Cr stress. Cr stress significantly inhibited plant growth, physiological and biochemical performance, gas exchange parameters, and chlorophyll synthesis, while increasing Cr accumulation in soil, roots, and shoots. In contrast, FA and HS applications, particularly in combination, significantly alleviated Cr toxicity. The combined treatment improved seedling growth, enhanced chlorophyll fluorescence efficiency, and promoted gas exchange under Cr stress. It also strengthened antioxidant defense, as indicated by increased activities of superoxide dismutase (SOD; 29%), peroxidase (POD; 41%), catalase (CAT; 36%), and ascorbate peroxidase (APX; 49%), which are crucial for reactive oxygen species (ROS) scavenging. Transmission electron microscopy analysis confirmed that FA and HS mitigated Cr-induced ultrastructure damage in leaf cells, whereas scanning electron microscopy revealed improved stomatal characteristics under combined treatment. Moreover, Cr accumulation in roots and shoots was significantly reduced. qRT‒PCR analysis showed that FA and HS regulated chlorophyll biosynthesis- and chloroplast-related genes, including CHLG (32.35%), CCD4 (71.24%), PsbO (93.30%), and PsbQ (105.90%), compared with the Cr-stressed control. Overall, the combined application of FA and HS effectively alleviates Cr-induced toxicity by reducing Cr accumulation, strengthening antioxidant defense systems, preserving leaf ultrastructure, and enhancing photosynthetic capacity and overall growth performance.

Previously, the crucial role of calcium-dependent protein kinase 3 (CPK3) in Cu2+-triggered immunity (CuTI) has been established in Arabidopsis. CPK3 promotes the phosphorylation of basic helix-loop-helix 107 (bHLH107), thereby activating the expression of 1-aminocyclopropanecarboxylic acid synthesis 8 (ACS8) and downstream defense responses. However, the precise mechanisms underlying the fine-tuning of CPK3-mediated phosphorylation of bHLH107 remain unclear. To elucidate the mechanism underlying the inactivation of the CPK3-bHLH107 module, we identified a 14-3-3 protein, GRF1 (GF14χ), among candidate bHLH107 interactors. Notably, the interaction between GRF1 and bHLH107 was weaker following Cu2+ treatment than under control conditions. In grf1 mutant plants, the transcript level of ACS8 was slightly increased, concomitant with a modest increase in resistance to Pst DC3000. GRF1 interacts with both CPK3 and bHLH107 to maintain an 'OFF' state. Cu2+ mediates the phosphorylation of CPK3, attenuating its interaction with GRF1 and promoting its dissociation from GRF1. Subsequently, CPK3 phosphorylates bHLH107 and GRF1, thereby switching the immune response to the 'ON' state. Furthermore, wild-type GRF1, the quadruple phospho-defective mutant GRF1S72/88/125/156A and the quadruple phosphomimetic mutant GRF1 S72/88/125/156D directly interact with unphosphorylated bHLH107, but not with the double phosphomimetic mutant protein bHLH107S62/72D. We also revealed that GRF1 attenuates CPK3 kinase activity. Collectively, our findings indicated that CuTI is precisely orchestrated through GRF1-mediated modulation of the CPK3-bHLH107 module in Arabidopsis.

NADPH oxidases (NOXs) are crucial enzymes for reactive oxygen species (ROS) generation in plants and play vital roles in growth, development, and stress responses. To elucidate the sequence characteristics of the NOX gene family and its low-temperature response patterns in melon (Cucumis melo L.), this study conducted genome-wide identification and expression profiling of NOX family members using bioinformatics analysis, RNA-seq transcriptome sequencing, and real-time quantitative PCR (RT-qPCR). The results revealed that eight NOX members were identified in the melon genome, distributed across six chromosomes. All members harbored conserved domains including Ferric_reductase, FAD_binding_8, NAD_binding_6, and NADPH_Ox, and the encoded proteins were generally basic and hydrophilic. Phylogenetic analysis classified the NOX proteins into five subgroups. Synteny analysis indicated the presence of only one pair of intraspecific duplicated genes in melon, which was under purifying selection. The promoter regions contained multiple hormone- and stress-responsive cis-acting elements, with CmNOX2 and CmNOX4 harboring low-temperature responsive elements. Following treatment at 4℃ for 24 h and 48 h, leaf relative electrolyte leakage (REL) increased from 28.33% to 42.67% and 52.67%, respectively; transcriptome analysis identified 5,633 and 6,882 differentially expressed genes (DEGs), respectively. Cold-responsive genes exhibited significant differential expression, with SLAC1 and CPK19 showing sustained upregulation. RT-qPCR results demonstrated that the expression of CmNOX2, CmNOX5, CmNOX6, and CmNOX7 was significantly downregulated after low-temperature treatment, whereas CmNOX4 expression was significantly upregulated at 48 h. Integrating promoter elements and expression characteristics, CmNOX4 may represent an important candidate gene involved in melon low-temperature response. This study systematically characterized the structure, evolution, and expression patterns of the melon NOX gene family, identified candidate genes responsive to low temperature, and provides a reference for further investigation into the mechanisms underlying melon cold adaptation.

Plants rely on both transcriptional reprogramming and post-translational machinery to regulate their responses to biotic stress. While transcriptome-wide dynamics underlying plant defense against insect herbivores have been extensively documented, systematic investigations into protein-level regulation and integrative analyses of these multi-layered regulatory networks remain limited. In this study, we employed quantitative proteomics and phospho-proteomics to characterize the protein-level responses of potato plants to attacks by Phthorimaea operculella. A total of 60 differentially expressed proteins (DEPs) and 56 differentially phosphorylated proteins (DPPs) were identified in potato leaves at 5 h post-herbivory. Among them, 88.3% of DEPs showed upregulated protein accumulation post herbivory, whereas 92.8% of DPPs showed downregulated protein phosphorylation post herbivory. Integrated transcriptomic and proteomic analyses revealed a multi-layered regulatory complexity in the herbivory response, with only approximately 50% of the observed proteomic changes being explained by transcriptional regulation. Notably, CDPK2 (Calcium-Dependent Protein Kinase 2) was found to be dually regulated at both transcriptional and phosphorylation levels following herbivory. Our functional analyses in Nicotiana benthamiana suggest that CDPK2 enhances plant herbivore resistance by promoting jasmonic acid (JA) biosynthesis and likely suppressing salicylic acid (SA) signaling activation during herbivory response. Collectively, this study provides integrated insights into mechanisms underlying potato herbivore defense and identifies an important regulatory factor.

Root exudates (RE) represent an important but often overlooked fraction of photosynthetically fixed carbon released into hydroponic nutrient solutions, creating a need for monitoring. Here, we developed adaptive microbial fuel cell (MFC)-based biosensors for translating the total COD of RE-organic acid mixtures into electrical signals. Anode biofilms were selectively acclimated with RE-associated organic acids, including malate, citrate, and pyruvate, with acetate as a control. After screening with real lettuce RE, citrate- and malate-acclimated MFCs were operated with RE-containing substrates for 88 days to assess sensing stability. Malate-acclimated MFCs showed consistent concentration-dependent responses, with a mean in-sample absolute relative error of 7.2%. Predictive performance was subsequently evaluated over 15 days using three parallel ma-MFCs and a previously established calibration curve. The mean absolute relative error across 45 repeated observations was 10.63% (SD, 7.66%). Multi-omics analysis showed that citrate- and malate-acclimated biofilms shared a fermentative-electrogenic route for RE sensing, but differed in auxiliary metabolic functions. Omics analyses suggested differences between citrate- and malate-acclimated biofilms in nitrogen-related metabolic potential and possible sulfur- and denitrification-associated pathways. These findings provide a basis for further investigation of the sensing mechanism. Overall, acclimated MFC biosensors offer a low-cost approach to estimating total COD of RE-organic acid mixtures during hydroponics cultivation through sequential batch measurements and support further development toward in situ, real-time, continuous monitoring.

Confronted with severe global challenges, including water scarcity, excessive use of chemical fertilizers and pesticides, and heavy metal contamination in soils, conventional agricultural technologies exhibit marked limitations in integrated water-fertilizer management and non-point source pollution control. Leveraging their excellent water retention capacity, intelligent sustained-release properties, and environmental responsiveness, hydrogels offer innovative solutions to advance sustainable agricultural development. This review comprehensively outlines the fundamental types, crosslinking mechanisms, and key functional properties of hydrogels, with a focused discussion on their agricultural deployment as high-efficiency soil conditioners, fertilizer vectors, and pesticide carriers; it deciphers the microscopic water-holding mechanisms under the tristate water model, delineates the divergent water-uptake and retention behaviors between ionic and non-ionic hydrogels, and clarifies the cyclic water-holding and release mechanisms of hydrogels during soil amelioration. Thise paper further synthesizes hydrogel-enabled environmental remediation applications, in which heavy metals and pesticide residues in soils and aquatic systems are removed via functional-group coordination adsorption or photocatalytic degradation; concurrently, hydrogels have been shown to activate plant systemic immunity through calcium-signaling pathways, thereby inducing broad-spectrum antiviral defense responses. Moreover, hydrogels can be integrated into precision agriculture frameworks to enable real-time monitoring of crop physiological status and to support targeted irrigation and fertilization management. This work also evaluates the role of hydrogels in promoting seed germination, root system development, crop metabolic regulation, and stress resilience, while introducing tailored application strategies across distinct plant growth stages. Their documented economic advantages include water conservation, enhanced crop yields, reduced dependence on synthetic fertilizers, and lower labor costs. Nevertheless, the large-scale implementation of hydrogels continues to face multifaceted challenges-particularly poor degradability and latent ecological risks, as conventional polyacrylamide (PAM)-based gels resist soil mineralization and retain potentially neurotoxic monomers, leaving a critical gap in multi-annual field data concerning their non-target interference with native soil aggregate evolution, pore distribution, and rhizospheric carbon-nitrogen footprints. Mechanistically, many hydrogels with tensile strengths below 1 MPa are highly susceptible to three-dimensional network collapse under high-salinity osmotic shock and tillage mechanical stress, exhibiting a precipitous drop in water retention after more than three wet-dry cycles due to deficient long-term structural stability. Compounding these technical gaps are elevated production costs and low farmer adoption, driven by the absence of texture-specific performance thresholds-such as an available water increment ≥ 40% for sandy soils-and the lack of established life-cycle cost models and farmer incentive mechanisms for bio-based hydrogels. Moving forward, hydrogel technology should pivot toward materials innovation and cost-reduction engineering to broaden its applicability, employ ≥3-year, multi-habitat regional trials to delineate ecological benefit-risk boundaries, and ultimately position hydrogels as pivotal enablers of sustainable, green agricultural paradigms.

Nitrogen plays a positive role in rice growth, whereas salt negatively affects it. However, rice varieties differ in nitrogen responses and whether nitrogen management can regulate the rice salt tolerance of all varieties remains largely unknown. We then investigated the effects of nitrogen levels on the responses of different rice varieties to salt stress through a comprehensive analysis including morphological, antioxidant physiological, and metabolomics‐related indicators. A hydroponic experiment was conducted with three rice cultivars (Hanyou73, Meixiangzhan2, and Xiangyaxiangzhan) grown under two nitrogen treatments (HN: full nitrogen Kamura B nutrient solution and LN: 75% nitrogen reduction Kamura B nutrient solution) and two salt treatments (W: 0 mmol L⁻¹ NaCl and S: 100 mmol L⁻¹ NaCl). This resulted in four treatments: HNW, HNS, LNW, and LNS. Variety, salt, and variety x salt significantly affected the total fresh and dry weights of rice seedlings. Interestingly, compared with the HNS treatment, the LNS treatment increased the total dry weight of Hanyou73 by 32.88%, decreased the total dry weight of Xiangyaxiangzhan by 17.28%, whereas no significant change was detected in Meixiangzhan2. In addition, nitrogen and salt affected the photosynthetic pigment content, soluble protein content, hydrogen peroxide content, and antioxidant response. Moreover, metabolic analysis revealed that the metabolism of tryptophan, D‐amino acid, and α‐linolenic acid varied with nitrogen level across different salt treatments. This study demonstrated that nitrogen availability differentially regulates salt tolerance in rice varieties. For specific varieties, low nitrogen levels are more effective in reducing reactive oxygen species (ROS) and minimizing cellular damage in rice seedlings.

Aflatoxin B₁ (AFB₁), produced by Aspergillus flavus, is a major threat to food safety and human health. In this study, we report that diethyl aminoethyl hexanoate (DA-6), a plant growth regulator, exhibits strong antifungal and anti-aflatoxigenic activities against A. flavus. DA-6 significantly inhibited mycelial growth, conidial germination, and sclerotia formation in a dose-dependent manner, while also effectively suppressing fungal colonization in stored rice bran and maize. Microscopic and biochemical analyses revealed that DA-6 disrupted plasma membrane integrity, leading to cytoplasmic leakage and hyphal collapse. Further analyses demonstrated that DA-6 caused severe oxidative-redox imbalance, characterized by excessive reactive oxygen species (ROS) accumulation and impaired antioxidant defenses, which were accompanied by mitochondrial dysfunction, reduced membrane potential, and ATP depletion. Transcriptomic profiling confirmed a global repression of the aflatoxin biosynthetic gene cluster (including aflR and aflS) and key developmental regulators (laeA and veA), resulting in the near-complete cessation of AFB₁ production. Additionally, functional characterization of two highly responsive Zn₂Cys₆ transcription factors (AFLA_059960 and AFLA_084720) further supported the involvement of oxidative stress adaptation pathways in the fungal response to DA-6-induced stress. Collectively, these findings provide mechanistic insights into the antifungal action of DA-6 and highlight its potential application for controlling fungal contamination and aflatoxin risks in agricultural commodities.IMPORTANCEPlant growth regulators (PGRs) are widely used in agriculture, yet their potential effects on fungal physiology and mycotoxin biosynthesis remain largely unexplored. In this study, we demonstrate that diethyl aminoethyl hexanoate (DA-6) caused extensive transcriptional repression of fungal developmental regulators and the aflatoxin biosynthetic gene cluster, accompanied by severe oxidative-redox imbalance and mitochondrial dysfunction. These findings reveal a previously unrecognized antifungal application of PGRs and provide new insights into their potential use for controlling toxigenic fungi and mycotoxin contamination in food and agricultural systems.

Syntrichia caninervis (S. caninervis) serves as a model organism for desiccation tolerance research because of its exceptional capacity to withstand 98% cellular water loss. Although jasmonic acid (JA) biosynthesis and signaling genes have been well characterized in vascular plants, their functional and evolutionary roles in bryophytes remain poorly understood. This study demonstrated that exogenous JA treatment significantly delayed the reduction in absolute water content (AWC) and the optimal photochemical efficiency of photosystem II (Fv/Fm) during dehydration in S. caninervis, while mitigating reactive oxygen species (ROS) accumulation through enhanced antioxidant enzyme activity. Multi‐omics analyses revealed the upregulation of JA pathway genes during early dehydration, coinciding with JA pathway protein accumulation. Notably, the genes involved in JA biosynthesis undergo phosphorylation and acetylation during dehydration. Genome‐wide analysis identified 34 JA pathway genes in S. caninervis that were uniformly distributed across chromosomes without sex chromosome association. Collinearity analysis confirmed the conservation of the JA pathway genes between S. caninervis and Physcomitrella patens (P. patens). Multiple stress‐ and phytohormone‐responsive cis‐elements were enriched in JA pathway genes, highlighting their regulatory adaptation to environmental stresses. In addition, RT‐qPCR analysis revealed that JA pathway genes responded to dehydration, salt, cold, and heat stress. Overall, these findings provide compelling evidence for the evolutionary conservation of JA pathway in terrestrial plants and advance our understanding of their functional mechanisms in desiccation tolerance.

Abstract Overexpression (OE) of phospholipase C ( PLC ) genes is known to increase the drought tolerance of various plant species. As an enzyme, PLC hydrolyses polyphosphoinosititides, which are inositol containing phospholipids with an important in cell signalling, and produces inositolpolyphosphates and phosphatidic acid, which have both been recognized as prominent second messengers in various cellular processes. How the OE of PLC leads to an increase in drought tolerance is unknown. Here, RNA-Seq analyses were performed on drought-stressed Arabidopsis thaliana wild-type (WT) plants and four distinct PLC - OE lines ( PLC2, -3, -5, or -7 ). A vermiculite-based assay was developed to measure relatively early responses in both root- and shoot tissues. In general, results showed that PLC-OE plants respond much less to drought than WT. Zooming in, focussing on gene expression responses found for all four PLC-OE lines, the most striking differences between WT and PLC-OE lines reflected genes involved in: i) phospholipid metabolism (potentially echoing increased PLC activity), ii) circadian rhythm (shifting towards a more night-like expression pattern for PLC-OEs), iii) light reactions (downregulated in PLC-OEs), iv) brassinosteroid signalling (upregulated in PLC-OEs), and v ) auxin signalling. Interestingly, several genes upstream of H 2 O 2 were found upregulated, while several ROS-induced genes were downregulated in PLC-OE lines. Though unable to pin point the primary cause of the improved drought tolerant phenotype in PLC-OE plants, several novel connections were identified, which are highlighted and discussed in more detail.

U-box-type E3 ubiquitin ligases (PUBs) are critical for signaling homeostasis and abiotic stress responses via substrate ubiquitination, yet their upstream transcriptional regulation remains largely unexplored, especially in alfalfa drought adaptation. Through GWAS of 171 alfalfa accessions, we identified a novel U-box gene, MsPUB6, on chromosome 3, which encodes a protein with E3 ubiquitin ligase activity and is induced by drought, cold, salt, and ABA. Overexpression and virus-induced gene silencing (VIGS) revealed that MsPUB6 positively regulates drought tolerance by enhancing antioxidant enzymes (SOD and CAT), reducing malondialdehyde (MDA) and reactive oxygen species (ROS) levels, and maintaining chlorophyll content and leaf water content. Further mechanistic investigation showed that the transcription factor MsDREB1C specifically binds to the CRT/DRE element in the MsPUB6 promoter and activates its transcription. CRISPR-Cas9-mediated MsDREB1C mutants exhibited a drought-sensitive phenotype similar to that of MsPUB6-silenced lines, accompanied by significantly reduced MsPUB6 expression. In contrast, MsDREB1C-overexpressing lines showed enhanced drought tolerance and upregulated MsPUB6 expression, and similarly promoted drought tolerance by alleviating oxidative damage and membrane damage, and by maintaining chlorophyll content and relative leaf water content. Collectively, this study reveals a novel regulatory module, MsDREB1C-MsPUB6, in which a DREB1 transcription factor positively regulates a U-box-type E3 ligase gene to contribute to drought tolerance in alfalfa. These findings not only provide important genetic resources for molecular breeding of drought-tolerant alfalfa but also expand our understanding of the functional diversity of plant U-box proteins.

Autophagy is an intracellular recycling pathway with profound impacts on development, growth, and stress tolerance in eukaryotes. Therefore, unravelling signalling mechanisms that modulate the process has broad applicability. Here, we characterised a small organic molecule as an enhancer of autophagy across diverse plant lineages. Through an in vivo photoaffinity proteomics approach in Arabidopsis thaliana seedlings, we identified Domain of Unknown Function 89 (DUF89) proteins as molecular targets. Hereafter, we refer to these proteins as Reactive Metabolite Damage-Control Phosphatases (RMDPs), which are metabolite-repair phosphatases that clear reactive products. The new autophagy modulator is called RMDP inhbitor-1 (RMDPi-1) due to its demonstrated inhibitory effect on AtRMDP1 activity in vitro. Binding was further confirmed through co-incubation that thermostabilised the protein. Crystal structures of AtRMDP1 were obtained via X-ray diffraction, confirming binding of RMDPi-1 in the catalytic site and revealing a flexible region near the binding pocket that was absent in previous models. Molecular docking predicted two possible binding modes consistent with the observed electron density. RMDPi-1 treatment and the phosphatase gene knockout increased autophagic flux in Arabidopsis and Chlamydomonas reinhardtii, establishing a conserved effect. The observed autophagy response is linked to a spike in reactive oxygen species (ROS). AtRMDP knockout also results in higher ROS tolerance and greater biomass in Arabidopsis seedlings. Targeted and untargeted metabolomic experiments revealed that both pharmacological and genetic suppression of RMDP leads to accumulation of glycating agents and phosphate sugars, associating loss of RMDP activity to the ROS stress that induces alternate ROS-scavenging mechanisms, and autophagy. Taken together, our integrated chemical genetics approach reveals enhanced autophagy via inhibition of RMDP family members, which impacts on plant stress tolerance via conserved metabolite damage-control functions.

Ustilaginoidea virens (Cook) Takahashi, the causal agent of rice false smut disease (RFS), causes severe losses in yield and grain quality worldwide. The ascomycete pathogen deploys a large arsenal of effectors to facilitate infection, but only a few have been functionally characterized. In this study, we characterized Uv7366, an apoplastic glycoside hydrolase family 12 (GH12) protein that was previously identified in the culture filtrate of U. virens. Purified Uv7366 triggered a reactive oxygen species (ROS) burst and strongly induced the transcription of multiple defense-associated genes, and pretreatment of rice (Oryza sativa L.) and Nicotiana benthamiana plants with Uv7366 enhanced their disease resistance. Uv7366 was strongly induced during host invasion and targeted deletion of this gene in U. virens substantially attenuated virulence on rice. Collectively, our findings demonstrate that Uv7366 facilitates U. virens infection while also being recognized by the plant innate immune system, providing new insights into the pathogenic mechanisms of U. virens and potential strategies for the biocontrol of RFS.

IntroductionDrought stress adversely affects photosynthesis, redox homeostasis and metabolic signalling in plants. Exogenous application of hormonal and nonhormonal elicitors, have emerged as promising strategies to improve plant resilience to changing climatic conditions.MethodsIn the current study, the effect of foliar application of gibberellic acid (GA3) and 6-benzylaminopurine (6-BAP) individually and in combination was assessed at different doses (0.2 and 0.4 mM) in pigeonpea (Cajanus cajan) under drought stress through integrated physiological, metabolomic and transcriptomic approaches.ResultsGrowth, photosynthetic performance and metabolic homeostasis were severely affected by drought stress. The combined application of 0.4 mM GA3+6-BAP resulted in 20.6%, 29.7% and 33.2% increase in the maximum quantum efficiency of PSII (Fv/Fm), ΔpH, and ΔΨ, respectively, compared to untreated drought-stressed plants, which shows that the stability of PSII and the energy regulation of the thylakoids improved with combined phytohormone application. Untargeted metabolite profiling revealed that drought reduced the abundance of metabolites belonging to carbohydrates, lipids and organoheterocyclic compounds respectively. While GA3+6-BAP led to the restoration of metabolites involved in carbon metabolism and antioxidative metabolism wherein highest accumulation of flavonoids, phenolics and terpenoids was noticed. Hormonal application induced 24 unique metabolic signatures including quercetin 3-(2-glucosylrhamnoside), camellianin A, 3-O-p-coumaroylquinic acid, byakangelicin. Besides, transcriptomic profiling demonstrated treatment-associated changes in the abundance of transcripts involved in photosynthesis, redox regulation and calcium-based signaling, suggesting coordinated stress signaling.ConclusionOverall, 0.4 mM GA3+ 6-BAP treatment proved to be the most effective among other treatments for the induction of drought stress tolerance in pigeonpea by dynamic transcriptional and metabolic regulatory mechanisms.

Calcineurin B‐like proteins (CBLs) are calcium sensors, and clarifying their evolutionary patterns and stress responses is critical for understanding adaptation in Magnoliaceae. Here, we characterize the CBL family in Magnoliaceae and test whether CBL4 shows conserved function in species‐specific cold responses. We identified 38 CBL genes from six Magnoliaceae species. Evolutionary analysis revealed that CBLs likely originated in green algae and mainly expanded in eudicots, while copy number in Magnoliaceae remained stable (6–7). Phylogenetic and structural analyses classified Magnoliaceae CBLs into four clades and revealed conserved EF‐hand domains and FPSF motifs. Analyses revealed purifying selection with frequent duplications, whereas positively selected lineages had fewer, suggesting functional divergence. GO/KEGG annotations linked Magnoliaceae CBLs to Ca²⁺‐mediated signaling, stress responses, and metabolism. In three Magnoliaceae species with contrasting cold tolerance (Magnolia biondii, Liriodendron chinense, and Magnolia sinica), qRT‐PCR showed an inverse cold‐induction gradient of CBL4: cold‐resistant M. biondii had the weakest upregulation, L. chinense was in the middle, and cold‐sensitive M. sinica had the strongest upregulation. Other CBL members exhibited species‐specific patterns. After equal heterologous expression of the CBL4 homologs from the three species in Arabidopsis, all lines exhibited similar enhancement of cold resistance. The results showed reduced ROS accumulation and electrolyte leakage, increased antioxidant enzyme activities, and improved photosynthetic performance under cold stress. CBL4 overexpression also enhanced the cold‐induced expression of ICE1–CBF–COR pathway genes. Together, our study refines the evolutionary framework of Magnoliaceae CBLs and highlights CBL4 expression‐level tuning as a practical route to enhance cold tolerance in woody plants.

Calcium signaling plays a central role in plant adaptation to abiotic stresses and is primarily mediated by calmodulin and its associated transcription factors. Calmodulin-binding transcription activators (CAMTAs) regulate stress-responsive gene expression, but their characteristics and functions remain largely unexplored in Aegilops tauschii Coss., the D-genome progenitor of bread wheat. In this study, a genome-wide identification and characterization of the CAMTA gene family was performed, followed by phylogenetic, structural, conserved domain, promoter cis-element, and expression analyses under drought stress. Five AetCAMTA genes were identified and classified into three phylogenetic groups. All proteins contained conserved CG-1 DNA-binding, ankyrin repeat (ANK), and IQ calmodulin-binding domains and exhibited similar exon-intron organization. Promoter analysis revealed abundant hormone- and stress-responsive cis-elements, particularly abscisic acid-responsive element (ABRE) and drought-responsive MYB-binding site (MBS) motifs, suggesting their involvement in drought-responsive signaling. Quantitative RT-PCR showed genotype- and stress-dependent expression patterns, with the drought-tolerant ecotype (TN-01-1747) exhibiting higher expression of AetCAMTA1, AetCAMTA2, and AetCAMTA3 than the drought-sensitive ecotype (TN-01-1559) under moderate drought stress. These findings provide new insights into the evolutionary and functional characteristics of AetCAMTA genes and identify promising candidates for improving drought tolerance in wheat through molecular breeding and biotechnological approaches.

BackgroundCalcineurin B-like (CBL) protein-mediated calcium signaling represents a core regulatory pathway underlying plant abiotic stress adaptation. Ziziphus jujuba var. spinosa (sour jujube) is a perennial woody species with remarkable saline-alkali and drought tolerance, yet the CBL gene family in this species has not been systematically characterized.MethodsWe performed genome-wide identification and characterization of the CBL gene family in sour jujube through integrated bioinformatic analyses, transcriptomic profiling, qRT-PCR, and yeast two-hybrid (Y2H) assays, with the annotation of one gene manually corrected based on molecular cloning and transcriptome validation.ResultsTen non-redundant ZjCBL genes were identified and grouped into three phylogenetic clades. Synteny and selection pressure analyses identified two collinear gene pairs that have undergone strong purifying selection. All ZjCBL proteins contain canonical EF-hand motifs and are predicted to be mainly localized to the plasma membrane, with ZjCBL4 and ZjCBL9 harboring additional N-terminal transmembrane helices. The promoter regions of ZjCBL genes are enriched in hormone- and stress-responsive cis-acting elements. ZjCBL1, ZjCBL2, ZjCBL3, and ZjCBL5 were highly expressed across multiple tissues, and ZjCBL1 exhibited sustained upregulation under long-term saline-alkali stress, concurrent with the accumulation of osmoprotectants. Y2H assays confirmed that ZjCBL1 interacts with two ZjCIPK proteins, exhibiting a stronger interaction signal with ZjCIPK13.ConclusionsThis study provides the first systematic characterization of the CBL gene family in sour jujube and revises the annotation of ZjCBL1, establishing ZjCBL1 as an important component of the ZjCBL-ZjCIPK signaling network and offering candidate gene resources for molecular breeding to enhance stress tolerance in fruit crops.

Copper speciation plays a central role in modulating redox reactivity and biological responses under oxidative stress conditions, for example in soybean (Glycine max (L.) Merr.) under salt stress. In this study, a series of Cu(II)-amino acid complexes (Cu(aa)2) containing glycine, serine, histidine, tryptophan and tyrosine were synthesized and characterized by elemental analysis, infrared spectroscopy, mass spectrometry, and ultraviolet-visible spectroscopy. Their reactivity toward superoxide was evaluated using the riboflavin-nitro blue tetrazolium assay and potassium superoxide kinetic assays as chemical models of reactive oxygen species (ROS) interaction. The complexes displayed ligand-dependent superoxide reactivity, with Cu(His)2 showing the highest kinetic response toward KO2-derived superoxide species. Selected compounds were subsequently evaluated in soybean plants subjected to acute salt stress to investigate the relationship between the copper coordination environment and physiological responses. Histochemical analyses revealed marked differences among treatments: whereas CuCl2 promoted increased ROS accumulation and phytotoxicity, Cu(aa)2 complexes reduced superoxide and hydrogen peroxide levels while maintaining lower toxicity. Among the evaluated compounds, Cu(His)2 exhibited the most consistent behavior, combining high chemical reactivity with effective ROS reduction in planta and moderate copper accumulation in leaf tissues. These findings demonstrate that ligand coordination strongly influences copper behavior, decoupling in vitro reactivity from biological outcome under stress conditions. Overall, the results establish a structure-reactivity-biological response relationship for Cu(II)-amino acid complexes and highlight the importance of copper speciation in the design of safer redox-active compounds for biological applications.

More frequent short-term episodes of extreme high temperatures caused by global warming seriously threaten plant growth and development around the world. Although heat stress can occur throughout the plant life cycle, exposure during the seedling stage severely impacts subsequent plant growth and productivity. In this study, we found that the Arabidopsis metacaspase mutant atmc1 exhibited a heat-sensitive phenotype at the seedling stage, and restoration of AtMC1 expression in the atmc1 substantially rescued the heat-sensitive phenotype, indicating an important role of AtMC1 in thermotolerance. To investigate the underlying molecular responses associated with AtMC1 under heat stress, RNA sequencing was performed to compare the transcriptomic profiles of wild-type and atmc1 seedlings under normal and heat treatment conditions. Gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified key genes related to heat acclimation, protein folding, and endoplasmic reticulum-associated degradation (ERAD). Genes involved in reactive oxygen species (ROS) homeostasis also displayed differential expression patterns under heat stress. In addition, 45 transcription factors belonging to the ERF, HSF, WRKY, NAC, and MYB families were differentially expressed between wild-type and atmc1 in response to heat stress. Protein-protein interaction analysis revealed 27 key heat-responsive genes, most of which were heat-induced but exhibited attenuated upregulation in atmc1. Collectively, our findings provide transcriptomic insights into the heat stress responses associated with loss of AtMC1 in Arabidopsis seedlings and provide a foundation for future mechanistic studies of AtMC1-mediated thermotolerance.

While direct‐seeded rice (DSR) offers a sustainable and resource‐efficient alternative to conventional puddled transplanting, its widespread adoption is severely constrained by the susceptibility of elite cultivars to anaerobic stress during germination, leading to poor crop establishment and substantial yield losses. To address this critical agricultural bottleneck, we conducted a comprehensive genetic evaluation of 96 rice genotypes, predominantly comprising traditional landraces from Tamil Nadu, India, to identify superior genetic donors and elucidate the molecular architecture underlying anaerobic germination (AG) tolerance. Phenotypic screening under controlled anaerobic conditions identified shoot and coleoptile elongation as key adaptive traits conferring survival advantage, with landraces Kudavazhai and Poongar demonstrating exceptional AG tolerance and representing invaluable genetic resources for future breeding initiatives. Genomewide association studies (GWASs) employing a high‐density 50 K SNP array revealed 50 significant marker–trait associations distributed across 21 linkage disequilibrium (LD) blocks spanning 11 chromosomes. The reliability of these associations is strongly supported by extensive colocalization with previously characterized quantitative trait loci (QTLs) for AG and related stress tolerance traits, including the major QTL qAG9 on chromosome 9, as well as QTLs controlling coleoptile length (qCL6b), mesocotyl development and root architectural traits (QRSA‐4, QRV‐4). These genomic regions harbour well‐characterized AG‐tolerance genes, most notably OsTPP7, which facilitates gibberellic acid‐independent starch mobilization, along with genes encoding alcohol dehydrogenase (ADH) and the metallothionein OsMT2B, collectively confirming their essential roles in anaerobic metabolic adaptation. Our investigation revealed an extensive repertoire of novel candidate genes critical for hypoxic survival, including energy metabolism regulators (ADP‐glucose pyrophosphorylase, fructose‐6‐phosphate‐2‐kinase and beta‐glucosidase), hormone biosynthesis and signalling components (ACC synthase mediating ethylene production, galactinol synthase governing raffinose family oligosaccharide synthesis), cellular stress signalling and detoxification systems (calmodulin‐like proteins, calcineurin B‐like proteins orchestrating Ca²⁺‐mediated responses, glutathione homeostasis regulators and superoxide dismutase) and polyamine biosynthesis enzymes (S‐adenosylmethionine decarboxylase). Haplotype analysis of critical LD blocks (1.1, 5.1 and 10.1) revealed functionally significant allelic variations that directly correlate with phenotypic performance. Notably, specific haplotypes of the calcineurin B‐like protein gene (Os10g0564800) within LD block 10.1 exhibited strong correlation with differential shoot elongation capacity, establishing its function as a molecular switch in Ca²⁺‐mediated stress response cascades involving the calcium‐dependent protein kinase CIPK15. These findings reinforce the complex polygenic architecture of AG tolerance, which is orchestrated through coordinated molecular networks integrating anaerobic fermentation pathways, carbohydrate mobilization systems, reactive oxygen species detoxification mechanisms and hormonal regulatory circuits. Through this integrated approach combining GWAS mapping, comprehensive candidate gene identification and functional haplotype analysis, we provide a detailed molecular framework to understand anaerobic germination tolerance, identify elite germplasm resources with superior adaptive capacity and deliver specific allelic targets to accelerate precision breeding of flood‐resilient, high‐yielding DSR varieties for sustainable rice production systems.

Plant immunity against pathogens involves multiple immune responses and intricate regulatory networks. However, how immune networks are deployed in fruit remains poorly understood. Here, we show that citrus fruit immune responses, including hydrogen peroxide (H2O2) production and callose deposition, are multiply regulated by transcriptional activation, phosphorylation, and ubiquitination. Citrus sinensis genes encoding nicotinamide adenine dinucleotide phosphate (NADPH) oxidase CsRBOHG and callose synthase CsCalS5, responsible for H2O2 production and callose deposition, respectively, are transcriptionally activated by CsWRKY46. Phosphorylation-enhanced activity of CsRBOHG by CsPBL9 enhances immunity. RING1-IBR-RING2 (RBR)-type E3 ligase CsARI1, acting as an immune brake, ubiquitinates CsRBOHG and CsCalS5 for degradation. Interestingly, CsARI1 also shows a moonlight function wherein it interacts with CsPBL9 in a non-ubiquitination manner, disrupting CsPBL9's interaction with CsRBOHG. This CsARI1-CsPBL9 interaction is stimulated by H2O2 as feedback. Moreover, H2O2 contributes to callose deposition, indicating an interplay between two immune responses. Our study reveals a tripartite regulatory hub orchestrating self-linked immunity in citrus fruit.

Plant cation/H+ exchanger 1 (CAX1) is a vacuolar Ca2+/H+ antiporter that plays a central role in calcium homeostasis. The initial 36 residues of CAX1 make up the amino-terminal regulatory region (NRR), this defining feature mediates autoinhibition and serves as a target for phosphorylation and protein-protein interactions. While CAX1 has been viewed as a transporter, recent genetic studies suggest a broader functional role. Loss of CAX1 enhances tolerance to anoxia and submergence stress, while synthetic biology reveals that transport-deficient amino-terminal modules of CAX1 influence stress responses. Notably, a dominant-negative amino-terminal construct phenocopies cax1 loss-of-function mutants, whereas a transport-deficient amino-terminal module lacking the autoinhibitory region restores anoxia sensitivity in a cax1 background. These observations are difficult to explain through altered calcium transport alone. In this review, we summarize current understanding of CAX1 structure, regulation, and physiological function and evaluate evidence supporting transport-independent activities of the amino terminus. We propose that the amino-terminal architecture of CAX1 could function as a control module that integrates environmental and cellular cues to influence stress responses. This framework expands the functional scope of CAX1 beyond ion exchange and might suggest that membrane transport proteins contribute directly to signaling networks.

Respiratory burst oxidase homologs (RBOHs) are core enzymes for reactive oxygen species (ROS) production in plants and play critical roles in stress signal transduction. In this study, we performed a genome-wide identification of the RBOH gene family in Nelumbo nucifera, comprehensively employed phylogenetic analysis, gene structure analysis, cis-acting element prediction, and other bioinformatics approaches, and examined their expression patterns under high-temperature stress using qRT-PCR. A total of ten NnRBOHs were identified, unevenly distributed across eight chromosomes. Phylogenetic analysis classified them into five subfamilies, with members within the same subfamily sharing highly similar gene structures and conserved motifs. The promoter regions contained abundant cis-acting elements associated with hormone, light, and stress responses. Under heat treatment, NnRBOHA and NnRBOHB were significantly upregulated, whereas the remaining NnRBOH family genes showed no significant changes in expression. These results indicate that different members of the NnRBOH family may play distinct roles during the heat-stress response. Collectively, this study clarifies the evolutionary characteristics of the NnRBOH family and identifies NnRBOHA as a potential key candidate gene for heat-tolerance genetic improvement, providing clear targets for subsequent functional validation and molecular breeding.

Cadmium (Cd) contamination, a major constraint on plant growth, causes a serious threat to ecological safety. Populus tomentosa is a promising woody species for Cd phytoremediation, but the mechanism of Cd phytoremediation by melatonin (MT) in this species remains unclear. To elucidate how MT confers Cd tolerance in P. tomentosa, four treatments (control, MT: 100 μmol·L-1 melatonin, Cd: 100 μmol·L-1 CdCl2, and MT + Cd, CM: 100 μmol·L-1 CdCl2 + 100 μmol·L-1 melatonin) on seedlings were conducted, and integrated analyses of physiological traits, noninvasive micro-test (NMT) data, transcriptomes, and metabolomes were further performed. Cd stress significantly inhibited seedling growth, reduced chlorophyll content, promoted excessive Cd accumulation, and triggered a burst of reactive oxygen species (ROS). Exogenous MT application reversed Cd-induced growth suppression by promoting root Cd2+ efflux while reducing Cd influx, decreasing Cd deposition in leaves, and activating superoxide dismutase and peroxidase to scavenge ROS under Cd treatment. Transcriptomic and metabolomic profiling suggested that metabolic pathways, including hormone signaling, glutathione metabolism, and phenylpropanoid biosynthesis, might be participated in the MT-mediated Cd response. Furthermore, three transcription factors, ERF098, bHLH041, and MYB308, were also identified as candidate regulators of MT-mediated Cd detoxification. Heterologous expression in yeast revealed that MYB308 not only enhanced Cd tolerance but also reduced Cd accumulation under Cd stress. These findings elucidate the multilayered regulatory network underlying MT-enhanced Cd tolerance in P. tomentosa, and provide genetic resources for breeding Cd-resistant tree varieties and theoretical guidance for remediating Cd-contaminated soils.

Soil salinization is a major abiotic stress limiting plant growth and crop productivity. MYB transcription factors play central roles in plant stress responses. Here, we investigated NtMYB78 using overexpression (OE) and knockout (KO) lines. Expression analysis revealed that NtMYB78 is strongly induced by salt stress. Phenotypic evaluation revealed that OE lines exhibited enhanced germination, biomass accumulation, and survival under salinity, whereas KO lines were highly sensitive. Physiological analyses indicated that NtMYB78 mitigates membrane damage and reactive oxygen species (ROS) accumulation, facilitated osmotic adjustment, protects the photosynthetic apparatus, and maintains Na+/K+ homeostasis. Further, transcriptomic profiling revealed that NtMYB78 orchestrates broad transcriptional reprogramming, regulating genes involved in stress defense, photosynthesis, ion transport, and phenylpropanoid metabolism. Molecular analyses using yeast one-hybrid, EMSA, and dual-luciferase reporter assays demonstrated that NtMYB78 binds to and activates the NtHCT promoter, while lignin measurements showed genotype dependent changes in lignin accumulation under salt stress. Together, these findings identify NtMYB78 as a positive regulator of salt tolerance that integrates stress responsive transcriptional reprogramming with physiological protection and modulation of lignin deposition. This gene represents a significant target for the development of salt tolerant tobacco varieties and offers a promising genetic resource for improving salt tolerance in other crop species.

Soil salinization acts as a major abiotic stress and severely hampers global agricultural productivity. Elucidating the molecular mechanisms underlying plant salt tolerance is of great significance for crop improvement. Remorin (REM) proteins are plant-specific membrane marker proteins that play a key role in stress signaling; however, their function in the salt tolerance response of sugar beet remains unclear. In this study, 14 BvREM gene family members were identified. Phylogenetic analysis revealed that this family can be divided into five subgroups and exhibits significant interspecies collinearity with Arabidopsis. Transcriptomic analysis showed that several BvREM genes were up-regulated under salt stress, with BvREM12 displaying greater expression fold change than most other family members. Using sugar beet line M14 as the gene resource material, we explored the potential function and proposed a working regulatory model for candidate gene BvREM12 in salt stress response. Subcellular localization results indicated that the BvREM12 protein is localized to the cell membrane and nucleus. Overexpression of BvREM12 significantly increased biomass accumulation in Arabidopsis, elevated antioxidant enzyme activities, decreased MDA levels, and increased proline content; a high K+/Na+ ratio was maintained, whilst Ca2+ levels were significantly higher than in the Col-0. Furthermore, qRT-PCR analysis revealed that several key salt-responsive genes were significantly up-regulated under salt stress. In summary, BvREM12 integrates the 'Ca2+-SOS3-SOS2-SOS1' signaling pathway with the 'H+-ATPase-AKT1' ion transport system to synergistically regulate Na+ efflux and K+ influx, thereby maintaining cellular ion homeostasis and redox balance, and significantly enhancing the plant salt tolerance. This study systematically characterized the sugar beet BvREM gene family and functionally dissected BvREM12. Different from previous studies on REM proteins mainly focusing on ROS scavenging at plasma membrane nanodomains, our work reveals that BvREM12, which is dual-localized to the plasma membrane and nucleus, coordinates the Ca2+-dependent SOS signaling cascade and H+-ATPase-AKT1 ion transport system to jointly maintain ion homeostasis and redox balance under salt stress. This work provides candidate genes and a mechanistic reference for molecular breeding of salt-tolerant sugar beet.

Salt stress critically impairs plant growth and development; therefore, elucidating salt-tolerance genes and their regulatory mechanisms is essential for advancing molecular breeding. In our previous study, a GRAS transcription factor, BpGRAS19, was identified within the salt-responsive gene regulatory network of birch (Betula platyphylla). Phylogenetic analysis and conserved domain prediction validated BpGRAS19 as a member of the GRAS family, which contains a highly conserved GRAS domain. Subcellular localization experiments and quantitative real-time PCR (RT-qPCR) analyses further showed that BpGRAS19 is a nuclear-localized protein with tissue-specific expression patterns, and its expression is significantly induced by salt stress in birch. Transient transformation assays confirmed that BpGRAS19 functions as a positive regulator of salt tolerance in birch. In contrast, CRISPR/Cas9-mediated knockout of BpGRAS19 remarkably reduced salt tolerance, which was associated with impaired reactive oxygen species (ROS) scavenging capacity. Yeast one-hybrid (Y1H) assays indicated that BpGRAS19 specifically binds to characterized (MBS, W-box) and novel cis-acting elements (UN1: ACCGAGC; UN2: CGACATC), thereby expanding the DNA-binding motif of GRAS transcription factors. Taken together, these results demonstrate that BpGRAS19 improves salt tolerance in birch by mitigating cell membrane damage and enhancing ROS scavenging efficiency. This study lays a foundation for understanding the molecular mechanisms underlying salt tolerance in woody plants and provides theoretical support for salt-resistant breeding of birch.

Palladium (Pd), a technology critical platinum group metal, is increasingly lost to the environment through anthropogenic activity, yet the molecular basis of Pd uptake and tolerance in plants remains poorly understood. Here, we examine the role of the Arabidopsis thaliana (Arabidopsis) COPT2 copper transporter in Pd transport and plant stress responses. Expression of COPT2 in Saccharomyces cerevisiae conferred Pd sensitivity, indicating a capability for Pd transport. Arabidopsis copt2 loss-of-function mutants accumulated less Pd in shoots and exhibited reduced reactive oxygen species (ROS) production, supporting a direct role for COPT2 in Pd distribution and associated oxidative stress. Our subsequent transcriptome profiling revealed extensive changes under Pd exposure, including downregulation of HMA2 and aquaporins, and upregulation of HMA7 , glutathione transferases, and glutamine synthetase GLN1;1 , consistent with the activation of metal detoxification and redox homeostasis pathways. Together, these findings identify COPT2 as a key transport protein mediating the uptake of Pd in Arabidopsis and suggest that Pd induces similar detoxification mechanisms as zinc (Zn), copper (Cu) and cadmium (Cd), providing a foundation for engineering plants for precious metal recovery.

Salt stress severely restricts the growth and development of pear trees, and DNA methylation may potentially modulate the salt tolerance of Pyrus betulaefolia by regulating ion transporter genes. Cyclic nucleotide-gated channel (CNGC) family genes are essential for plant ion transport and salt stress adaptation; however, the epigenetic regulatory relationship between DNA methylation and CNGC genes underlying pear salt tolerance remains unclear. In this study, 26 PbCNGC genes were systematically identified from P. betulaefolia, which possess the conserved motifs characteristic of the CNGC family and can be classified into five subclades. PbCNGC members exhibit distinct spatiotemporal expression patterns in ordinary and salt-tolerant genotypes. Under salt stress, core members PbCNGC3, PbCNGC4, PbCNGC10, and PbCNGC14 displayed typical fluctuating up-and-down expression patterns across roots, stems, and leaves, with distinct spatiotemporal specificity in their expression peaks. In contrast, PbCNGC19 and PbCNGC20;1 were exclusively induced in roots under salt stress. Quantitative results showed marked genotypic differences in salt responsiveness. At 4 h of salt treatment, the root transcript levels of PbCNGC4 and PbCNGC14 were upregulated by 8.27-fold and 6.76-fold in the salt-tolerant genotype, respectively, which were considerably higher than those in the ordinary genotype (2.08-fold and 4.50-fold). Obvious genotypic differences in mCHH methylation modifications of PbCNGC4 and PbCNGC14 were detected after salt treatment, and pharmacological experiments confirmed that DNA methylation negatively modulates the transcription of these two genes. Yeast functional complementation assays further demonstrated that PbCNGC4 and PbCNGC14 act as functional Na+ and K+ permeable cation influx channels. Combined with methylation analysis results, this study reveals a potential regulatory cascade in which DNA methylation may inhibit the transcription of PbCNGC4 and PbCNGC14, modulating their ion-transport capacity, effectively reduces Na+ overaccumulation, sustains cellular K+ retention to alleviate salt-induced ionic toxicity, and may ultimately shape the salt tolerance of P. betulaefolia. These findings enrich the understanding of epigenetic regulation of plant salt tolerance and provide valuable candidate genes and theoretical references for salt-tolerant molecular breeding in pear trees.

Cold stress, including chilling and freezing, severely limits global wheat (Triticum aestivum L.) production. Although many cold-responsive genes have been identified, the mechanisms that connect early cold sensing to transcriptional regulation and downstream physiological adaptation are still not fully understood. By generating TaCOLD1-2D overexpression and CRISPR/Cas9 knockout lines in wheat and integrating protein interaction assays, transcriptional regulation analyses, physiological measurements, and haplotype analysis of 143 Chinese wheat accessions, we characterized the TaSAP7-A2/TaCOLD1-2D/TaCML5-7B regulatory module. TaSAP7-A2 functions as a transcriptional repressor that directly binds the TaCOLD1-2D promoter under normal conditions. Cold exposure rapidly downregulates TaSAP7-A2, relieving this repression and elevating TaCOLD1-2D expression. The increased TaCOLD1-2D then recruits TaCML5-7B in a Ca2+-dependent manner. This cascade activates ABA biosynthesis and signalling, promotes rapid stomatal closure, maintains ROS homoeostasis, and fine-tunes CBF-COR gene expression, conferring robust tolerance at both seedling and booting stages without affecting growth under non-stress conditions. A natural TaSAP7-A2 promoter haplotype (Hap-7A-2) with weakened repressive activity shows strong artificial selection in high-latitude cultivars. These results uncover an efficient derepression strategy that integrates early cold perception with Ca2+-ABA-mediated protective responses. The module provides valuable genetic targets for developing climate-resilient wheat while avoiding the growth penalties associated with constitutive defence activation.

Low-temperature stress restricts plant growth, development, and geographical distribution. The C-repeat binding factor (CBF) is a core transcriptional regulator in plant cold stress responses, but its role in the thermophilic "Manaohong" cherry (Cerasus pseudocerasus Lindl.) remains elusive. Here, a cold-induced transcription factor gene, CpCBF6, was isolated and characterized from this species. Phylogenetic analysis showed its protein shares the highest homology with orthologs from Prunus persica and Prunus dulcis in Rosaceae. CpCBF6 localized to the nucleus, and its transactivation function mainly depends on the N-terminal and C-terminal domains. Forward and reverse genetics confirmed that CpCBF6 positively regulates cold tolerance. Heterologous overexpression in Arabidopsis thaliana enhanced cold tolerance, while virus-induced gene silencing (VIGS)-mediated knockdown in "Manaohong" cherry increased cold sensitivity. Physiological and biochemical analyses revealed that CpCBF6 scavenges reactive oxygen species (ROS) by enhancing superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, thereby reducing malondialdehyde (MDA) accumulation. Concurrently, CpCBF6 promoted the synthesis of soluble sugars, soluble proteins, and proline, alleviating oxidative and osmotic stress. Yeast one-hybrid and dual-luciferase assays demonstrated that CpCBF6 directly targets and activates promoters of the cold-responsive genes CpCOR27 and CpCOR47. This study provides the first systematic functional characterization of CpCBF6 in "Manaohong" cherry, elucidating its molecular mechanism in enhancing cold tolerance via activating CpCORs expression, strengthening antioxidant defense, and improving osmotic adjustment, thus offering a key candidate gene and theoretical foundation for cold tolerance breeding in fruit trees.

Soil salinization severely constrains crop yields and quality. Apocynum venetum L. is an important halophytic plant; however, its young seedlings exhibit limited salt tolerance. To elucidate the mechanism by which exogenous quercetin alleviates salt stress in A. venetum seedlings. The present work integrates phenotypic observation, physiological measurement, and transcriptome analysis. There were four treatment groups: HCK (control), HCKN (salt stress), Q3 (quercetin), and Q3 + N (quercetin + salt stress). These findings demonstrated that under salt stress, exogenous quercetin significantly improved phenotypic traits, including plant height, stem, root, and leaves. Quercetin application reduced malondialdehyde (MDA) content to maintain cell membrane stability and restore the photosynthetic system. It also enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), thereby promoting the accumulation of proline and soluble proteins and alleviating salt stress damage. Transcriptomic analysis identified 21 differentially expressed genes (DEGs) modulated by quercetin to alleviate salt stress. These DEGs were enriched in pathways associated with glycerophospholipid metabolism, plant hormone signal transduction, and secondary metabolite biosynthesis. Notably, genes such as PIOX (flavonoid synthase) and TPPD (terpenoid phenol synthase) were specifically up‐regulated. This upregulation facilitates the synthesis of salt‐stress‐mitigating secondary metabolites, including flavonoids and terpenoids, which contribute to reducing reactive oxygen species (ROS) and, subsequently, alleviate oxidative damage. The reliability of the RNA‐seq data was confirmed by quantitative real‐time polymerase chain reaction (qRT‐PCR). Collectively, these findings demonstrated that quercetin alleviates salt stress through a dual mechanism: enhancing antioxidant defenses and modulating key molecular pathways.

A critical regulator of plant tolerance to abiotic challenges is the AP2/ERF transcription factor superfamily, which encompasses four main subfamilies: AP2, ERF, RAV, and Soloist. In this study, we successfully isolated and cloned a new ERF transcription factor, named FaERF105, from Fragaria × ananassa. Subcellular localization assays demonstrated that the FaERF105 protein resides within the nucleus. Overexpressing FaERF105 conferred enhanced tolerance to drought and cold stress in both Arabidopsis thaliana and F. × ananassa. Physiologically, the transgenic lines maintained higher levels of proline and chlorophyll while exhibiting robust antioxidant enzyme activities (SOD, POD, and CAT). Conversely, the accumulation of oxidative damage markers, such as MDA and ROS (O₂ ⁻ and H₂O₂) was significantly mitigated compared to unloaded line (UL) and wild‐type (WT) controls. Taken together, the data demonstrate that FaERF105‐mediated regulation is essential for plant adaptation to adverse environmental conditions, specifically drought and cold.

Soil salinization severely impacts cotton yield and quality. Excess salt stimulates excessive accumulation of reactive oxygen species (ROS), resulting in oxidative damage and impairment of cell membrane integrity. Therefore, enhancing the antioxidant capacity of cotton is a key strategy to improve salt tolerance. In this study, we systematically characterized the salt tolerance function of GhVIP1, a bZIP family transcription factor. Overexpression (OE) of GhVIP1 enhanced tolerance to salt stress in Arabidopsis, as evidenced by higher germination rates, root length, chlorophyll content, and total antioxidant capacity (T-AOC), along with lower malondialdehyde (MDA) and ROS contents compared to the wild-type (WT). Conversely, virus-induced gene silencing (VIGS) of GhVIP1 resulted in salt-sensitive phenotypes in cotton. Yeast one-hybrid (Y1H) and dual-luciferase (LUC) reporter assays further confirmed that GhVIP1 directly binds to the promoter of GhMYB44 and activated its transcription in vitro. However, genetic analysis revealed that GhMYB44 functions as a negative regulator of salt tolerance, as its silencing significantly enhanced ROS-scavenging capacity and upregulated the expression of ROS-related genes. Notably, silencing GhVIP1 unexpectedly led to elevated GhMYB44 transcript levels in cotton, suggesting the existence of a complex in vivo regulatory network involving additional intermediate factors. Collectively, our results demonstrate that GhVIP1 positively regulates salt tolerance by enhancing ROS detoxification, partially through its interplay with the antagonistic GhMYB44 pathway. This study provides new genetic resources and a theoretical basis for molecular breeding of salt-tolerant cotton.

Humic substances (HS) are widely recognized as plant biostimulants, yet the molecular mechanisms underlying their mode of action remain incompletely defined. Here, we used RNA sequencing to investigate the early transcriptional responses of rice roots exposed for only 4 h to vermicompost‐derived humic acid (HA). Our results reveal a rapid and pronounced transcriptional reprogramming consistent with the establishment of a eustress‐like physiological state. HA treatment induced 231 genes, whereas only seven were repressed, indicating a predominantly stimulatory effect on gene expression. The induced genes were significantly enriched in functional categories related to redox homeostasis, glutathione metabolism, oxidoreductase and peroxidase activities, and cellular detoxification, suggesting an immediate adjustment of intracellular redox balance and antioxidant capacity. Concomitantly, pathways associated with phenylpropanoid metabolism, oxylipin biosynthesis, and jasmonate‐mediated signaling were activated, together with the induction of transcription factors from the WRKY and C2H2 zinc finger families, supporting early regulatory control of defense‐related networks. The coordinated activation of redox‐ and hormone‐associated pathways indicates that HA rapidly modulates ROS‐dependent signaling and integrates it with jasmonate‐centered responses. Importantly, this transcriptional signature is consistent with a eustress‐like state in which defense and adaptive mechanisms are mobilized without evidence of acute stress injury. Collectively, our findings establish a mechanistic framework for HS action in which HA functions as a chemical eustressor that rapidly reconfigures redox–hormone crosstalk, positioning reactive oxygen species signaling as a central integrative hub underlying the biostimulant and adaptive effects of humic substances in plants.

Abstract Leaf stripe disease, caused by the seed-borne fungal pathogen Pyrenophora graminea , poses a major threat to hulless barley production on the Qinghai–Tibet Plateau. In this study, a novel resistance locus, QY-156 , was identified in the Tibetan hulless barley landrace Pengcuolinzongqingke. Bulked segregant analysis coupled with whole-genome resequencing (BSA-WGRS), combined with KASP marker-based fine mapping and pangenome analysis, localized QY-156 to a 101-kb interval on chromosome 6H (533.393–533.494 Mb). Genetic analysis demonstrated that resistance is controlled by a single dominant gene. Expression analysis of candidate genes revealed that HORVU.MOREX.r3.6HG0622460.1 was strongly induced in the resistant genotype following P. graminea infection, whereas HORVU.MOREX.r3.6HG0622470.1 , encoding a GLR2.8-like protein, was significantly upregulated in the susceptible parent. Bioinformatic and structural analyses indicated that the protein encoded by HORVU.MOREX.r3.6HG0622470.1 is a glutamate receptor-like protein containing conserved PBP1_GABAb_receptor_plant, GluR_Plant, and Lig_chan domains, with predicted roles in signal transduction. These findings suggest that QY-156 represents a previously unreported resistance locus and that multiple genes within this interval may coordinately contribute to the regulation of stress responses. The tightly linked molecular markers and candidate genes identified in this study provide valuable resources for marker-assisted selection and map-based cloning of resistance genes in hulless barley.

Plants allocate fixed carbon between growth and defense based on environmental constraints, yet how light and nutrient interactively regulate this trade‐off remains unclear. Here we demonstrate that high‐light/low‐nutrient combination synergistically enhances phenylpropanoid synthesis in Agastache rugosa via ROS/RNS signaling and substrate competition cascades. Plants were grown under factorial light (0% and 50% shade) and nutrient (40–160 mg kg⁻¹) levels in a nested design. Path analysis revealed that nutrient supply strongly promotes free amino acid accumulation (β = 0.789), which negatively regulates chalcone synthase activity (β = −0.412), confirming substrate competition between protein synthesis and phenylpropanoid production. Simultaneously, high‐light increases hydrogen peroxide and inhibits nitric oxide levels (β = −0.811), yet these ROS/RNS molecules function as complementary signaling mediators that positively regulate shikimic acid synthesis and phenylalanine ammonia‐lyase activity (β = 0.789). This dual regulatory mechanism increased shikimic acid and chalcone synthase activity under high‐light/low‐nutrient conditions by 6‐fold and 5‐fold, respectively, than low‐light/low‐nutrient treatment, resulting in increases in flavonoid and ascorbic acid content. Principal component analysis confirmed light conditions explain 52.5% of metabolic variance, with nutrient availability modulating response magnitude. These findings establish that environmental stress combinations elicit non‐additive metabolic responses through integrated substrate competition and ROS/RNS signaling networks, providing a mechanistic framework for optimizing bioactive compound production in medicinal plants.

Saline irrigation is a major constraint on vegetation establishment in arid coal-mining landscapes. Nitraria tangutorum Bobrov is a xerohalophytic shrub established in the Dananhu mine-restoration area of Hami, Xinjiang, but its physiological responses to long-term saline irrigation remain insufficiently characterized. In this field study, approximately two-year-old plants were maintained for approximately two growing seasons under freshwater drip irrigation or irrigation water containing 8 or 12 g L-1 total dissolved solids. Unlike short-term controlled salinity assays that evaluate individual response pathways, the present field study integrates root and shoot responses across osmolyte accumulation, oxidative injury, redox regulation, nitrogen metabolism, phytohormone signaling, and lipid remodeling under long-term mixed-salt irrigation. Increasing salinity significantly reduced shoot total chlorophyll content, whereas shoot carotenoid content showed a nonsignificant numerical increase. Soluble sugars, proline, and soluble proteins increased, while total free amino acids declined. Superoxide anion (O2-), hydrogen peroxide (H2O2), and malondialdehyde (MDA) increased progressively, demonstrating oxidative injury. The protein concentrations of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) increased, whereas glutathione peroxidase (GSH-PX) and glutathione reductase (GR) declined. Concurrent decreases in ascorbic acid/ascorbate (AsA) and reduced glutathione (GSH), together with increases in dehydroascorbic acid (DHA) and oxidized glutathione (GSSG), indicated progressive oxidation of the cellular redox environment. Nitrate reductase (NR) declined under salinity, while glutamine synthetase (GS) and glutamate synthase (GOGAT) increased at 8 g L-1 but decreased at 12 g L-1. Growth-associated hormones declined, whereas stress-associated hormones increased. Together, these responses reveal a dose-dependent transition from co-occurring biochemical adjustment and oxidative injury at 8 g L-1 to broader redox and metabolic disruption at 12 g L-1 under long-term field irrigation. These variables provide candidate indicators of downstream salinity response, but ion homeostasis, plant water status, growth, survival, and long-term performance must be evaluated before salt tolerance or irrigation thresholds can be established.

BackgroundThis study examined how different subspecies of Triticum turgidum (T. durum, T. polonicum, T. turanicum) respond to cold and freezing, assessing their water status, stress responses, and antioxidant system, with particular focus on the structure and function of glutathione reductase (TtGR).MethodsTtGR genes were first identified from the T. turgidum genome using publicly available genomic resources such as Ensembl Plants. Promoter regions (~2 kb upstream) were analyzed to identify cis-regulatory elements using PlantCARE. Gene classification was performed based on predicted subcellular localization and conserved domain features. Plants were subjected to cold acclimation and freezing treatments, and physiological, biochemical, and enzymatic parameters were measured.ResultsBioinformatics analyses identified four TtGR genes in the T. turgidum genome. The genes in two groups: cytosolic (Class I) and chloroplastic (Class II). Gene structure analysis showed a conserved exon-intron organization, while motif analysis confirmed the presence of Nicotinamide Adenine Dinucleotide Phosphate (NADPH)-binding and redox-active domains across all TtGR proteins. Several regulatory sequences in the promoters are involved in cold (DRE), abscisic acid (ABRE), and stress (STRE) responses, indicating that TtGR genes are dynamically regulated in response to environmental changes. Physiological analyses showed that freezing treatment reduces leaf water content in all genotypes, leading to turgor loss, hydrogen peroxide (H2O2) accumulation, and increased malondealdehyte (MDA) levels. However, tolerance mechanisms addressing water stress and membrane damage differ among genotypes. At the biochemical level, activation of the antioxidant defense system occurs in all genotypes. T. turanicum displays strong defense by significantly increasing enzyme activities, ensuring that the ascorbate-glutathione cycle continues under stress. By contrast, T. polonicum, although showing increased overall enzyme activities, experiences a dramatic drop in glutathione reductase (GR) activity at freezing temperatures, which restricts reduced glutathione (GSH) regeneration and creates a functional bottleneck in the antioxidant cycle. T. durum fails to sustain enzyme activities over the stress period, leading to an intermediate-sensitive response. Thus, whereas T. turanicum effectively maintains antioxidant function during freezing, T. polonicum and T. durum exhibit less efficient stress responses, either through enzymatic bottlenecks or a lack of sustained defense.ConclusionsOne of the most striking findings of this study is the observed dissociation between TtGR gene expression levels and enzyme activities. Low temperature limits the link between transcription and enzyme function. The primary determinant of low-temperature tolerance in T. turgidum subspecies is the sustainability of GR enzyme activity and GSH regeneration under freezing conditions.

The interaction between plants and microbes is a complex network of signaling pathways that plays a pivotal role in plant health and resilience to environmental stressors. Recently, polyamines (PAs), including putrescine, spermidine, and spermine, have emerged as crucial mediators in the crosstalk between plants and microbes. These small molecules are not only involved in cellular growth and differentiation but also significantly influence the plant's response to biotic and abiotic stresses. Research indicates that PAs can enhance the expression of defense-related genes, thereby increasing resistance to pathogens such as Pseudomonas syringae and Hyaloperonospora arabidopsidis. Furthermore, PAs interact with reactive oxygen species (ROS) to form positive feedback loops that amplify defensive responses during pathogen attacks. Additionally, the modulation of polyamine metabolism has been shown to improve crop resilience under stress conditions, suggesting their potential as biostimulants in agricultural practices. This review explores the multifaceted roles of PAs in plant-microbe interactions, highlighting their implications for stress tolerance and crop protection. Understanding the molecular mechanisms underpinning polyamine-mediated crosstalk could lead to the development of innovative strategies for enhancing crop productivity and sustainability in the face of increasing environmental challenges.

Leaf senescence is a critical developmental transition that drives nutrient remobilization, profoundly impacting plant fitness and forest productivity. In dioecious plants, divergent reproductive costs drive sex-specific life-history strategies, yet how these fundamental differences are rewired at the senescence level remains largely unknown. Here, integrating physiological, hormonal, metabolomic, and transcriptomic analyses, we reveal a striking sexual dimorphism in the senescence trajectories of the perennial tree Populus deltoides. Males execute a "fast-recycling" strategy characterized by accelerated canopy yellowing, massive reactive oxygen species (ROS) bursts, rapid nitrogen export, and synergistic surges in abscisic acid (ABA) and salicylic acid (SA), which collectively support enhanced vegetative growth. In contrast, females adopt a "maintenance-and-defense" strategy, retaining higher chlorophyll levels and mitigating oxidative stress through enhanced antioxidant metabolism to prolong leaf longevity. By constructing hierarchical regulatory networks, we identify a male-biased HD-ZIP/MYB transcriptional cascade that acts as a master amplifier of senescence signals and is associated with downstream effector genes (such as PLIP2, ATGAP1, and ASG1) involved in lipid degradation and cell death. Our findings provide a comprehensive multi-omics framework demonstrating that sex shapes the fundamental paradigm of leaf senescence, highlighting an evolutionary trade-off between rapid nutrient remobilization and prolonged organ maintenance.

KEY MESSAGE: Calcium plays a role in modulating aquaporin expression, reducing oxidative stress, preserving chlorophyll content, and maintaining membrane integrity, suggesting it may enhance eucalypt resilience to water restriction. Drought is a significant abiotic stress that severely impacts the growth and productivity of forests worldwide. This study investigates the role of calcium (Ca²⁺) supply in modulating aquaporin (AQP) gene expression and mitigating oxidative stress in eucalypt (Eucalyptus urophylla × Eucalyptus camaldulensis) under water restriction. Three-month-old eucalypt seedlings were subjected to water restriction, with or without Ca²⁺ supplementation. We measured mRNA expression levels of five aquaporin isoforms (PIP1;2, PIP1;3, PIP2;1, PIP2;2, and PIP2;7), chlorophyll content, and markers of oxidative stress [e.g., hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and the activities of catalase (CAT) and superoxide dismutase (SOD)]. Our findings reveal that initial water restriction upregulated the expression of PIP1;2, PIP2;1, PIP2;2, and PIP2;7 in roots, which likely facilitated water uptake and maintained cellular hydration. However, under prolonged water limitation, plants supplemented with Ca²⁺ showed downregulation of these PIPs, possibly as a protective mechanism to reduce water loss. In contrast, plants without Ca²⁺ exhibited an upregulation of PIP1;2, PIP1;3, and PIP2;7 under prolonged water deficit. Ca²⁺ supplementation also played a crucial role in preserving chlorophyll content and reducing oxidative damage under water limitation. Plants supplemented with Ca²⁺ had lower levels of H₂O₂ and MDA, reduced oxidative damage and membrane permeability, alongside increased CAT activity, which suggests an enhanced oxidative response. Our results provide new insights into how Ca²⁺ enhances eucalypt responses to water deprivation, enhancing our understanding of the function of Ca²⁺ in drought responses.

Salt stress is one of the major abiotic factors limiting rice growth, development and yield. In this study, a member of the AP2/ERF transcription factor family, OsERFSR1, was cloned from rice. Overexpression of OsERFSR1 (OE) significantly reduced salt tolerance, with survival rates of 53.2% compared with 70.2% in wild-type (WT) plants, whereas CRISPR/Cas9-mediated knockout (ko) markedly enhanced salt tolerance, with survival rates of 54.6% compared with 28.2% in WT, suggesting that OsERFSR1 acts as a negative regulator of salt stress tolerance. Physiological analyses revealed that, under salt stress, the OE lines accumulated significantly higher levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2), but exhibited lower activities of antioxidant enzymes and slower accumulation of osmotic regulatory substances compared with WT plants. Conversely, the ko lines displayed opposite physiological trends. Transcriptome analysis identified differentially expressed genes among the three genotypes that were mainly enriched in tryptophan metabolism and plant hormone signal transduction pathways. Taken together, our findings suggest that OsERFSR1 is correlated with salt tolerance in rice, and may modulate this process through tryptophan metabolism and hormone signaling pathways, thereby affecting the downstream antioxidant defense system. This study provides a promising candidate target gene for improving salt tolerance in rice through gene editing approaches.

Ursolic acid (UA) is a natural pentacyclic triterpene present in many fruits, vegetables and medicinal plants. We tested whether adding UA to the freezing extender improves the quality of frozen-thawed goat sperm. Ejaculates from six bucks were pooled at each collection, and the pooled sample was split into six equal aliquots diluted in Andromed® extender either without UA (UA-0, control) or with 0.5 µM (UA-1), 2 µM (UA-2), 5 µM (UA-3), 15 µM (UA-4) or 30 µM (UA-5). Samples were frozen using a standard protocol and thawed at 37 °C for 30 s. Post-thaw sperm quality was assessed by computer-assisted sperm analysis (CASA) for motility, the hypo-osmotic swelling test for membrane integrity, and flow cytometry for acrosome status (FITC-PSA/PI), phosphatidylserine externalization (Annexin V-FITC), mitochondrial membrane potential (Rhodamine 123) and intracellular reactive oxygen species (H₂DCFDA). Adding UA changed progressive motility (PR) and mitochondrial activity (MA), and both peaked at 0.5 µM. PR at 0.5 µM was significantly higher than at 15-30 µM, and MA at 0.5-2 µM was significantly higher than at 30 µM; however, no UA concentration differed significantly from the control. Total motility, membrane integrity (HOST), acrosome integrity, phosphatidylserine externalization and intracellular ROS did not differ among the treatments. In short, the response was dose-dependent: sperm frozen with 0.5 µM UA retained the best PR and MA, the high concentrations were harmful, and an improvement over the control itself could not be demonstrated. Confirming a fertility benefit will require in vitro fertilization and in vivo insemination trials.

The alpha/beta hydrolase DANGEROUS MIX 3 (DM3), a proline aminopeptidase in Arabidopsis thaliana , contributes to stress resilience through both metabolic regulation and immune signaling. Its functions are partitioned within its oligomeric structure as a trimer-of-dimers, in which the immune regulatory switch resides at the dimer interface. While the enzymatic activity of DM3 is not necessary for immune response, its activity to free prolines is critical to promote tolerance to salt and drought stress. However, how this activity is regulated has remained unclear. Here, we show that DM3 undergoes dynamic and reversible assembly into higher-order filaments in a salt-sensitive manner. The cryo-electron microscopy (EM) structure of filamentous DM3 reveals that the three-stranded helical filament arises from rearrangements of a planar hexamer into a tilted hexameric configuration. Notably, within these filaments, one of the catalytic residues is flipped out to disrupt the catalytic geometry, rendering the enzyme inactive. These findings establish filament assembly as a mechanism to sequester DM3 in an inactive state and suggest that the transition between distinct oligomeric states enables DM3 to coordinate its roles in biotic and abiotic stress responses. Significance statement Proteins with multiple interaction interfaces can assemble in different ways to regulate their function, but how this flexibility controls plant stress responses remains poorly understood. The Arabidopsis protein DM3 has dual roles as a metabolic enzyme and an immune regulator, and these functions are determined by its assembly state. Here, we show that DM3 reversibly assembles into higher-order filaments in a salt-sensitive manner. This transition disrupts its active site, switching off its enzymatic activity. Our findings reveal how structural plasticity in protein assemblies enables a single protein to coordinate multiple stress-response functions, highlighting a general mechanism for regulating protein activity in plants.

Passiflora edulis seeds are frequently discarded as agro-industrial residues despite being a source of bioactive compounds such as piceatannol (PIC). Their valorization through ethanolic extraction aligns with circular economy principles and enables the investigation of antioxidant, anti-inflammatory, and antidiabetic potential relevant to metabolic disorders associated with oxidative stress, inflammation, and impaired glucose homeostasis. Therefore, this study compared the biological activities and toxicological profiles of P. edulis seed ethanol extract (PESE) and PIC using in vitro and in vivo assays, including human and nonhuman models, and hyperglycemia induced during embryogenesis. The zebrafish procedures were approved by the Animal Use Ethics Committee (CEUA; protocol No. 13/2022). LC-MS identified PIC among the main constituents of PESE, which exhibited a high total phenolic content and relevant antioxidant capacity. PIC showed greater antioxidant potency than PESE against DPPH•, HOCl, and O2 •-. It also inhibited α-amylase and α-glucosidase, with IC50 values of 11.8 and 3.1 μg/mL, respectively, outperforming acarbose against α-glucosidase, and activated glucokinase. In contrast, PESE inhibited PTP1B (IC50 = 29.8 μg/mL), an effect not observed with PIC alone. Both samples reduced ROS production in human neutrophils, with PIC displaying high potency in the luminol assay (IC50 = 0.12 μg/mL). In zebrafish embryos, PESE was less embryotoxic than PIC, with LC50 values of 39.3 and 6.2 μg/mL, respectively. In the hyperglycemic zebrafish model, both samples significantly reduced total free glucose and oxidant activity (p ≤ 0.05) and induced distinct antioxidant gene-expression responses: PESE upregulated nfe2l2a, sod1, sod2, and cat, whereas PIC upregulated nfe2l2a, gpx1a, and cat. These findings demonstrate that PESE and PIC act through multiple complementary mechanisms to counteract hyperglycemia and oxidative stress, highlighting the therapeutic potential of an underutilized byproduct while emphasizing the importance of developmental toxicity assessment for the safe advancement of plant-derived agents.

Aims High-temperature (HT) stress significantly impairs plant growth by impairing antioxidant enzyme activities and the photosynthetic apparatus. Melatonin (ME) is a novel plant hormone that plays diverse roles in regulating tolerance to abiotic stresses. This study examined the role and potential of ME in mitigating the negative impacts of heat stress on water spinach plants. Methods Different concentrations of ME (50, 100, 150, and 200µM) were foliar-sprayed for 12 days (every 3 days). After ME pretreatment, water spinach plants were exposed to heat stress (40 °C) for 96hours. Results HT stress dramatically decreased biomass production by hindering pigment concentration, lowering gas exchange parameters, impairing root architecture, and altering antioxidant enzyme activities. In contrast, foliar application of ME (100µM) significantly improved biomass production, photosynthetic performance, and root architecture. In addition, ME (50-200µM) effectively reduced the accumulation of H2O2 and O2•-, restored cellular membrane integrity by reducing MDA accumulation, and regulated the ascorbate-glutathione cycle. Furthermore, foliar application of ME considerably increased secondary metabolite concentrations (anthocyanin, flavonoids, and phenols), and these secondary metabolites strongly controlled ROS overproduction. Conclusions Our study found that ME (100µM) may effectively mitigate heat-induced stress in water spinach by enhancing the photosynthetic machinery, regulating redox homeostasis, reducing cellular membrane damage, and modulating antioxidant defense and the AsA-GSH cycle.

Hydrophobic nano-calcium superphosphate (NPS1) is a novel nano-fertilizer that has shown great potential for enhancing plant growth and suppressing foliar diseases. However, the mechanisms underlying these effects remain unclear. We investigated the responses of grape leaves and phyllosphere microbiota to NPS1 under both open-field and rain-shelter cultivation systems. NPS1 application significantly reduced leaf wetness duration and the disease severity of downy and powdery mildew, increased leaf-associated phosphorus retention, which remained elevated at later sampling stages, while enhancing vine growth, increasing SOD and POD activities, and inhibits several grape pathogens in vitro. Integrated transcriptomic and metabolomic analyses identified substantial changes in gene expression and metabolite accumulation following NPS1 treatment, characterized by the upregulation of stress-responsive genes (WRKY41, PEROXIDASE, and EREBP) and enrichment of pathways related to phenylpropanoid and secondary metabolite biosynthesis. Exogenous validation experiments indicated that coumaric acid and lignin were associated with enhanced seedling growth and restricted pathogen growth in vitro. Furthermore, amplicon sequencing revealed significant shifts in the composition, diversity, and co-occurrence patterns of phyllosphere microbial communities, accompanied by reduced relative abundances of pathogen-associated taxa (e.g., Erysiphe and oomycetes). Notably, representative beneficial isolates (e.g., Sphingomonas spp. and Bacillus spp.) exhibited stronger pathogen inhibition when combined with NPS1 than when applied alone. Collectively, these findings indicate that NPS1 alters the leaf surface conditions through reduced leaf wetness, associated with coordinated changes in plant physiological responses, metabolite accumulation, and phyllosphere microbial communities, which may be linked to reduced disease severity and improved grapevine performance.

IntroductionSterility mosaic disease (SMD) represents a critical biotic constraint that severely limits pigeonpea yields throughout South Asia. The disease is incited by Pigeonpea sterility mosaic emaravirus (PPSMV), which is actively transmitted by the eriophyid mite vector Aceria cajani.MethodsHost-pathogen interactions were comprehensively analyzed using a multi-disciplinary approach. This included field screening, molecular and microscopic diagnostics along with biochemical, spectroscopic and elemental profiling.ResultsOver two growing seasons, genotypes ICPL 20096-1 and ICPL 99090 exhibited high resistance, while the susceptible check (ICP 8863) exceeded 90% disease incidence. Molecular analysis confirmed PPSMV-2 as a geographically related strain of Emaravirus toordali and Emaravirus cajani. SEM revealed severe trichome deformities and collapsed stomatal guard cells in infected leaves. Biochemically, advanced disease infection increased stress injury, phenol content, peroxidase and polyphenol oxidase activities while depleting chlorophyll and soluble sugars. This reflects triggered progressive physiological impairment and active biochemical defenses. Fourier Transform Infrared Spectroscopy (FTIR) analyses revealed prominent shifts in protein, lipid and polysaccharide spectral bands (N-H, C-H, C=O, C-O-C stretching) which tentatively point to membrane damage and modified primary metabolic state following infection. Concurrently, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) data demonstrated a noticeable reduction in tissue concentrations of Na, Mg, Al and K. This elemental depletion included a drop in calcium levels that may have altered downstream calcium signaling pathways and cellular structural integrity accompanied by decreased manganese concentrations that suggest a potential inhibition of manganese-activated enzymes required for carbohydrate metabolism.DiscussionCollectively, these baseline findings identify candidate spectral and mineral biomarkers unique to resistant genotypes while reinforcing the genetic dominance of PPSMV-2. This exploratory profile of SMD associated elemental and spectral shifts serves as a template for validating resistance traits across a wider range of pigeonpea genotypes.

Cadmium pollution severely suppresses potato growth, impairs photosynthesis, and disrupts cellular redox balance, threatening crop yield and food safety. Exogenous small-molecule regulators represent low-cost strategies to alleviate heavy-metal phytotoxicity, yet systematic comparisons of N-acetyl-L-cysteine (NAC), L-tryptophan (Trp), and melatonin (MT) on Cd-stressed potatoes remain insufficient. In this study, two rounds of concentration-gradient screening using morphological and physiological assays identified 5 μM of melatonin as the optimal treatment to alleviate cadmium-induced damage in potato seedlings. This concentration significantly restored shoot height, root length, fresh weight, and chlorophyll content. Relative to the Cd-only control, 5 μM of MT reduced MDA and cadmium accumulation by 51.88% and 49.26%, respectively, while increasing proline content by 40.99%. Transcriptomic analysis revealed that MT reversed Cd-induced suppression of photosynthesis and carbon fixation pathways and activated carbohydrate metabolism. Weighted gene co-expression network analysis identified four key modules significantly correlated with Cd tolerance and MT-mediated recovery. Eight hub genes, including StANNAT1 (Ca2+-ROS homeostasis), StNRT1.1 (nitrogen transport), StSWEET10 (carbon allocation), and StHMG1 (MVA pathway), were validated by qPCR, confirming their involvement in Ca2+-ROS signaling, membrane integrity, and photosynthetic recovery. Mechanistically, MT rebalanced cellular redox status, promoted Cd sequestration, optimized carbon-nitrogen distribution, and enhanced antioxidant defense. These findings demonstrate that exogenous MT alleviates Cd toxicity through coordinated regulation of multiple pathways, providing a practical strategy for safe potato cultivation in Cd-contaminated soils and establishing a molecular framework for understanding MT-mediated heavy-metal tolerance in crops.

Summary statement Our study reveals that microtubules regulate alfalfa ( Medicago sativa ) seed vigour by modulating reactive oxygen species (ROS) homoeostasis. Candidate microtubule‐associated proteins (MAPs) were identified through transcriptome analysis, among which MsWDL4 was functionally verified to alleviate ageing‐induced germination inhibition.

Background Mechanical signals are important regulators of cellular responses in plants. They guide plant development and can activate defense and repair mechanisms. Yet, the molecular mechanisms by which plants perceive, transduce, and interpret mechanical signals are still poorly understood. This is in part due to the lack of methods to apply local, precise and non-damaging mechanical forces to plant cells. Micro-indentation is highly suitable for this purpose, yet available instrumentation is often expensive and difficult to combine with high-resolution microscopy. Results We designed an open-source and affordable modular indentation device, consisting of 3D printed elements, 3 commercially available piezo motors, and a variety of indentation needles. Due to its modularity, the setup can be readily adapted to meet experimental requirements and works on all microscopes with bespoke adaptors. We show that the setup can be used to explore both rapid and slower touch responses, exemplified by visualizing calcium waves and actin patches induced by touch. We also show that the setup is compatible with various plant species and tissues and can be combined with high-resolution functional imaging. Conclusions The simple and flexible design of the indentation device presented in this paper ensures that any lab with a 3D printer can build their own setup at low cost and with minimal time investment. The system has a wide range of applications for live plant tissue, making indentation experiments and thereby plant mechanobiology studies, more accessible.

Cadmium (Cd), a highly toxic and mobile heavy metal, has emerged as a severe environmental concern in global agroecosystems, posing a substantial threat to human health. Although prior studies have established that ZAT6 and ZAT10 positively regulate Arabidopsis tolerance to Cd toxicity, the underlying molecular mechanisms remain largely elusive. The present study provides evidence that a class I TCP transcription factor, TCP9, significantly enhances Arabidopsis tolerance to Cd toxicity through the direct activation of ZAT6 and ZAT10 expression. The real-time quantitative PCR (RT-qPCR) analysis indicates that the expression of TCP9 was induced under Cd toxicity. Meanwhile, the tcp9 mutant exhibited heightened sensitivity to Cd toxicity, accompanied by elevated Cd accumulation in both shoots and roots. Notably, the complemented lines exhibited phenotypic characteristics analogous to those observed in the wild-type (WT) plants. Further physiological and biochemical analyses revealed that, in comparison to WT, the tcp9 mutant displayed elevated hydrogen peroxide (H2O2) accumulation and reduced contents of catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD) under Cd toxicity. Furthermore, TCP9 directly interacted with the promoters of ZAT6 and ZAT10 in vitro, facilitating their transcription and consequently enhancing plant tolerance to Cd toxicity. Overall, our findings showed that TCP9 enhances Cd tolerance via modulating ZAT6 and ZAT10, thereby identifying TCP9 as a potential key target for improving plant tolerance to Cd toxicity.

IntroductionSoil salinity poses a major constraint to cereal production, yet the genetic basis underlying salt tolerance in foxtail millet (Setaria italica (L.)) remains largely elusive.MethodsTo dissect these tolerance mechanisms, we integrated phenotyping, physiological assays, transcriptomics, and population genomics across 18 extreme salt-tolerant panel (STP) and 18 salt-sensitive panel (SSP) accessions selected from over 500 germplasm resources, with evaluations conducted under NaCl treatment.ResultsUnder salt stress, STP accessions exhibited better growth and higher chlorophyll content (RCC: 5.9 vs. 3.0 on day 2) alongside lower membrane damage (REC: 14% vs. 24%) and ROS accumulation compared to SSP accessions. Transcriptome profiling identified a tolerance-associated module, STP007, which was enriched for AP2/ERF and IDD motifs and contained candidate transcription factors including YABBY and WRKY. Population genomics revealed that despite a relatively homogeneous genetic background, localized selective sweeps on chromosomes 3 and 6 were associated with salt tolerance. By integrating transcriptomic interaction networks with population evolutionary signatures, five core candidate genes involved in abscisic acid biosynthesis and redox homeostasis were identified, highlighting a probable genetic basis for salt tolerance.DiscussionIn summary, these findings suggest a dual-layered mechanism for foxtail millet salt tolerance, likely coordinated by localized adaptive selection at core genomic nodes and dynamic plasticity within transcriptional networks. This model offers insights into mitigating the growth-stress trade-off, presenting potential candidate targets for breeding resilient cereal crops.

Abstract Pectins are major components of the plant cell wall that contribute to multiple foundational functions of the cell. Homogalacturonan (HG) is the most common type of pectin that undergoes significant structural changes when binding to calcium, a mechanism that subsequently alters cell wall dynamics during development and in response to stress. In this study, we employed the unicellular streptophyte alga, Penium margaritaceum , to analyze the topography of its unique pectin lattice located on the outside of its cell wall. Using a variety of microscopy-based technologies, we show the sensitivity of lattice architecture to calcium levels. When allowed to recover, the cell uses multiple lattice recovery mechanisms that are parts of uni- and bipolar expansion as well as a repair system. The production of the HG lattice also requires light as it drives the production of photosynthetic carbon for pectin biosynthesis machinery. When placed under an accelerated light dark cycle, the topography of the HG exhibits a banding pattern. This work demonstrates the malleability of HG in response to external chemical and physical signals and the use of this unicellular alga in the study of pectin dynamics of plants.

Sesame is an important oilseed crop, but its germination stage growth is increasingly limited by heat stress under climate warming. However, the molecular responses underlying heat tolerance in sesame germination stage remain unclear. In this study, a heat-tolerant genotype (G14) and a heat-sensitive genotype (G9) were compared under different durations of high-temperature treatment using phenotypic, metabolomic, and transcriptomic analyses. The results showed that G14 maintained better growth than G9 under both normal and heat stress conditions, with a more pronounced advantage under high temperature. G14 also accumulated lower levels of hydrogen peroxide (H2O2) under heat stress, suggesting reduced oxidative damage and enhanced heat tolerance. Metabolomic analysis showed that differentially accumulated metabolites (DAMs) in G14 were mainly enriched in lipid metabolism, antioxidant systems, and secondary metabolism, which were activated at early stages and maintained throughout heat stress. In contrast, DAMs in G9 were primarily associated with hormone signaling, carbon metabolism, energy metabolism, and vitamin metabolism. Transcriptomic analysis further showed that heat stress induced extensive changes in gene expression. KEGG enrichment analysis suggested that differentially expressed genes (DEGs) in G14 were mainly involved in maintaining key physiological processes, including photosynthesis, protein processing, and lipid metabolism, whereas those in G9 were predominantly associated with defense and signaling pathways. Integrated metabolomic and transcriptomic analyses supported these patterns and indicated a close coordination between gene expression and metabolic changes. Transcription factor analysis identified members of the ERF and bHLH families as candidate transcription factors associated with the heat stress response in sesame. These findings provide new insights into the molecular responses underlying heat tolerance during sesame germination.

Solanesyl diphosphate synthase (SPS) is essential for plastoquinone-9 (PQ-9) biosynthesis and has emerged as a promising target for herbicide discovery. The commercial SPS herbicides bind to the dimer interface of the protein; however, substrate-competitive SPS inhibitors are still rare, particularly in agronomically relevant weed and crop orthologs. Here, we show that zoledronic acid (ZOL) inhibits Arabidopsis thaliana SPS1 with an IC50 value of 2.55 μM in an Mg2+-dependent manner. Crystal structures of ZOL-bound SPS from the weed Aegilops tauschii (AeSPS) and the crop Sorghum bicolor (SbSPS) revealed that the bisphosphonate moiety is anchored in the catalytic pocket through an Mg2+-water coordination network and conserved aspartate-rich motifs, showing substrate-competitive inhibition. ZOL induced chlorosis, root-growth inhibition, and reactive oxygen species (ROS) accumulation in A. thaliana. Transcriptomic analyses further showed that ZOL and aclonifen affected shared antioxidant-related processes, but with markedly different response magnitudes. ZOL showed good herbicidal activity against S. viridis, D. sanguinalis, V. persica, and C. album at 1000 g a.i./ha. Together, this work establishes ZOL as a mechanistically distinct SPS inhibitor and provides a structural basis for the development of competitive SPS-inhibiting herbicides.

A dual-mode sensing strategy is presented by integrating a magnetic Fe3O4/CoFe2O4/Ce-BDC (denoted as FCF/Ce-BDC) nanozyme with TMB/H2O2 chromogenic chemistry to construct peroxidase-mimetic hybrids. The FCF/Ce-BDC catalyst efficiently oxidizes 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2), generating a blue product (oxTMB, λmax = 651 nm). The subsequent reaction of sulfide under acidic conditions scavenges reactive oxygen species (ROS), predominantly hydroxyl radicals (•OH), and reduces oxidized 3,3',5,5'-tetramethylbenzidine (oxTMB), leading to a pronounced decrease in absorbance at 651 nm and a visually discernible color transition from blue to colorless. This dual-signal response enables quantitative determination of sulfide via both conventional spectrophotometric absorbance measurement and smartphone-based RGB colorimetry, achieving detection limits of 0.29 μM (absorbance mode) and 7.43 μM (RGB mode), respectively. We fabricated three portable, field-deployable sensors for on-site sulfide detection: a hydrogel film, a cotton fabric strip and a cotton swab. The hydrogel sensor leverages ion-mediated crosslinking to modulate reaction kinetics and ensure reproducible color development, whereas the cotton-based substrates exploit capillary-driven fluid transport to enrich analyte concentration, thereby enhancing sensitivity and mitigating matrix interference. This study not only advances the rational design of multifunctional nanozymes but also delivers a validated, application ready framework for their safe and selective use in environmental water matrices and human whole blood, enabling real time, cost effective, and operationally simple tracking of target pollutants without compromising analytical reliability.

Graphene oxide (GO), a two‐dimensional nanomaterial, has shown potential for improving plant stress tolerance. However, its involvement in, and mechanism of, regulating the drought stress response in apple plants remains unclear. In this study, we investigated the effects of GO on drought tolerance of M9‐T337 plants under both short‐term and long‐term drought conditions. Results revealed that under short‐term drought conditions, 0.1 and 1 mg L⁻¹ GO significantly alleviated drought‐induced damage by reducing electrolytic leakage and MDA contents, while enhancing antioxidant enzyme activities and ROS scavenging. Under long‐term drought conditions, 0.1 and 1 mg L⁻¹ GO improved photosynthetic rate and promoted root system development, thereby enhancing plant drought tolerance. Additionally, in M9‐T337 plants, GO elevated the levels of γ‐aminobutyric acid, proline, phenylalanine, arginine, and histidine, and upregulated the expression of MdCAT2, MdPOD2, MdDREB2A, MdERF1, and MdABI1. Taken together, this study connects GO with drought tolerance in apple plants, providing evidence that GO effectively enhances the drought tolerance of M9‐T337 plants. These findings offer a promising strategy for the sustainable cultivation of apple in water‐scarce regions through the application of nanomaterials.

Climate change is intensifying episodes of high irradiance and thermal stress, posing a major threat to the sustainability of citrus production. Although reflective particle films have emerged as mitigation tools, their protective effects at the anatomical and ultrastructural levels under field conditions remain insufficiently understood. Here, sweet orange plants (Citrus sinensis (L.) Osbeck cv. Valencia) were exposed to full sunlight, shaded or treated with kaolin or calcium carbonate particle films, and leaves were examined using light and electron microscopy, alongside foliar nutrient analysis. Exposure to full sunlight caused disruption of thylakoid membranes and mitochondrial ultrastructural alterations consistent with stress‐associated metabolic imbalance, increased plastoglobule accumulation, and alterations in oil cavities, indicating sustained photo‐oxidative pressure. These effects were accompanied by marked anatomical changes, including cell hyperplasia and reduced intercellular air spaces, together with elevated foliar K and Mg concentrations that may reflect stress‐driven changes in leaf structure and ion homeostasis. In contrast, both particle film treatments preserved cellular and organelle integrity comparable to that of shaded plants, demonstrating that the structural injury observed under full sunlight is directly attributable to excess irradiance. This study provides mechanistic evidence of the limits of citrus leaf structural tolerance to high irradiance and establishes a detailed anatomical and ultrastructural baseline to support the development of more effective mitigation strategies under adverse field climate conditions.

Plants maintain energy balance under salinity stress through increased respiration and energy use, processes also associated with reactive oxygen species generation. Although respiration imposes a high energy cost, mitochondrial respiration and the tricarboxylic acid (TCA) cycle activity are vital for ATP production and for providing electron donors that drive ion exclusion and ROS detoxification. This study examined the molecular basis of salinity-induced respiratory responses in barley using physiological, biochemical, metabolomic and proteomic analyses. Salt exposure resulted in sodium accumulation, decreased photosynthesis and biomass, and increased respiration. Metabolite profiling indicated activation of the TCA cycle, while proteomics showed increased abundance of all targeted TCA enzymes, including phosphoenolpyruvate carboxylase isoforms and succinate dehydrogenase. Enhanced pyruvate oxidation and accumulation of downstream metabolites are consistent with a central role for the classical TCA cycle in barley's salinity response. Conversely, reduced levels of 2-oxoglutarate and succinate, together with non-detection of key GABA shunt enzymes (SSADH, GDH), are consistent with limited GABA shunt contribution under the conditions examined, although we cannot exclude dynamic GABA cycling that does not result in net accumulation. The absence of detectable arginine and ornithine, unlike their salt-induced increase in wheat, further suggests that the GABA shunt may contribute less to barley's salinity response under these conditions. Overall, the combined metabolomic and proteomic evidence supports an interpretation that barley preferentially relies on enhanced mitochondrial respiration and the canonical TCA cycle under these experimental conditions, with a lesser contribution from GABA shunt metabolism than in wheat. As neither metabolite pool sizes nor protein abundances measure pathway flux directly, this is presented as an interpretation of the combined datasets rather than a demonstration of the relative fluxes through the two pathways. These results point to a species-specific divergence in respiratory and osmotic adjustment strategies under salinity and invite future investigation into how key compatible solutes such as glycine betaine, an alternative osmolyte with a known relationship to GABA metabolism, contribute to barley's salinity tolerance.

Innovative antibacterial agents, which need to be urgently developed, should provide a new path that avoids pathogenic bacteria drug resistance. A promising strategy to treat bacterial infections consists of implementing nanoparticles (NPs). Several nanomaterials, including metal NPs, induce oxidative stress in living cells by producing reactive radicals at their surface. The oxidative stress thus raised is a potent mechanism of antibacterial action. Owing to their reactive oxygen species (ROS)-related ability, metal NPs could contribute to the targeted bioactivity of preparations designed on their basis. However, totally inorganic nanomaterials frequently suffer from relatively low selectivity with respect to bacterial targets, limited dispersibility, and undesirable side effects linked to ambiguous toxicity. For the development of bacteria drug resistance, the incorporation of specific ligands responsible for bacterial eradication into NPs seems to be necessary. The synergistic action of NPs with natural materials demonstrates complementary effects and improved antibacterial capacity. The modification of NPs by natural organic supporting agents extracted from plants could potentially resolve some of the existing issues related to bacterial drug resistance. The purpose of the current work is to review the ROS-mediated antibacterial manifestation strategy of metal NPs conjugated with botanical bactericidal substances. Different aspects of the metal toxicity of NPs and the compromised selectivity of ROS for bacterial pathogens are also considered.

BackgroundFusarium pseudograminearum is a highly destructive fungal pathogen that threatens the production and quality of wheat and barley worldwide. Controlling this toxin-producing pathogen is a particularly significant challenge. Streptomyces spp. produce diverse secondary metabolites with promising potential for plant disease management. Therefore, in this study, we evaluated the antifungal activity of roflamycoin, a secondary metabolite produced by Streptomyces alfalfae XN-04, against F. pseudograminearum.ResultsRoflamycoin strongly inhibited the mycelial growth of F. pseudograminearum, with an EC50 value of 2.49 μg/mL. Conidial germination and conidiation were completely inhibited at 1.0 and 2.0 μg/mL, respectively. Scanning and transmission electron microscopy revealed marked morphological and ultrastructural damage to the cell walls and plasma membrane of F. pseudograminearum hyphae and conidia. Fluorescence microscopy combined with different dyes showed that roflamycoin induced reactive oxygen species accumulation and altered mitochondrial membrane potential. Additional analysis showed that roflamycoin destroyed the membrane of hyphae, resulting in exosmosis of cell contents and reduced ergosterol content. Plant infection assays further demonstrated that roflamycoin suppressed disease development on wheat coleoptiles, seedlings, and heads.ConclusionThese findings provide mechanistic insights into the antifungal activity of roflamycoin and support its potential as a lead compound against F. pseudograminearum. © 2026 Society of Chemical Industry.

Key messageOur study revealed that SlTCP16 can transcriptional activation and interaction with Sl4CL3, thereby enhancing the tomato's resistance toPstDC3000. This resistance is associated with an increased accumulation of lignin. Lignin deposition serves as a critical physical barrier against bacterial pathogens. However, the precise regulatory networks orchestrating this defense response in tomato remain incompletely understood. We identify the transcription factor SlTCP16 as a novel positive regulator of tomato immunity against Pseudomonas syringae pv. tomato (Pst DC3000). We show that Pst DC3000 infection strongly induces SlTCP16 expression. Functionally, SlTCP16 overexpression significantly reduced reactive oxygen species (ROS) accumulation and bolstered disease resistance, while SlTCP16 knockout mutants showed the opposite performance. Mechanistically, we uncover a dual regulatory mode, SlTCP16 not only directly activates the transcription of the lignin biosynthetic gene Sl4CL3 by binding to its promoter but also physically interacts with the Sl4CL3 protein. Sl4CL3 acts downstream of SlTCP16, and overexpression of Sl4CL3 enhanced disease resistance in the sltcp16 mutant. Collectively, our data suggest that SlTCP16 contributes to disease resistance by regulating Sl4CL3 through transcriptional activation and protein-protein interaction. This regulatory module is associated with increased lignin accumulation.

Alzheimer's disease (AD) is a neurodegenerative disorder, which is characterized by several features, such as the deposition of amyloid β (Aβ) fibrils in the brain, leading to the formation of plaques and secondly tau-associated neurofibrillary tangles, resulting in the degeneration of the brain cells. Another feature of AD is mitochondrial dysfunction and oxidative stress, which are linked to the excessive production of reactive oxygen species (ROS). Under physiological conditions, ROS homeostasis is well controlled by the ROS generating system and the cellular antioxidant network. This antioxidant network is in part controlled by nuclear factor erythroid 2-related factor 2 (Nrf2), and its activation protects tissues against oxidative stress and chronic inflammation. Since oxidative stress, inflammation, and impaired proteostasis are significantly involved in the pathogenesis of AD, the KEAP1-Nrf2 system has emerged as a promising therapeutic target for the disease. Caenorhabditis elegans is an invaluable model organism among others for neurodegenerative disease research, due to its short life cycle, transparent body, and fully mapped nervous system and many transgenic C. elegans models have been developed to study different aspects of AD. This review attempts to provide comprehensive understanding of the molecular characteristics and pathological basis of AD, including the altered Nrf2-ARE signaling in AD and the molecular architecture and regulation of the Nrf2-SKN-1 pathway in C. elegans. An overview of plant-derived natural products and their effect on Nrf2/SKN-1 activation. Several classical and novel transgenic strains are described, translating findings from C. elegans to mammalian models and humans, including clinical translations and ongoing trials, as well as personalized medicinal approaches are discussed.

Curcumin, a plant-polyphenolic compound, has been reported to induce autophagy and autophagic cell death in insect cells but its impact on cell cycle regulation is not well understood. In this study, we investigated the short-term effects of curcumin on the cell proliferation, cell cycle progression and autophagy-associated cellular responses in the Sf9 insect cells of Spodoptera frugiperda. Short-term treatment of Sf9 cells with low-dose curcumin (5-15 µg/mL) resulted in a continuous decrease in cell proliferation in a dose and time dependent manner. Flow cytometric analysis showed a significant increase in the number of cells in the G 1 phase, indicating G 1 cell cycle arrest. This was also supported by nuclear morphology analysis using DAPI staining. Importantly, the growth inhibition caused by curcumin was not associated with apoptotic features or DNA fragmentation. Treatment with curcumin evoked a time-dependent increase in intracellular reactive oxygen species without significant mitochondrial membrane depolarization, indicating a controlled stress response instead of mitochondrial-dependent apoptosis. Scanning electron microscopy revealed significant surface morphological remodeling including cell shrinkage and membrane irregularities consistent with structural reorganization associated with autophagy. Molecular docking analysis using conserved insect cell cycle regulatory proteins supported a potential interaction of curcumin with G1 phase cyclin-dependent kinases. Taken together, the findings demonstrate that curcumin induces autophagy-linked G1 cell cycle arrest in Sf9 cells, extending previous observations of curcumin-induced autophagy and nucleophagy. This study highlights autophagy-mediated cell cycle regulation as a key mechanism underlying curcumin-induced growth suppression in insect cells and provides insight into the cellular actions of botanical bioactive compounds relevant to insect physiology.

Climate change has intensified drought frequency and severity, threatening global food security, particularly for staple crops like maize, wheat, and soybean. As central secondary metabolites, flavonoids orchestrate cellular redox homeostasis, stress signal transduction, and metabolic plasticity during plant drought responses. Previous reviews are confined to isolated molecular cascades and separate treatment of flavonoid metabolism, rhizosphere microecology, and agronomic mitigation strategies, failing to establish an integrated multi-scale regulatory framework. To fill this fragmented gap, this review integrates advances in plant physiology, microbial ecology, and nanobiotechnology to construct a cross-scale framework of flavonoid-mediated drought resistance in major food crops. We systematically summarize species-specific flavonoid regulatory networks and metabolic reprogramming triggered by drought-induced oxidative stress, dissect rhizosphere microbiome effects on flavonoid biosynthesis and drought signaling, and elaborate novel mechanisms whereby nanomaterials reshape flavonoid metabolism and boost drought tolerance via tuning ROS homeostasis. Furthermore, this work integrates soil amendment and precision irrigation to decipher synergistic drought-resistance crosstalk among agronomic practices, crop metabolism, and root-associated microbiota. Collectively, this review unifies molecular, microbial, technological, and agronomic perspectives to establish a multi-scale, interdisciplinary framework for crop drought adaptation. It delivers fundamental theoretical support for climate-resilient agriculture and outlines priority research avenues to safeguard global food security.

Nitrogen (N) is an essential nutrient that strongly influences plant growth and development, and its availability can affect plant susceptibility to fungal pathogens. Poplar is a tree species widely distributed around the world. It is severely threatened by Melampsora larici‐populina Kleb., a biotrophic pathogen which is the most destructive foliar pathogen of poplar trees and leads to significant biomass losses. Research on the interaction between Populus and M. larici‐populina remains limited. We assessed the impact of N deficiency on foliar disease development caused by rust and to elucidate underlying physiological and molecular mechanisms in poplar, thereby providing insights into the relationship between plant nutrition and disease susceptibility. We performed an analysis of host physiological traits and plant‐pathogen interaction transcriptomics in poplar trees under N deficiency and rust infection. Notably, N‐deficient poplar trees showed reduced rust disease development, whereas N‐sufficient poplar trees infected with rust accumulated higher levels of reactive oxygen species (ROS) and nitric oxide (NO). Both N deficiency and rust infection increased secondary metabolite accumulation in poplar leaves. Furthermore, dual RNA‐seq analysis of the poplar–rust interaction identified numerous differentially expressed genes (DEGs) in poplar trees. The positive correlation in gene expression between N‐deficient and rust‐infected poplar suggests that similar response pathways are activated under both conditions. In contrast, an adequate N supply increased susceptibility to the pathogen. Under N‐sufficient conditions, rust infection preferentially activated biotic stress‐response genes while suppressing growth. N‐deficient poplar conditions accumulated more antioxidant compounds, enhancing resistance by creating a less favorable environment for pathogen development. Consistently, genes in M. larici‐populina associated with pathogen invasion and proliferation were down‐regulated in N‐deficient poplar leaves. Integrated physiological and transcriptomic analyses indicate that defenses induced by N deficiency can effectively suppress rust development and slow disease progression. These findings provide new insights into the poplar‐rust interactions and offer a basis for optimizing nutrient management to improve poplar resistance.

Main conclusionExogenous regulators mitigate waterlogging stress via a three-tier framework; integrated strategies boost field waterlogging resilience effectively. Waterlogging, an increasingly critical constraint on crop productivity with climate change amplifying extreme precipitation, limits rhizospheric O2 diffusion to induce hypoxia. This rapidly impairs mitochondrial respiration, forces a shift to low-efficiency glycolysis and fermentation, and accumulates potentially toxic by-products. Upon postanoxic stress, plants face an oxidative burst that compromises membrane integrity, suppresses photosynthesis, and destabilizes yield. Despite inherent adaptive programs (e.g., aerenchyma formation, adventitious rooting), most crops remain vulnerable to prolonged/recurrent waterlogging, necessitating practical interventions complementing genetic improvement. Here, we synthesize evidence that exogenous inputs-including phytohormones, osmoprotectants, antioxidants, gaseous signaling molecules, mineral nutrients, and beneficial microorganisms-mitigate injury by coordinating early signaling, metabolic maintenance, and rhizosphere stabilization. We integrate these effects into a three-tier framework: (i) resetting hypoxia perception/response thresholds, (ii) sustaining energy/redox homeostasis via balanced mitochondrial function and fermentation, and (iii) converting short-term tolerance to sustained recovery through morphological remodeling and rhizosphere improvement. Finally, we outline a translational strategy coupling exogenous regulation with functional microbiomes, targeted genetic improvement, and agronomic management to enhance field robustness and reduce environment-driven "effect drift". Highlights Exogenous regulators improve plant-waterlogging tolerance via a three-layer regulatory framework. A combined application of exogenous substances, microbiome, genetics, and agronomy enhances field waterlogging resistance. Hypoxia response, energy metabolism, and ROS homeostasis are core regulatory targets for stress alleviation.

Plants adapt to recurrent drought through transcriptional memory, yet the upstream regulators remain largely unknown. This study integrated expression genome-wide association study (eGWAS) across 115 Arabidopsis thaliana ecotypes with differential methylome profiling to identify these regulators. Focusing on the memory genes LKR, HIS1-3, and DREB1A, eGWAS identified signaling and epigenetic loci involved in ABA/JA responses and DNA methylation. Methylome profiling by whole-genome bisulfite sequencing of ecotypes contrasting in drought tolerance, and in the superinduction of memory genes revealed significantly greater methylation variation during the second drought (D2) than during the first (D1), highlighting the role of epigenetic reprogramming in memory maintenance. Functional validation using T-DNA mutants demonstrated specific modulation of the D2/D1 induction ratio without affecting initial drought responses. Mutants of LKR eGWAS-delineated genes AT1G56660, AT2G19120, AT4G16490, and DEG3, those of HIS1-3 eGWAS genes AT1G14220, AT2G24960, AT3G10845, AT3G19340, CNGC10, EMB2770, GRF7, and RPP2A, and DREB1A eGWAS genes AT1G67000, AT3G61610, AT5G62110, HK2, JMJ12, and LUP5 abolished respective memory gene induction. The eGWAS and methylome approaches converged on DNA repair, chromatin modification, vesicular transport, and proteostasis as core memory hubs. These findings reveal a genetic-epigenetic interplay that coordinates transcriptional memory, priming plants for rapid reactivation of stress pathways during recurrent drought.

Saline-alkali stress severely impairs plant growth by disrupting osmotic balance, inducing oxidative damage, and suppressing metabolic processes. This study investigates the efficacy of biostimulants including Trametes odorata extract (TOE), camphor, and xanthine, in mitigating Na2CO3-induced stress in wheat plants. Wheat (Triticum aestivum cv. Sakha 95) was grown under controlled conditions, with saline-alkali stress (100 mM Na2CO3) and weekly foliar treatments applied. After 60 days, comprehensive growth, biochemical, and molecular assessments were conducted. TOE exhibited a rich phytochemical profile, with soluble carbohydrates and alkaloids as major constituents in addition to high nitrogen and essential micronutrients, alongside significant antioxidant capacity. Na2CO3 stress reduced plant height, shoot water content, and photosynthetic pigments. Biostimulant applications reversed these effects, with TOE restoring Chl a and b, and xanthine enhancing carotenoids. Na2CO3 stress increased osmolytes (amino acids, proline, glycine betaine) but decreased soluble proteins. Biostimulants regulated these levels, with xanthine restoring soluble proteins to near-control values. Oxidative stress markers (H2O2, and MDA) were relieved by all biostimulants, with xanthine most effective in decreasing MDA. Antioxidant enzyme activities (SOD, APX) and stress-responsive gene expression (SOD, and CAT) were elevated under stress but differentially modulated by biostimulants, with TOE restoring homeostasis, while xanthine further enhanced MAPK and BADH expressions. TOE acted via ROS scavenging and nutrition, camphor boosted glutathione defense, while xanthine was associated with upregulated MAPK expression and enhanced flavonoid biosynthesis. Collectively, TOE, camphor, and xanthine effectively alleviated saline-alkali stress by enhancing antioxidant defenses, osmotic regulation, and photosynthetic efficiency, demonstrating their potential as sustainable crop stress mitigators.

Main conclusionWheat resilience depends on coordinated signaling, reproductive protection, and source-sink regulation, providing a framework to breed robust trait combinations that stabilize yield under combined heat and drought. Climate change is increasing the frequency and severity of heat and drought events, posing a major threat to wheat productivity, yield stability, and food security. Because these stresses often coincide in the field, their combined effects can impair growth, reproductive development, grain filling, and final yield more severely than either stress alone. Wheat resilience under such conditions depends on coordinated physiological adjustment and molecular regulation that sustain cellular homeostasis, protect reproductive tissues, and preserve yield-related traits. This review synthesizes current knowledge on the physiological and molecular bases of wheat resilience to heat and drought, with emphasis on their combined effects. We discuss major physiological responses, including photosynthetic adjustment, stomatal regulation, canopy cooling, osmotic balance, antioxidant defense, membrane stability, and source-sink coordination. We also examine key regulatory pathways involved in stress perception and adaptation, including calcium and reactive oxygen species signaling, mitogen-activated protein kinase cascades, phytohormonal crosstalk, transcriptional regulation, heat shock proteins, late embryogenesis abundant proteins, and osmoprotective and redox-associated pathways. In addition, we highlight the growing contribution of transcriptomics, proteomics, metabolomics, and phenomics to the identification of candidate genes, biomarkers, and adaptive traits. Finally, we consider how mechanistic insights can be translated into wheat improvement through molecular markers, genomic selection, gene editing, and climate-realistic phenotyping. An integrated understanding of stress signaling and adaptive trait deployment will be essential for developing wheat cultivars with improved resilience and yield stability under future climates.

The discharge of cationic azo dyes from textile industries poses significant environmental challenges due to their toxicity and resistance to conventional treatment methods. This study developed a sustainable adsorbent by modifying cotton textile waste biochar (CTWB) with hydrogen peroxide (H2O2) to enhance the removal of basic red 46 (BR46). Among the modified materials, biochar treated with 10% H2O2 (CTWB@HO10) exhibited the best performance. Under optimized conditions (pH 8, adsorbent dosage of 0.03 g/25 mL, and contact time of 90 min, initial BR46 of 52.82 mg L-1), CTWB@HO10 achieved a maximum adsorption capacity of 38.34 mg g-1 and a removal efficiency of 87.10%, which were substantially higher than those of pristine CTWB (31.69 mg g-1 and 72.00%, respectively). The adsorption kinetics were satisfactorily described by both pseudo-first-order and pseudo-second-order models, with the pseudo-first-order model showing the closest agreement with the experimental data. Diffusion analyses based on the Weber-Morris and Boyd models suggested that BR46 adsorption involved both boundary-layer diffusion and intraparticle diffusion. The equilibrium data were best described by the Sips isotherm, suggesting adsorption on energetically heterogeneous surfaces. Mechanistic analyses suggest that electrostatic attraction, hydrogen bonding, and π-π interactions, and diffusion processes collectively contribute to BR46 adsorption, which may be facilitated by the increased density of oxygen-containing functional groups, and improved accessibility of adsorption sites after H2O2 modification. Furthermore, machine learning models were developed to predict adsorption capacity, with the support vector machine (SVM) model showing the highest predictive performance among the evaluated models (R 2 = 0.956, RMSE = 2.263, and MAE = 1.662 within the available dataset). SHAP analysis provided additional insight into the relative influence of the operating variables, suggesting that adsorbent dosage and contact time were among the variables most strongly associated with the predicted adsorption capacity. These findings suggest that H2O2-modified cotton textile waste biochar may be a low-cost, sustainable adsorbent for removing cationic dyes, particularly in applications where material availability, waste valorization, and process simplicity are important considerations.

The increasing prevalence of aluminum oxide nanoparticles (Al2O3-NPs) in various industries poses potential risks to agricultural systems, highlighting the need to better understand their impact on plant health. However, studies investigating their phytotoxicity, particularly in biofuel crops, remain limited. This study investigated the effects of Al2O3-NPs on an important biofuel crop, Camelina sativa (C. sativa), focusing on phenotypic variability, biochemical responses, and gene expression dynamics. Results indicate a non-linear dose-response relationship, with higher concentrations of Al2O3-NPs significantly inhibiting root length and leaf number, while lower concentrations promoted leaf length and shoot fresh weight. Biochemical analyses revealed increased oxidative stress at higher nanoparticle concentrations, as evidenced by elevated hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels. Gene expression analysis showed significant upregulation of stress response genes such as AECC1 and AKT1, suggesting the activation of adaptive molecular responses under Al2O3-NPs exposure. This study elucidates the complex interactions between Al2O3-NPs and plant systems, highlighting both inhibitory and stimulatory effects on growth, biochemical responses, and gene expression in C. sativa. These findings contribute to the broader understanding of nanoparticle phytotoxicity and support the development of safer and more sustainable agricultural application of nanotechnology.Supplementary informationThe online version contains supplementary material available at https://doi.org/10.1007/s13205-026-05045-x.

Low light is a major environmental constraint that limits crop productivity by restricting photosynthetic carbon assimilation and disrupting photosynthetic function. Nitric oxide (NO) is an important signaling molecule involved in plant stress responses; however, its role in regulating photosystem stability under low-light conditions remains unclear. In this study, tomato seedlings were treated with the NO donor sodium nitroprusside (SNP) and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) to investigate the effects of NO-related treatments on growth, photosynthetic performance, photosystem function, electron transport, ROS accumulation, and antioxidant defense under low-light conditions. Low light markedly suppressed growth and impaired the photochemical activities of both photosystem II (PSII) and photosystem I (PSI), as indicated by decreases in the maximum quantum efficiency of PSII (Fv/Fm), the maximal P700 oxidation capacity (Pm), and electron transport rates, together with enhanced reactive oxygen species (ROS) accumulation. Exogenous NO application significantly improved photosystem performance under low-light conditions. NO enhanced PSII and PSI photochemical performance, improved excitation energy distribution, and restored photosynthetic electron transport, while strengthening antioxidant enzyme activities, thereby reducing excitation pressure and oxidative damage. In contrast, PTIO treatment aggravated photosystem impairment under low light. These findings indicate that NO contributes to maintaining photosystem function and photosynthetic electron transport under low-light conditions, highlighting its potential role in improving low-light tolerance in tomato.

Melatonin (MT) is a secondary metabolite that plays a role in environmental stress response in plants. However, the physiological and molecular mechanisms by which MT alleviates excessive copper (Cu) toxicity in rice (Oryza sativa) remain unknown. Herein, exogenous MT application increased root and shoot biomass and inhibited the expression of Cu uptake-associated genes, thereby reducing the Cu uptake capacity of the root and Cu concentration in vivo and effectively enhancing Cu stress tolerance compared with Cu treatment. Transcriptome analysis revealed the essential transcription factor CARBON STARVED ANTHER (OsCSA) as responsive to Cu stress. Compared with the wild type, OsCSA overexpression lines exhibited significantly enhanced Cu tolerance, which was manifested by increased MT content, up-regulated expression of the reactive oxygen species (ROS) scavenging genes CATALASE C and ASCORBATE PEROXIDASE, elevated activities of catalase, superoxide dismutase, and ascorbate peroxidase, and decreased hydrogen peroxide and malondialdehyde content. OsCSA-knockout plants showed the opposite results, and exogenous MT rescued its excessive Cu-sensitive phenotype. Further experiments established that OsCSA directly binds to the GTTA/TAAC element of CAFFEIC ACID O-METHYLTRANSFERASE 12 (OsCOMT12) promoter, up-regulating its expression. Under Cu stress, significant increases in Cu concentration and ROS accumulation were also observed in loss-of-function oscomt12 mutants, consistent with the results observed in OsCSA-knockout lines. Additionally, knockout of OsCOMT12 in the OsCSA-overexpression background abolished the positive effect of OsCSA on Cu stress tolerance. These findings establish the OsCSA-OsCOMT12 regulatory module as a new mechanism for MT-mediated Cu stress response in rice, providing a target for genetic improvement of Cu-tolerant crops.

Soybean cyst nematode ( Heterodera glycines , SCN) is one of the most damaging pathogens of soybean worldwide. Virulence diversity among SCN populations and reliance on a limited number of resistance sources present major challenges for durable SCN management. Here, we investigated a wild soybean ( Glycine soja ) genotype (WsR), that is resistant to two SCN populations, race 2 (HG type 1.2.5.7) and race 5 (HG type 2.5.7), and compared its transcriptional and metabolic responses with those of the susceptible genotype (WsS). WsR exhibited a substantially stronger defense-associated transcriptional response to both SCN populations, including preferential induction of genes associated with Ca²⁺/calmodulin and salicylic acid signaling. Integration of transcriptomic and time-resolved metabolomic analyses revealed convergence on phenolic metabolism, with enhanced accumulation of phenolic acids, flavonoids, and isoflavonoids in resistant WsR. Genes involved in phenolic biosynthesis and modification were also preferentially induced in WsR, linking transcriptional reprogramming with the observed metabolic response. Importantly, two resistance-associated phenolic compounds, 4-hydroxybenzaldehyde and 2,3-dihydroxybenzoic acid, directly increased mortality of SCN second-stage juveniles from both populations in a concentration-dependent manner. By connecting resistance-associated transcriptional responses and metabolic reprogramming with direct activity of specific phenolic compounds against SCN, our study provides functional evidence linking phenolic metabolism to chemical defense against SCN. Together, these findings identify enhanced phenolic chemical defense as a major component of resistance to multiple SCN populations in wild soybean and highlight wild soybean as a valuable source of molecular and biochemical diversity for SCN resistance.

This study hypothesized that phosphorus-iron (P-Fe) modification improves the pore structure and surface chemical properties of biochar, thereby enhancing its adsorption capacity for salt ions and CO2. When incorporated into saline-alkali soil, this modification improves soil fertility and enriches beneficial microbiota, ultimately directly and indirectly reducing soil salinity and alkalinity while enhancing carbon sequestration capacity. Hence, three types of P-Fe-modified biochar (P1-Fe@WBC, P2-Fe@WBC, and P3-Fe@WBC) were prepared using wheat straw biochar modified with three phosphate and iron salts, and the effects of P-Fe-modified biochar on saline-alkali soil remediation and carbon sequestration were systematically investigated. The results suggest that P-Fe modification substantially improved the pore structure of biochar, forming FeO and POC functional groups, which enhanced CO2 sequestration performance by 8.49-25.05%. Through Ca-Na exchange mechanisms and surface immobilization effects, P-Fe-modified biochar reduced soil exchangeable sodium percentage (ESP) by 21.97-61.54%. Additionally, it increased soil organic carbon (SOC) by 1.40-2.65-fold and soil inorganic carbon (SIC) by 0.31-1.33-fold. Furthermore, P-Fe-modified biochar enriched beneficial bacterial species such as Burkholderia-Caballeronia-Paraburkholderia and Mesorhizobium, thereby enhancing salt excretion potential mediated by ABC transport proteins. More importantly, iron incorporation may promot soil carbon transformation through enrichment of Acidiferrimicrobium, suggesting potential for stable carbon sequestration. These findings provide a feasible and effective approach for ecological restoration and carbon sequestration in saline-alkali soils.

Plants have to deal with a wide variety of unfavourable and unavoidable environmental conditions, among which low temperature is one of the most harmful. The temperature conditions determine plant growth, development, and geographic distribution. Recently, climate change results in an increase in the frequency of unfavourable weather phenomena, including sudden frosts. Exposure to low temperature results in disturbance of cellular metabolism and structure, leading, among other negative effects, to the occurrence of oxidative stress. Photosynthetic apparatus is especially prone to damage caused by reactive oxygen species (ROS) formed because of its overexcitation occurring at low temperatures under light. Among the mechanisms of acclimatisation to cold stress, an important one is an increase in the share of polyunsaturated fatty acid chains (PUFA) in membrane lipids to maintain membrane fluidity. However, PUFA are very susceptible to lipid peroxidation. Therefore, antioxidant protection of lipids is crucial to provide tolerance to cold stress. Prenyllipids, such as carotenoids, isoprenoid chromanols, and isoprenoid quinols, are potent lipophilic antioxidants. Additionally, carotenoids play a crucial role in photoprotection. In this review we tackle the role of lipophilic antioxidants in low temperature-induced stress. Firstly, we describe ROS properties and action, sites of their formation, and the ways of their detoxication. Then, we summarise the current knowledge concerning chemical structures, occurrence, biosynthesis, and function of prenyllipid antioxidants. The third part is devoted to the effects of low temperature stress on plants and the mechanisms enabling acclimatisation and adaptation to cold and frost. The final part focusses on the literature data concerning the role of prenyllipid antioxidants in the response to cold stress.

Global warming severely restricts the cultivation and landscape application of heat-sensitive alpine rhododendrons. Rhododendron simsii, an important parent of hybrid azaleas, exhibits strong heat tolerance and wide environmental adaptability. Heat Shock transcription Factors (HSFs) act as core regulators governing plant heat tolerance. Screening and functional characterization of HSF genes in R. simsii can supply candidate genes for genetic improvement of heat-sensitive Rhododendron species. In this study, a total of 22 HSF family members were identified from the whole-genome sequence of R. simsii, one of which encodes a gene rapidly and markedly induced by heat stress, designated RsHSFA3. Subcellular localization assay verified that RsHSFA3 localizes to the cell nucleus. Heterologous overexpression of RsHSFA3 significantly elevated basal heat tolerance and acquired heat tolerance in transgenic Arabidopsis. CRISPR/Cas9-mediated gene-edited callus lines of R. simsii targeting RsHSFA3 were generated. Physiological measurements demonstrated that RsHSFA3 positively regulates heat tolerance by boosting reactive oxygen species (ROS) scavenging capacity and preserving cell membrane stability. Integrative analyses of DAP-seq, yeast one-hybrid and dual-luciferase reporter assay validated that RsHSFA3 binds to the promoters of heat-responsive genes including RsHSP70, RsHSP30, RsAPG2, RsXPO1 and RsHSP80 to activate their transcription. Yeast two-hybrid and bimolecular fluorescence complementation assays further revealed physical interactions between RsHSFA3 and RsHSP70 as well as RsHSP82. Collectively, RsHSFA3 synergistically enhances heat tolerance in R. simsii via activating the expression of heat shock proteins and facilitating ROS detoxification. The findings provide valuable genetic resource and theoretical foundation for directed heat-tolerance breeding of Rhododendron varieties.

Cotton fiber length and seed vigor are crucial traits determining cotton farming efficiency and commercial value. Cotton fiber elongation and seed vigor establishment exhibit high spatiotemporal overlap and a close relationship. However, the underlying regulatory mechanisms orchestrating these two developmental processes remain poorly understood. Here, we identified an antagonistic module comprising radical-induced cell death 1 protein (GhRCD1) and transcription factor GhMYC3, which participates in this synergistic development. GhRCD1 positively regulated fiber elongation but negatively regulated seed vigor, whereas GhMYC3 had the opposite effects. Further studies demonstrated that GhRCD1 interacted with GhMYC3 through its RST domain, thereby attenuating GhMYC3-mediated promoter binding and transcriptional repression of GhGPX4, GhPER25, GhPER63, GhKCS12, GhCUT1, and GhFAD7A-1. In elongating fibers, higher GhRCD1 levels and lower GhMYC3 levels activate this module, which attenuates reactive oxygen species (ROS) signaling and increases linolenic acid and very-long-chain fatty acid (VLCFA) levels, thereby promoting fiber elongation. In ripening seeds, lower GhRCD1 and higher GhMYC3 repress this module, which inhibits peroxidase (PER)-mediated ROS-cell wall demethylesterification-seed mechanical properties pathway, thus contributing to seed vigor formation. The genetic evidence further supported the GhMYC3 epistasis effect on GhRCD1 during fiber elongation and seed maturation coordination. Together, our findings elucidate a trade-off mechanism orchestrated by the antagonistic GhRCD1-GhMYC3 module, which coordinates cotton fiber elongation and seed vigor formation through their specific expression profiles and downstream regulatory networks in developing fibers and seeds. This study provides a basis for synergistic improvement of cottonseed and fiber quality.

Calcium (Ca) deficiency can impair fruit development even under optimal soil Ca levels due to its transpiration-dependent transport. Because fruits exhibit significantly lower transpiration rates than leaves, any further reduction in fruit transpiration during development can limit Ca delivery, leading to lower fruit Ca content and diminished quality. Foliar application of Ca offers a potential strategy to mitigate these effects; however, its low mobility in the phloem often limits treatment efficacy. To better understand this phenomenon, we employed X-ray fluorescence spectroscopy (XRF) to investigate the penetration and transport of foliar-applied Ca, using strontium (Sr) as a physiological tracer. Based on the prevailing paradigm that Ca is largely immobile in the phloem, we evaluated the influence of osmotic regulators, sucrose, mannitol, glycerol, and potassium, on Ca transport. Results showed that Sr was effectively translocated to distal tissues. While potassium and mannitol had no significant impact on transport kinetics, sucrose and glycerol showed a trend toward enhancing Sr movement. XRF imaging of leaf tissue revealed that Sr was primarily transported through the apoplast toward the leaf margin. Moreover, foliar application of Sr combined with sucrose significantly increased Sr accumulation in seeds and in the apical portion of tomato fruits. These findings suggest that sucrose has the potential to actively enhance foliar-applied Ca mobility by acting as an osmotic regulator, accelerating short-range apoplastic movement within leaf tissues and long-distance systemic translocation to the fruit.

Plant-derived lignans are increasingly investigated as components of feed supplements in poultry production due to their antioxidant, anti-inflammatory and intestinal barrier-supporting properties. However, direct evidence linking lignan supplementation to improved nutrient digestibility remains limited, and the individual contributions of magnolol and honokiol to the biological effects of such additives are not yet fully understood. This study therefore examined the effects of a wood-derived lignan feed supplement containing both compounds on growth performance, nutrient utilization and intestinal health in broiler chickens, alongside targeted in vitro mechanistic investigations. In a 35-day broiler feeding trial, 180 birds in 6 pens per treatment received a control diet or the lignan supplement at 0.1 g/kg feed. Supplementation increased body weight by 90.8 g and reduced feed-to-gain ratio from 1.41 to 1.32. Apparent ileal digestibility of crude protein, crude ash, calcium and phosphorus increased by 2.8 %, 3.9 %, 5.1 % and 2.2 %, respectively. Jejunal gene expression analysis revealed a significant downregulation of IL-8, while TNF-α and IL-10 remained unaffected. To further investigate underlying mechanisms, magnolol and honokiol were evaluated in intestinal epithelial and immune cell models. Transepithelial transport was documented for both lignans in the Caco-2 model, with magnolol achieving higher apparent permeability. Magnolol enhanced wound closure speed in IPEC-J2 cells, reflecting a capacity to support epithelial restitution after injury. In contrast, honokiol reduced intracellular reactive oxygen species in epithelial cells and strongly suppressed nitric oxide production in LPS-stimulated macrophages, pointing to a dominant role in redox and inflammatory signaling. Both compounds inhibited NF-κB activation, though honokiol acted more broadly across TLR4-dependent and -independent pathways. These in vitro findings provide possible explanations for the in vivo effects, although their contribution to the observed responses in broilers remains to be established. Overall, magnolol and honokiol provide complementary and mechanistically distinctive activities in vitro and their combined use in a single feed supplement may therefore represent a promising strategy for improving nutrient utilization and productive performance in poultry production.

Clubroot disease is caused by the obligate biotrophic protist Plasmodiophora brassicae and severely limits Chinese cabbage (Brassica rapa subsp. pekinensis) production. Infection is accompanied by extensive reprogramming of host carbon metabolism and starch accumulation in enlarged roots. ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed step of starch biosynthesis, but the upstream regulation of the Chinese cabbage small-subunit gene BrAGPS1 remains poorly understood. We cloned the promoter regions of BrAGPS1a (BraA07g007740.3.5C) and BrAGPS1b (BraA01g014740.3.5C) from the inbred line 'Chiifu-401-42'. PlantCARE analysis predicted core promoter motifs and putative cis-regulatory elements associated with hormone, stress, and light responses. A root cDNA library prepared from 1, 3, and 5 weeks post-inoculation (wpi) was screened by yeast one-hybrid (Y1H) using selected promoter fragments as baits to recover candidate promoter-associated proteins. The screen yielded 76 nonredundant library hits, 15 of which were annotated as proteins with potential transcriptional, DNA-binding, histone-binding, or chromatin-associated functions. Integration with an RNA-sequencing (RNA-seq) time course at 1, 2, 3, and 5 wpi identified 28 Y1H-derived genes that were differentially expressed at one or more time points. Five candidates-a calcium-dependent protein kinase (CPK), a basic leucine zipper (bZIP) transcription factor, a three-high-mobility-group-box (3×HMG-box) protein, and two Alfin1-like plant-homeodomain (PHD-domain) proteins-were selected on the basis of functional annotation and infection-responsive expression. Quantitative real-time PCR (qRT-PCR) confirmed distinct treatment- and time-dependent expression patterns but did not test DNA binding or regulatory function. This study provides a prioritized set of BrAGPS1 promoter-associated candidates and testable hypotheses for investigating the transcriptional control of starch metabolism during clubroot development. One-to-one Y1H back-assays, electrophoretic mobility shift assays, promoter transactivation tests, and genetic analyses are required to establish direct binding, regulatory direction, and biological function.

Melanoma continues to be one of the most challenging diseases in oncology, given its aggressiveness and increased resistance to existing therapies. Therefore, the identification of novel therapeutic strategies is of considerable interest, with medicinal plants representing a promising source of bioactive compounds. In the present study, the phytochemical profile and the in vitro biological activity of a lyophilized aqueous leaf extract of Eriobotrya japonica (Thunb.) Lindl. cultivated in Romania were investigated. The extract was characterized by determining the total phenolic content, antioxidant capacity, and polyphenolic composition using UHPLC-MS/MS. Its cytotoxic potential and cytocompatibility were evaluated at concentrations of 50, 100, 200, 300, 400, and 500 µg/mL in two human melanoma cell lines (RPMI 7951 and A375) and in the human keratinocyte cell line HaCaT. Cell viability and morphological alterations were assessed in all three cell lines. Subsequently, the study focused on assessing the effects of the extract on melanoma cells by evaluating lysosome integrity and the intracellular production of reactive oxygen species (ROS). The impact on mitochondrial membrane potential, as well as on mitochondrial and nuclear morphology, was also determined. Finally, the HET-CAM test was carried out to assess the extract's preliminary irritant potential at 500 µg/mL. The results showed that the extract presented a high content of phenolic compounds and high antioxidant capacity. The most abundant compounds in the extract were chlorogenic acid, rutin, 3,5-dihydroxybenzoic acid/3,4-dihydroxybenzoic acid, and p-coumaric acid/trans-p-coumaric acid. With regard to biological activity, the treatment induced low cytotoxicity in HaCaT cells including at 500 µg/mL. At the same time, it produced a dose-dependent reduction in the viability of melanoma cells, accompanied by impaired lysosomal integrity. It was observed that the extract increased ROS levels and mitochondrial membrane depolarization, thereby inducing morphological changes at the mitochondrial and nuclear levels. However, across all performed assays, the most pronounced effects were observed starting at a concentration of 300 µg/mL. The HET-CAM assay classified the extract as non-irritant at 500 µg/mL. These findings suggest that Eriobotrya japonica leaf extract exerts antimelanoma effects associated with oxidative stress and mitochondrial dysfunction while maintaining a favorable preliminary safety profile.

BackgroundEnvironmental temperature critically shapes feeding behavior in ectotherms, yet the molecular mechanisms that integrate temperature stress signals with adaptive feeding decisions remain poorly understood. In this study, we explored a neuro-antioxidant circuit that links temperature sensing to feeding modulation via reactive oxygen species (ROS) signaling.ResultsTo dissect the molecular basis of temperature-dependent feeding plasticity, we characterized larval phenotypes under different thermal regimes, and showed that the feeding-promoting neuropeptide F (NPF) and the salivary gland-enriched gustatory receptor 4 (Gr4) form a positive feedback loop for larval feeding and growth. NPF and Gr4 exhibited a gland-specific and temperature-bidirectional expression pattern. At 38 °C, their transcript levels were suppressed in the mandibular gland; at 18 °C, they were induced in the labial gland, relative to a temperature of 28 °C. Extreme temperatures alter ROS levels, which in turn regulate Gr4 expression. Furthermore, we identify catalase (CAT) as the dominant antioxidant enzyme in salivary glands, whose expression and activity are differentially modulated across glands and temperatures. Intriguingly, Gr4 and CAT, as a bidirectional regulatory relationship, together maintain redox homeostasis. Disruption of either component elevates ROS and impairs feeding and growth across temperatures.ConclusionThis study reveals a previously unrecognized NPF-Gr4-CAT regulatory axis. This dual-regulatory axis, prioritizing energy intake in the cold and antioxidant defense under heat or host plant challenge, reveals how insects integrate environmental threats to balance survival and growth. These findings provide a mechanistic basis for understanding how adaptive behavioral plasticity evolves in response to complex ecological stressors. © 2026 Society of Chemical Industry.

Haloxylon ammodendron, a drought- and salt-tolerant desert shrub, is a valuable plant resource in arid ecosystems. Laccase plays a crucial role in lignin biosynthesis and adaptation of plants to abiotic stress, including heavy metals and organic pollutants; however, the specific functions of laccase genes in the highly stress-tolerant H. ammodendron remain largely unexplored. Therefore, this study focused on a novel laccase gene, HaLAC15, cloned from H. ammodendron, to investigate its role in mitigating the effects of copper (CuSO4), phenolic acids (sinapic acid, syringic acid, vanillic acid), and 2,4,5-trichlorophenol (TCP) stress. The results indicated that HaLAC15 encoded a secreted laccase protein. Overexpression of HaLAC15 in Arabidopsis enhanced growth and water status under CuSO4 stress. This improvement was associated with increased activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as reduced accumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA), thereby indicating a protective effect against oxidative damage and membrane lipid peroxidation. Furthermore, HaLAC15 overexpression increased lignin accumulation in roots, potentially limiting Cu2 + uptake and translocation. Mg, Mn, and Fe transport coefficients were also higher in HaLAC15-overexpressing lines. HaLAC15 overexpression conferred increased tolerance to phenolic acids, likely due to enhanced sinapic acid and syringic acid degradation. Overexpression of HaLAC15 also exhibited improved growth under TCP stress. Overall, these findings highlight the potential of HaLAC15 in enhancing plant stress resistance to Cu2+, phenolic acids and TCP, and its possible application in phytoremediation strategies for contaminated soils.

Potassium (K) deficiency severely constrains maize productivity, yet the molecular mechanisms underlying genotypic differences in K tolerance remain poorly understood. A comparative physiological and iTRAQ-based quantitative proteomic analysis was conducted on two maize inbred lines with contrasting K-deficiency tolerance (Ktm, tolerant; Ksm, sensitive) following 3 days of K starvation (0 mM K), using roots as the primary analytical target. Physiological assessments revealed that under K deficiency, Ktm exhibited higher root vitality (Ktm decreased by 16.65% vs. Ksm by 31.54%) and larger root volume but lower electrolyte leakage compared with Ksm. Proteomic profiling identified 93 and 126 differentially abundant proteins (DAPs) in Ktm and Ksm, respectively. Integrative analysis indicated that Ktm responsed to K deprivation though coordinated downregulation of glycolytic enzymes, differential ROS accumulation with enhanced catalase (CAT) and peroxidase (POD) upregulation, and increased the abundances of cell wall-reinforcing proteins (dirigent proteins and cinnamyl alcohol dehydrogenase). In contrast, Ksm exhibited a less efficient stress response characterized by impaired ROS signaling, H2O2 accumulation, and compromised membrane permeability. These results suggested that under short-term K starvation, K tolerance in maize was associated more closely with the efficiency of metabolic reprogramming and ROS homeostasis than with differences in root K content. Our findings provide a mechanistic framework for understanding genotypic variation in K adaptation and identify candidate protein markers for breeding K-efficient maize varieties.

Populus euphratica downregulates the transcription factor PeNAC029 during prolonged salinity, thereby promoting long-term acclimation to salt stress. To elucidate this regulatory network, we identified PeIDD5, an INDETERMINATE DOMAIN (IDD) family member, as a direct transcriptional activator of PeNAC029. This study investigates the role of the PeIDD5-NAC029 transcriptional module in the salt tolerance of two contrasting poplars, P. euphratica (salt-tolerant) and P. × canescens (salt-sensitive). PeIDD5 directly binds the conserved cis-element TTTGTCC in the promoter regions of PeNAC029 and PcNAC029. In P. × canescens, PeIDD5 overexpression increased PcNAC029 transcript abundance, whereas virus-induced gene silencing of PeIDD5 in P. euphratica leaves reduced PeNAC029 transcript levels. Notably, overexpression of PeIDD5 or PeNAC029 in P. × canescens reduced phospholipase D (PLD) and phosphatidic acid (PA) levels, impairing growth and photosynthesis, increasing Na+ accumulation, and diminishing antioxidant activity relative to wild-type controls after treatment with 150 mM NaCl for 15 d. Conversely, silencing the PeIDD5-PeNAC029 module in P. euphratica leaves enhanced PLD activity and PA accumulation, thereby promoting Na+ and ROS homeostasis under short-term salt stress (150 mM NaCl, 48 h). Thus, PeIDD5 activates PeNAC029 and PcNAC029, suppressing PLD activity and reducing PA accumulation, which disrupts ionic and redox homeostasis in salt-stressed poplars. In P. euphratica, salt-induced suppression of PeIDD5 reduces PeNAC029 levels, thereby promoting long-term salt adaptation. Therefore, genetic engineering to suppress the PeIDD5-NAC029 regulatory pathway represents a promising approach to developing salt-tolerant poplars.

ABSTRACT Plant‐parasitic nematodes (PPNs) cause major crop losses, while current nematicides face increasing regulatory restrictions and often show inconsistent efficacy. We tested whether nematode‐associated cues activate rhizosphere microbiomes and cuticle‐associated fungi to produce suppressive metabolites that impair nematodes and stimulate host defence. Maize rhizosphere microbiomes from different soils and fungal isolates recovered from nematode cuticles were exposed to Meloidogyne hapla , after which cell‐free filtrates were assessed for juvenile mortality, root invasion, gall formation, egg production and reactive oxygen species (ROS) accumulation in tomato. Nematode‐conditioned microbiome filtrates increased M. hapla juvenile mortality relative to nonconditioned controls across soils, although the magnitude of this effect varied with soil origin. Several fungal isolates also showed suppressive activity against Pratylenchus penetrans in maize and M. hapla in tomato, with distinct outcomes across biological assays. Among them, Akanthomyces sp. F20/JKI73389 was selected for mechanistic follow‐up: stimulation by M. hapla , or by nematode‐derived molecules, induced F20 to release a filtrate that reduced nematode root invasion and triggered a strong ROS response in host tissue. UHPLC‐MS analysis further revealed a distinct nematode‐induced metabolite profile in F20, including six candidate features that were absent from non‐stimulated controls and were provisionally associated with enhanced nematode mortality. These results support a model in which nematode‐derived cues alter the metabolite output of indigenous rhizosphere microbes and associated fungi in ways linked to nematode suppression and host defence, highlighting their potential as environmentally compatible tools for PPN management.

Plant-derived exosome-like nanoparticles (P-DENs) have emerged as promising bio-nanocarriers with antimicrobial potential. In this study, we isolated and characterized exosome-like nanovesicles from Rosa damascena and evaluated their antibacterial efficacy against Salmonella enterica, a significant foodborne pathogen. Rosa damascena-derived exosomes-like nanovesicles (R-DENs) exhibited 8.9 × 109 particles/mL in nanoparticle tracking analysis. Growth assays revealed significant inhibition of bacterial proliferation (63.52%). Propidium iodide (PI) staining in flow cytometry demonstrated concentration-dependent bacterial cell death (65.78%), while scanning electron microscopy (SEM) visualized distinct ultrastructural damage to the bacterial envelope, confirming membrane disruption as a primary antibacterial mechanism. Consistent with these effects, R-DENs markedly reduced biofilm formation (69.88%) and suppressed exopolysaccharide (EPS) production (69.10%). Elevated intracellular reactive oxygen species (ROS) levels (up to 65.37%) further indicated oxidative stress as a major antibacterial contributor. Moreover, R-DENs treatment impaired bacterial adhesion (77.88%) and invasion (67.37%) in L929 fibroblasts, correlating with attenuation of pathogenicity. Gene expression analysis confirmed significant downregulation of biofilm- and virulence-associated genes (fimA, csgD, bcsA, rsmA, and rpoS), linking the phenotypic changes to molecular interference with pathogenic pathways. Collectively, these findings demonstrate that R-DENs exert multifaceted antibacterial activity through membrane disruption, biofilm and EPS inhibition, ROS generation, and suppression of virulence traits, underscoring their potential as natural, biocompatible nanotherapeutics against drug-resistant bacterial infections.

This study aimed to develop and characterize a new food system for vegan consumption, using a commercial soy protein isolate (SP) enriched with a calcium salt (CaCl₂) to form emulsion‐filled protein gel with glucono delta‐lactone (GDL), based on emulsions containing 2%, 4%, or 6% soybean oil. Key formulation parameters, such as protein concentration, the protein‐to‐oil ratio, and gelling agent ratios, were optimized using static light scattering (SLS) and Turbiscan analysis. Samples prepared with 2% and 6% SP, 4% oil phase, and a GDL/SP ratio of 0.4 were chosen for more in‐depth analysis. The effects of calcium addition were investigated, and the structural properties of the matrices were characterized using confocal laser scanning microscopy (CLSM) and small‐angle X‐ray scattering (SAXS). The results revealed that incorporating 0.3% of CaCl₂ into the 6% SP‐stabilized emulsion led to a decrease in the size of the elements that dispersed light in the emulsion‐filled protein gel, as evaluated by SAXS, promoting the formation of a homogeneous emulsion‐filled protein gel network at high soy protein concentrations, as shown by CLSM, due to molecular synergistic mechanisms between GDL and CaCl₂. In conclusion, these findings demonstrate that calcium addition is a promising strategy for designing novel and homogeneous protein matrices with enhanced stability, thereby advancing the development of functional plant‐based emulsion‐filled protein gels suitable for vegan applications.

Orobanche cumana Wallr. is an obligate root parasite that mainly infects sunflower (Helianthus annuus L.), causing significant yield losses. Peroxidases are known to be involved in plant responses to abiotic and biotic stresses, while its role during parasitism has not been elucidated. This study investigated the role of OcPOD10, a peroxidase encoding gene from O. cumana, in the parasitic interaction between sunflower and O. cumana. OcPOD10 was specifically expressed in haustorium, an interface between sunflower root and O. cumana. Overexpression of OcPOD10 promoted parasite attachment, whereas silencing of OcPOD10 reduced broomrape attachment and induced haustorium necrosis. Exogenous application of peroxidase inhibitors resulted in a phenotype consistent with that of OcPOD10-silenced plants. Further mechanistic analysis revealed that silencing of OcPOD10 led to excessive ROS accumulation at the O. cumana infection site, subsequently triggering haustorium necrosis. These findings indicate that OcPOD10 acts as a positive regulator in O. cumana parasitism, maintaining haustorium integrity by scavenging ROS and thereby facilitating parasitism on sunflowers. Our study not only provides new insights into ROS-mediated parasitic mechanism but also offers a theoretical basis and potential target for breeding sunflower varieties resistant to O. cumana through the strategies such as host-induced gene silencing (HIGS) of OcPOD10.

Zinc (Zn) is an essential plant micronutrient, but excessive soil Zn causes toxicity, inhibiting growth and reducing crop yields. Elucidating plant adaptation mechanisms to Zn stress is critical for improving performance in Zn‐rich environments. While multiple genes/proteins regulating plant Zn homeostasis are known, proteins with unknown domains (e.g., the DUF506 family) remain functionally unclear. This study explored the role of MdDUF506 in apple (Malus domestica) responses to Zn stress. Results showed that MdDUF506 overexpression altered the expression of heavy metal stress‐related genes. Under Zn stress, MdDUF506‐overexpressing apple plants had 32% lower hydrogen peroxide (H₂O₂) and 21% lower superoxide anion (O₂ ⁻) content than wild‐type (WT) plants. This reduction in reactive oxygen species (ROS) improved growth and significantly enhanced Zn tolerance in both apple plants and their callus. Additionally, upregulated CAT, POD, and SOD in MdDUF506‐overexpressing plants may increase ROS‐clearing enzyme activity, aiding adaptation to adverse environments. In summary, MdDUF506 may positively regulate apple Zn stress responses by modulating ROS‐related gene expression, and its overexpression confers excellent Zn tolerance. This study clarifies DUF506 family function and provides a potential target for improving Zn stress resistance in woody fruit crops.

Plant architecture results from the integration of developmental regulatory networks and environmental signaling pathways. In soybean, the erect growth habit of cultivated soybean (Glycine max) contrasts markedly with the vine growth habit (VGH) of wild soybean (Glycine soja). However, the genetic and transcriptional mechanisms underlying this divergence in gibberellin-mediated stem elongation remain poorly understood. Therefore, this study aims to investigate the genetic, transcriptional, and hormonal basis underlying the differences in growth habit between Williams 82 (erect growth habit) and PI 366121 (VGH). PI 366121 exhibited accelerated stem elongation at the V3 growth stage, indicating a developmentally regulated onset of the VGH. Quantitative trait locus (QTL) mapping using a high-density bin-based linkage map identified a major QTL on chromosome 14, encompassing 20 candidate genes. At the V3 growth stage, Williams 82 exhibited transcriptional expression patterns associated with enhanced photodamage repair and ROS detoxification, whereas PI 366121 exhibited coordinated activation of photoprotective, circadian, and gibberellin-responsive regulatory networks. Weighted gene co-expression network analysis was used to identify an ELIP-mediated regulatory module associated with the VGH. Furthermore, the co-expression of ELIP and GASA22 genes supports a functional relationship between chloroplast redox homeostasis and gibberellin-mediated stem elongation. We propose that ELIP-mediated photoprotection maintains photosystem II redox homeostasis, suppresses CBF-dependent GA2ox gene expression, and sustains bioactive gibberellin accumulation, thereby promoting GASA22 activation and stem elongation. This ELIP-GASA22 regulatory module provides a molecular framework connecting chloroplast redox status with gibberellin signaling and morphological plasticity in wild soybean.

ABSTRACT Calcium oxalate (CaOx) crystals are widespread in plants, yet their physiological roles remain debated. Although calcium sequestration is the most widely accepted function, some studies propose that CaOx crystals can serve as a carbon source, implying calcium release. These studies report decreases in CaOx crystals under water stress and other CO₂-limiting conditions, suggesting that crystals are oxidized to CO₂ to sustain the Calvin cycle. To test this hypothesis, we subjected 39-day-old Amaranthus cruentus plants to 10 days of water stress, followed by rewatering and a 16-day recovery period. Crystal abundance was analyzed by polarization microscopy, first in fixed leaves and then in optically cleared leaves. In control plants, CaOx crystals were present from the leaf-primordium stage and increased throughout leaf expansion, remaining abundant until late senescence. Water stress accelerated this pattern, and rewatering had no detectable effect. Optical clearing was intended to improve crystal visualization but unexpectedly dissolved crystals in some leaves. Serendipitously, we found that crystal solubility decreased with leaf age in control plants. Water stress accelerated this loss of solubility, whereas rewatering restored solubility to levels characteristic of younger leaves. Natural senescence increased CaOx crystal abundance and rendered them increasingly recalcitrant; water stress accelerated both processes. These findings challenge the carbon-source hypothesis.

Circadian rhythms have traditionally been considered to be outputs of genetic transcription-translation feedback loop (TTFL) oscillators. However, the discovery of circadian peroxiredoxin oxidation rhythms in anucleate red blood cells suggests that cells also possess an autonomous circadian redox rhythm. The conservation of this rhythm across all lineages of life, as well as in TTFL-defective backgrounds in multiple organisms, further indicates its importance. Rather than functioning as an isolated system, the redox rhythm is bidirectionally coupled to the genetic circadian clock. Perturbation of one oscillation may alter amplitude or shift period of the other, depending on biological context. In animals, the interplay between the redox rhythm and the genetic clock is complex, and the biological significance of the redox oscillation has yet to be established. In plants, immune-related redox perturbation by salicylic acid can reinforce the genetic clock, while the redox rhythm itself gates immune-induced programmed cell death toward morning as a circadian output. We propose that the redox rhythm reflects an intrinsic metabolic cycling in which cells alternate between the high-metabolic states that generate energy, reducing power, and reactive oxygen species (ROS), and the lower-metabolic states that favor detoxification, repair, and restoration of redox homeostasis. In this model, the redox rhythm is driven by the feedback between glycolysis, pentose phosphate pathway, ROS production via electron transport chains, antioxidant capacity, and cellular repair, and becomes entrained to the external cues via diurnal energy generation such as photosynthesis in green lineages.

This study explores the bioinorganic potential of selenium nanoparticles (SeNPs) biosynthesized by Bacillus sp. (AKS_bp1, PQ824612) in mitigating hexavalent chromium [Cr(VI)] toxicity in chickpea (Cicer arietinum L.). SeNPs were produced via microbial reduction of sodium selenite and characterized by nanoscale size (~ 100-150 nm), negative surface charge (- 20 mV), and biomolecular capping. Under controlled hydroponic Cr(VI) exposure (0-100 ppm), SeNP treatment significantly reduced Cr accumulation in plant tissues, with decreases of 24% in roots and 45% in shoots, indicating restricted translocation and improved metal homeostasis. This suggests a Se-mediated modulation of Cr mobility within the plant system. At the physiological and biochemical levels, SeNPs alleviated Cr-induced oxidative stress, as evidenced by reductions in superoxide (65%) and hydrogen peroxide (52%) levels, along with enhanced antioxidant defense and photosystem II efficiency (a 21% increase in photochemical performance). Improved chlorophyll fluorescence and stomatal regulation further indicated restoration of cellular functionality under metal stress. Importantly, SeNPs exhibited intrinsic redox activity with catecholase-like catalytic behavior (kcat = 3.11 × 106 s- 1), suggesting a potential role in electron-transfer processes relevant to oxidative stress modulation. Overall, bacteriogenic SeNPs mitigate Cr(VI) toxicity through a combination of reduced metal uptake, altered intracellular metal dynamics, and attenuation of reactive oxygen species. These findings highlight the bioinorganic functionality of SeNPs as redox-active nanomaterials influencing metal-plant interactions and oxidative stress pathways.

BackgroundMeloidogyne incognita poses a severe hazard to worldwide agricultural productivity, and emergence of multidrug resistance in this root-knot nematode has further exacerbated this challenge. Benzaldehyde, a volatile organic compound, is a promising nematicidal compound; however, its mode of action against M. incognita remains poorly elucidated. The present study aimed to systematically elucidate the nematicidal mechanism of benzaldehyde against M. incognita.ResultsIn vitro nematicidal assays demonstrated that benzaldehyde showed a potent nematicidal activity against second-stage juveniles (J2s) and markedly inhibited J2s hatching from eggs. Microscopic observations revealed that J2s treated with 100 mg/L benzaldehyde developed vacuolar structures after 72 h, with a greater abundance of vacuoles observed in the 400 mg/L treatment group. Notably, higher concentrations of benzaldehyde significantly suppressed the activity of glutathione S-transferase (GST), causing reactive oxygen species (ROS) overproduction and nematode death. Molecular docking and dynamics simulations confirmed that benzaldehyde could bind steadily to GST, thus weakening its antioxidant performance. Furthermore, pot experiments showed that benzaldehyde effectively reduced gall formation on cucumber roots caused by M. incognita.ConclusionThis study uncovers a novel GST-targeted inhibitory mechanism of benzaldehyde, which shows promising prospects for the exploitation of eco-friendly nematicides. Meanwhile, it proposes an effective measure to curb the progressive multidrug resistance of M. incognita and alleviate severe agricultural economic losses. © 2026 Society of Chemical Industry.

Cytokinin response factors (CRFs), a subfamily attached to the transcription factor AP2/ERF family, are extensively involved in the responses to abiotic stresses in plants. However, the role of CRFs in cold tolerance remains poorly characterized in Capsicum. In this study, the cold tolerance of 14 accessions was screened in Capsicum, and it was found that accession C3 displayed strong cold tolerance, while accession C88 exhibited opposite characteristics. Transcriptome profiling revealed that 2223 genes, including 897 up‐regulated and 1326 down‐regulated genes, were commonly differentially expressed (DEGs) in response to cold treatment in both C3 and C88. It was interesting that the plant‐pathogen interaction was among the most significant pathways. Notably, the expression of CaCRF6 was obviously up‐regulated in C3 (92.80‐fold) than that in C88 (4.04‐fold) at cold treatment, which suggested that CaCRF6 was closely related to cold tolerance in Capsicum. Subsequently, CaCRF6 was silenced by Virus‐induced gene silencing to study its function at cold stress, and the results showed that silencing CaCRF6 reduced cold tolerance of C3, accompanied by the increases in content of malondialdehyde, electrolyte leakage, and reactive oxygen species, as well as a decrease in the maximal photochemical efficiency of PSII. These results indicate that CaCRF6 plays a critical role in coping with cold stress in Capsicum, which provides new insights into understanding cold tolerance in Capsicum.

Calcium (Ca2+) is an essential mineral for plants, functioning both as a nano- to micromolar signaling ion in gene regulation and, at millimolar levels, in nutrition-supporting structural integrity and enzyme activation. These functions depend on adequate fertilization. However, Ca2+ problems stem mainly from hydraulic access and local availability within specific tissues and compartments rather than bulk supply; thus, deficiency can occur even in Ca2+-rich soils due to soil immobilization and physiological constraints. Because Ca2+ is poorly phloem-mobile and moves mainly via transpiration-driven xylem flow, its distribution is uneven. By binding to cell walls, calcium also enhances plant stability and fruit firmness. Here we review deficiency symptoms, management strategies, developmental roles, soil interactions, detoxification, and recent fertilizer approaches.

Salinity disrupts plant redox balance, ion homeostasis, and water relations, and exogenous polyamines have been widely investigated as potential mitigators of these effects. However, it remains unclear which polyamine-mediated responses are reproducible across independent studies and whether redox protection consistently translates into downstream physiological recovery. We therefore conducted an integrated systematic review, evidence map, and publication-family-aware quantitative synthesis to evaluate the effects of exogenous putrescine, spermidine, and spermine in salt-stressed plants. We systematically searched Scopus and Web of Science through 9 August 2026, and eligible studies compared exogenous-polyamine-treated plants with corresponding salt-stressed controls. The review was not prospectively registered. Of 1093 records, 647 unique records were screened, and 179 reports were assessed in full text, yielding 34 retained reports and 444 complete quantitative contrasts. Multilevel random-effects models estimated reductions of 19.2% in malondialdehyde (95% confidence interval [CI], 12.9-25.1%) and 25.6% in hydrogen peroxide (H2O2; 95% CI, 15.5-34.6%). Superoxide showed a protective mean response but remained uncertain because its 95% CI included the null. In contrast, antioxidant-enzyme responses varied widely across studies, with no consistent cross-family increase in superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), or peroxidase (POD/POX) activity. Conservative independent-family syntheses supported favorable ion balance, modestly higher relative water content, and greater dry biomass, whereas total chlorophyll remained uncertain and net photosynthesis was highly heterogeneous. No clear superiority of putrescine, spermidine, or spermine was detected. Overall, exogenous polyamines most consistently reduced oxidative damage and stabilized ion-water homeostasis, whereas antioxidant-enzyme activity and photosynthetic recovery were more context-dependent. Interpretation is limited by small independent-family counts, incomplete reporting in some studies, and the absence of a formal study-level risk-of-bias assessment. By integrating systematic review, evidence mapping, and family-aware quantitative synthesis, this review provides an evidence-based hierarchy that distinguishes reproducible polyamine-mediated responses from context-dependent outcomes and helps define priorities for future salinity-tolerance research.

In plants, GOLDEN2‐like (GLK) transcription factors have become the focus of research on drought tolerance mechanisms due to their pivotal role in coordinating photosynthesis, stomatal movement and antioxidant defense. However, the roles of GLK transcription factors in Paeonia lactiflora (P. lactiflora), particularly those associated with Bacillus and drought stress, remain poorly understood. Here, we isolated the GARP family gene PlGLK1 from P. lactiflora. PlGLK1 is a nuclear‐localized protein specifically induced by Bacillus and drought stress, and it harbors transcriptional activation activity. Functional studies on herbaceous peony revealed that PlGLK1‐overexpressing lines showed significantly elevated catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) activities, proline and soluble sugar contents, wider stomatal aperture, and reduced H₂O₂, O₂ ⁻ and malondialdehyde (MDA) levels relative to wild‐type plants, whereas VIGS lines showed the opposite trends. Importantly, these two pathways, the stomatal regulation pathway and the ROS scavenging pathway, could further enhance drought resistance following Bacillus inoculation. Yeast one‐hybrid (Y1H), dual‐Luciferase Reporter (Dual‐LUC) and electrophoretic mobility shift assays (EMSA) revealed that PlGLK1 modulated the expression of PlWRKY40, a dehydration stress‐responsive gene in plants. Our findings suggest that PlGLK1, induced by Bacillus, plays a crucial role in enhancing dehydration stress tolerance in herbaceous peony plants by promoting stomatal closure and enhancing ROS scavenging capacity.

Metacaspases are cysteine proteases, found in every group of organisms except metazoa. They are structural orthologs of caspases, which orchestrate apoptosis in metazoa. Like caspases ( C ysteine-dependent ASP artate-directed prote ASES ), paracaspases, and orthocaspases, metacaspases are class C14 proteases, with a hemoglobinase fold and catalytic cysteine residue. However, metacaspases cleave after arginine or lysine instead of aspartate, leading some to call for metacaspases to be renamed. Furthermore, most metacaspases are activated by calcium, they do not target the same range of proteins as caspases, and they tend to cut target proteins multiple times rather than at a single, canonical site. The Candida albicans metacaspase, Mca1p mediates cell death in response to various stresses but also possesses pro-life functions such as clearing protein aggregates and lifespan extension. Many plant and protist metacaspases lack cell death roles and mediate development, differentiation and immunity. This article highlights a reversed effect of Mca1p on growth inhibition by acetic acid, hydrogen peroxide and amphotericin B and on virulence of C. albicans , when injected into Galleria mellonella (wax moth) larvae, depending on whether cells originate from exponential or stationary phase culture. This reversal of metacaspase function hints at the protein’s dual nature and at the conditions that drive the switch from its pro-survival to its pro-death role.

Cadmium-contaminated rice poses a serious threat to public health, necessitating rapid screening methods. Unfortunately, traditional cadmium detection techniques are often cumbersome and expensive, making them unsuitable for on-site applications. In this study, we developed a graphene field-effect transistor (GFET) biosensor with Deoxyribozyme (DNAzyme) probes in binding mode at the graphene channel. By rationally redesigning a traditional catalytic DNAzyme, we suppressed its autocatalytic cleavage activity while fully retaining its high-specificity binding affinity for Cd2+, thus creating a pure binding-mode probe. The probe was subsequently immobilized on the graphene channel of a GFET sensor to capture Cd2+. The surface charge change induced by Cd2+ accumulation is efficiently converted by the GFET into measurable electrical signals, enabling rapid and accurate quantitative detection of Cd2+ in rice samples with an ultra-low limit of detection (LOD) of 0.3 μg/L and a recovery rate up to 94.74%. This study provides a promising platform for rapid, accurate, and on-site detection of heavy metal contamination in agricultural products.

Abstract The experiment was conducted at the research station affiliated with the Department of Horticulture and Landscape Architecture, College of Agriculture, University of Diyala, during the autumn season of 2025. The aim of this study was to evaluate the effect of foliar spraying with potassium and nano-calcium, and the interaction between them, on certain growth characteristics and chemical composition of the red cabbage. The experiment was conducted using a completely randomized block design with three replicates, and included treatments with conventional potassium at a concentration of 0, 250 and 500 mg L⁻¹ and with nano-calcium at concentrations of 0, 1 and 2 mg L⁻¹, as well as the interaction between them. The results showed that the spray treatments outperformed the control treatment in most of the traits studied, as the treatments led to a significant increase in plant height, number of leaves, chlorophyll content and anthocyanin pigment, as well as an increase in the leaf content of the nutrients nitrogen, phosphorus and potassium. Furthermore, the interaction treatment between potassium and nano-calcium at a concentration of 2 ml/L recorded the highest values for most of the traits studied.

As climate variability and water scarcity intensify, subsurface drip irrigation (SDI) has emerged as a key water-saving technology across arid and semiarid regions. However, in practice, the wetted soil volume around each emitter remains close to saturation, disrupting the continuity of the soil gas phase and restricting oxygen diffusion into the root zone. Subsequently, the water and oxygen supplies may compete for the same pore space, which is an intrinsic constraint of SDI. Aerated SDI, particularly systems that use micro- and nanobubbles (MNBs), has shown potential to improve crop yield and water-saving performance, although the effects vary with soil, crop, and management conditions. A unified, cross-scale account of the influence of these engineered gas-liquid inputs on root system architecture (RSA) remodeling remains lacking. Therefore, based on the optimal foraging theory (OFT), we propose an oxygen-mediated root foraging cost framework as a working mechanism hypothesis. This framework treats oxygen not as a growth-promoting factor per se, but as a candidate regulatory variable that may modulate the metabolic cost of root resource acquisition; the sensing-signaling-response chain linking local oxygen gradients to architectural decisions remains to be causally validated. By altering foraging costs, oxygen availability may alter how roots perceive, trade off, and invest in heterogeneous underground resources. Guided by this framework, this review first traces how SDI has evolved technologically, from supplying water through the liquid phase to coupling gas and liquid through MNBs. It then examines how local oxygen gradients may be sensed through upstream oxygen-sensing pathways (including the N-end rule pathway), reactive oxygen species (ROS), nitric oxide (NO), and downstream ethylene-auxin interactions, and how these signals may shape patterns of RSA investment under localized SDI conditions, and proposes the root foraging-accessible domain (FAD) as a candidate concept for evaluating the spatial compatibility between root foraging and the coupled water-oxygen environment. The present evidence supports this conceptual framework, but field-scale causal validation, separation of oxygen effects from confounding factors (nutrients, mechanical impedance, microbial activity), and techno-economic evaluation of aerated SDI remain necessary before the framework can guide irrigation practice.

It is of utmost importance to investigate the effect of carbon-based nanostructures on plants in order to fully unlock their potential for enhancing productivity and stress tolerance. In this study, a comprehensive assessment of the effect of pre-sowing treatment of wheat seeds with fullerenol (PHF [C60(OH)24], 0.1 mg/L) solution on a number of physiological and biochemical parameters in seedlings under optimal and low temperature (LT, 4 °C, 5 days) conditions was carried out. Seedlings primed with PHF differed from control variant in accelerated growth (by 27-34%), increased biomass (by 10%), larger area of chloroplasts and mesophyll cells (by 40%), more intensive photosynthesis (1.5-fold higher), increased content of chlorophyll a (by 11%), proteins (by 28%) and ascorbic acid, reduced level of MDA and H2O2, decreased SOD activity (by 45%) and a higher level of COR-genes (WCOR15, WCOR726) transcription (p b (by 22%) and proportion of chlorophyll in LHC (by 11%), as well as increasing the expression of RBCS (fourfold higher), content of proline, and COR-gene expression (p < 0.05). All these changes are adaptive and expand the adaptive potential of plants. It is concluded that nanopriming with PHF is an active metabolic modulator that reduces ROS generation and enhances cold acclimation of wheat.

γ-Aminobutyric acid (GABA) is a signaling metabolite involved in plant stress responses; its role in cadmium (Cd) stress adaptation in oilseed crops remains poorly understood. Here, we investigated the effects of exogenous GABA on Cd stress responses in two Brassica species, oilseed rape (Brassica napus, Bn) and Indian mustard (Brassica juncea, Bj). GABA alleviated Cd toxicity, as indicated by improved chlorophyll retention and reduced Cd accumulation by 22-29% across the two species. Moreover, GABA altered Cd subcellular partitioning and was accompanied by elevations in ascorbate (ASA) and glutathione (GSH) contents, respectively, together with reduced superoxide anion radical (O2-) and hydrogen peroxide (H2O2) accumulation. GABA also altered fatty acid compositions, suggesting changes in membrane lipid metabolism under Cd stress. Transcriptome analysis identified 2502 and 352 upregulated genes under GABA + Cd treatment (relative to the control) in Bn and Bj, respectively, of which 2245 and 219 were uniquely detected in the GABA + Cd treatment groups. Moreover, GABA supplementation was associated with transcriptional changes in genes related to redox metabolism and ABC transporter-associated processes. Collectively, these findings suggest that GABA supplementation was associated with improved Cd tolerance in Brassica species, accompanied by changes in Cd subcellular partitioning, non-enzymatic antioxidant metabolism, fatty acid profiles, and stress-responsive transcriptome reprogramming.

Rising atmospheric CO2 is altering carbon-nitrogen interactions in C3 crops, with tomato (Solanum lycopersicum L.) showing enhanced carbon assimilation but frequently reduced nitrogen acquisition and assimilation. Nitrate reductase (NR), the rate-limiting enzyme in nitrate reduction, plays a central role by integrating nitrate assimilation with carbon metabolism and nitric oxide (NO) signaling. This review summarizes current knowledge of NR regulation in tomato under elevated CO2 (eCO2), focusing on post-translational mechanisms and their contribution to photosynthetic acclimation. Elevated CO2 modulates NR activity through interconnected changes in photorespiration, carbohydrate-mediated feedback, redox regulation, source-sink dynamics, and nitrogen availability. While eCO2 generally suppresses leaf nitrate assimilation by reducing photorespiratory support, root-zone CO2 enrichment can transiently stimulate root NR activity, highlighting tissue-specific regulation. Multi-omics studies further demonstrate extensive metabolic and molecular reprogramming affecting carbon skeleton supply, amino acid biosynthesis, and nitrogen assimilation. In addition, NR-dependent NO production links nitrogen metabolism with stomatal regulation through ABA-independent H2O2-NO signaling. Despite these advances, the roles of NR phosphorylation, 14-3-3 protein interactions, and redox-mediated regulation under eCO2 remain poorly understood. Overall, NR functions as a key metabolic and signaling hub coordinating carbon and nitrogen metabolism under future climate conditions. Understanding these regulatory mechanisms will facilitate strategies to improve nitrogen-use efficiency, sustain photosynthesis, and enhance tomato productivity under elevated atmospheric CO2 while identifying priorities for future physiological, molecular, and multi-omics research.

Leaf rust (LR), caused by Puccinia triticina, is a major constraint to global wheat production. Identifying quantitative trait loci (QTLs), conferring adult-plant resistance (APR), and developing breeder-friendly markers are essential for durable disease control. In this study, a recombinant inbred line (RIL) population derived from Liang66-S × Hengmai28 was evaluated for leaf rust severity across four environments. Maximum disease severity (MDS) showed continuous variation and moderate to high correlations among environments, indicating polygenic inheritance. Five APR QTLs were mapped on chromosomes 2BL, 3BS, 3BL, 7BL and 7DL, explaining 4.5-9.8% of the phenotypic variance (PVE) with LOD scores of 2.9-7.9. Among these, QLr.DFI-2BL, QLr.DFI-3BS, and QLr.DFI-7BL correspond to previously reported APR loci, whereas QLr.DFI-3BL and QLr.DFI-7DL represent potentially novel loci. QLr.DFI-3BL was detected across all four environments, while QLr.DFI-3BS and QLr.DFI-7BL showed the highest R2 (7.7-8.5% and 8.4-9.8%). Multiple defense-related genes within the QTL intervals for LR were identified, including the putative NBS-LRR, the putative BTB/POZ-MATH, F-box, receptor-like kinase, polyphenol oxidase, calcium-dependent protein kinase, peroxidase, and ethylene-responsive transcription factors. Based on the flanking markers, five KASP markers were developed. K-LR-3BS (for QLr.DFI-3BS) and K-LR-7BL (for QLr.DFI-7BL) successfully genotyped a natural population with 120 accessions, with call rates of 100% and 94.2%, and showed significant association with reduced leaf rust severity. However, further validation in diverse genetic backgrounds and populations is required before they can be considered for routine marker-assisted selection. These results provide two KASP markers as promising markers that require further validation in diverse backgrounds and highlight QLr.DFI-3BS and QLr.DFI-7BL as promising targets for fine mapping and eventual cloning of APR genes in wheat.

SUMMARY In Brassicaceae, self-incompatibility is initiated when the pollen ligand SP11/SCR activates the cognate stigma receptor kinase SRK. Although this interaction is usually considered at a single pollen-papilla interface, individual papilla cells encounter mixed pollen loads. Here, using a self-incompatibility-reconstituted Arabidopsis thaliana system, we show that self-pollen recognition transiently delays the hydration and germination of neighboring nonself pollen. The delay was stronger when two self-pollen grains were present but largely disappeared when nonself pollen was applied 2 h after self pollen. Repollination after removal of a self-pollen grain nevertheless revealed persistent inhibition at the original contact site. Live imaging with Lifeact-tdTomato showed that self-pollination transiently broadened F-actin orientations throughout the papilla cell, followed by recovery toward the longitudinal organization of unpollinated cells. Upon self- and non-self-dual pollination, actin-bundle orientation returned toward the papilla long axis prior to the delayed hydration of the non-self-pollen. These observations support the view that the nature of the self-recognition signaling produces a reversible, papilla-wide state that temporarily reduces compatible-pollen acceptance, while local self-rejection persists at the original contact site.

Cadmium (Cd) contamination has threatened plant health and resulted in a notable reduction of crop yields. Cladosporium sphaerospermum is known as a plant growth‐promoting fungus that modulates plant development under abiotic conditions. However, the molecular mechanisms underlying C. sphaerospermum‐mediated alleviation of Cd toxicity in wheat is unknown. By integrating physio‐biochemical, transcriptomic and metabolomic analysis, the beneficial roles of C. sphaerospermum in moderating Cd‐induced (100 μM CdCl₂) toxicity in wheat seedlings (Triticum aestivum L.) were studied here. C. sphaerospermum enhanced plant growth under Cd conditions by reducing MDA content and increasing antioxidant enzyme activities to re‐establish redox homeostasis. Expression of wheat genes in MAPK pathways, plant hormone signal transduction, glutathione metabolism and cysteine metabolism pathways were notably differentially regulated by C. sphaerospermum under Cd conditions. The metabolites involved in oxidative phosphorylation, amino acid metabolism and tRNA biosynthesis were significantly modulated by C. sphaerospermum under Cd conditions. Integrated transcriptome and metabolome analysis indicated that the genes and metabolites in glutathione metabolism and cysteine metabolism pathways were crucial for C. sphaerospermum‐mediated mitigation of Cd toxicity in wheat. The findings provide new insights into the molecular mechanisms of fungus‐regulated wheat tolerance to Cd toxicity. C. sphaerospermum, as a promising and eco‐friendly plant growth‐promoting fungus, is suggested to be applied in Cd contaminated soil for food safety production.

Abstract Root rot is an important red clover disease caused by Fusarium avenaceum . Severe attacks significantly reduce the persistence of this forage crop. In this study, we used contrasting accessions to identify important defense factors against the F. avenaceum pathogen initially using RNA-seq. We identified 251 genes that were uniquely induced in the more resistant accession Formica, including several receptor-like kinases and UDP-glycosyltransferases. Four candidates were shortlisted on the basis of their expression patterns and predicted defense-related functions for further evaluation in a new transient expression assay using red clover leaves. TpUGT89B1 and TpLRR-RLK most effectively reduced fungal colonization and were subsequently used in stable transformation in the background of the susceptible accession SW 1678002. Constitutive expression of either TpUGT89B1 or TpLRR-RLK significantly enhanced resistance to F. avenaceum , with TpUGT89B1 providing the greatest reduction in fungal biomass and disease symptoms. Sequence comparison and structural modeling revealed that TpUGT89B1 possesses the conserved GT-B fold and characteristic features of a plant family 1 UDP-glycosyltransferase. Homology-based STRING analysis, supported by RT‒qPCR, revealed that TpUGT89B1 is involved in flavonoid biosynthesis through its co-expression with TpCHS , TpCYP75B1 and TpFLS1 . Collectively, the results of this study provide integrated transcriptomic information and structural prediction of TpUGT89B1, suggesting that it functions in defense through glycosylation-associated specialized metabolism.

Phosphate (Pi) starvation severely limits plant growth, and the transcription factor PHR1 is a central regulator of the phosphate starvation response (PSR). However, its role in modulating the secreted proteome remains poorly understood. Here, we performed comparative secretome profiling of wild-type (WT) and phr1 mutant Arabidopsis seedlings under Pi-sufficient (+P) and Pi-deficient (-P) conditions using LC-MS/MS. We quantified 2077 proteins from culture media across four biological replicates. Pi starvation induced 394 differentially abundant secreted proteins in WT, while the phr1 mutation severely attenuated this response, particularly down-regulating 213 proteins under -P conditions. PHR1 was essential for the starvation-induced secretion of phosphate-acquisition enzymes, including GDPD6, PAP1, and RNS1, and its role in the promotion of extracellular phosphatases was confirmed by acid phosphatase activity assays. Additionally, PHR1 modulated redox-related and defense-related secreted proteins, linking nutrient stress with oxidative and immune signaling. Integrative transcriptomic analysis revealed partial concordance between protein and mRNA levels, suggesting both transcriptional and post-transcriptional regulation. Collectively, our findings establish PHR1 as a vital regulator of the Pi-starvation-induced secretome and provide new insights into plant adaptation to phosphorus limitation through coordinated protein secretion.

Identifying substrates of protein phosphatases has been technically challenging and has hampered progress in the field of plant sciences. Small molecule inhibitors of protein phosphatases have aided in uncovering classes of phosphatases that target substrates, but that too has severe limitations. Here, we describe a method that enriches phosphorylated substrates using TiO2 and phospho-tyrosine antibodies in phosphatase knockout lines of Arabidopsis thaliana. When compared to wild-type plants, this approach permits identification of putative substrates and specific phosphorylation sites by mass spectrometry, allowing for further in vitro or functional validation. The key to the approach described here is the use of phosphatase knockout lines to maintain substrates in a phosphorylated state and using phospho-tyrosine antibodies to enrich for tyrosine phosphorylated peptides. Key features • Requires genetic knockout lines for the protein phosphatase of interest to uncover increased phosphorylation of putative substrates compared to wild type. • Phospho-peptides are (quantitatively) compared using mass spectrometry. • Tyrosine phosphorylated peptides are immunoprecipitated after TiO2 phospho-peptide enrichment. • Putative substrates identified with this protocol can later be validated with in vitro assays using synthetic phospho-peptides and/or phospho-proteins.

Abstract Background Under global warming, important food crops such as rice are severely impacted by abiotic stresses. However, few studies have explored the mechanisms underlying abiotic stress responses from a systems biology perspective, limiting the identification of breeding targets for developing novel rice varieties with enhanced stress tolerance. Results Utilizing rice RNA-seq data under aridity, alkaline, and cold stress conditions, we proposed gsGNA (General Stress-resistance Gene Regulatory Network), a universal computational pipeline designed to identify plant stress-resistance regulators. This pipeline employs six ensemble strategies to integrate the predictive outputs of six machine learning and deep learning algorithms, thereby systematically constructing high-confidence gene regulatory networks (GRNs) of rice under abiotic stress. Comparative analysis demonstrated that the XGBoost ensemble method consistently exhibited stable predictive performance across all three stress conditions. Subsequent network analysis identified 20 core transcription factors (TFs) that are shared among the three stresses. Functional enrichment analysis revealed that these TFs and their target genes cooperatively regulate key biological pathways, including hormone signal transduction, the MAPK signaling pathway, and primary metabolism. Network module analysis indicated that GRNs under different stresses are highly conserved in their overall architecture but exhibit significant divergence at the functional module level, reflecting an organizational pattern characterized by a "conserved core and divergent periphery". Additionally, through a reverse screening strategy based on core pathway target genes, 45 potentially novel regulators were predicted using the GRNs, providing new clues for further dissecting rice abiotic stress responses. Conclusions By applying the proposed gsGNA pipeline, we systematically mapped the GRN landscape of rice in response to common abiotic stresses, elucidated its network organizational patterns, and identified a set of candidate key TFs. These findings provide a theoretical foundation and a valuable target repository for rice stress-tolerance breeding.

Following pollination, Arabidopsis pollen grains rapidly hydrate through the transfer of water from the stigma to the pollen. Several stigma regulators of pollen hydration have been identified, and the corresponding mutants generally support milder defects in wildtype Col-0 pollen hydration, signifying the involvement of other unidentified factors in this process. Here, we uncovered a role for the stigma-specific mechanosensitive channel gene, MscS-Like 7 (MSL7), in supporting pollen hydration. While the msl7 mutant stigmas were found to support reduced hydration of wildtype Col-0 pollen, the phenotype was quite mild and very similar to that observed for other published pollen hydration mutants. Thus, we conducted a detailed comparison of different pollen hydration mutants on the stigma side (receptor kinases, PIP aquaporins) and pollen (PCP-Bs, MSL8) to compare pollen hydration mutant phenotypes and look for any additive effects of combining different mutants. Overall, all combinations resulted in the same mild hydration defect with no additional reductions in pollen hydration and no impact on pollen germination. This is in contrast to that observed for self-incompatible (SI) pollen from a transgenic Arabidopsis SI Col-0 line which shows very little pollen hydration and no pollen germination as part of the SI pollen rejection response. Together, these findings suggest that the regulation of compatible pollen hydration is quite complex and that there are likely other unknown mechanisms involved.

Leaf senescence is a pivotal developmental program in plants, precisely regulated by diverse signals including salicylic acid (SA). The Arabidopsis VQ protein AtVQ25 has been previously characterized as a positive regulator of SA-mediated leaf senescence, functioning through interaction with AtWRKY53 to relieve the transcriptional self-repression of AtWRKY53 at its own promoter. However, the upstream regulatory mechanisms governing AtVQ25 itself remain elusive. In this study, a yeast library screening was performed, and the mitogen-activated protein kinases AtMPK1 and AtMPK2 were identified as interacting partners of AtVQ25. The direct physical interaction was validated by yeast two-hybrid (Y2H), luciferase complementation imaging (LCI), pull-down, and co-immunoprecipitation assays (Co-IP). Furthermore, AtVQ25 was shown to be directly phosphorylated by AtMPK1/AtMPK2, which enhanced its protein stability and retarded its degradation, thereby positively modulating leaf senescence progression. Genetic analyses revealed that the function of AtVQ25 in SA-mediated leaf senescence depends on the functional integrity of AtMPK1/AtMPK2. Collectively, these findings establish AtMPK1/AtMPK2 as upstream interactors of AtVQ25 that coordinate SA-mediated leaf senescence through phosphorylation-dependent enhancement of AtVQ25 protein stability, providing novel insights into the upstream regulatory circuitry of VQ proteins.

Heat stress significantly impacts rice productivity, making understanding of molecular mechanisms crucial for crop improvement. Here, we report a regulatory pathway in rice that integrates a membrane-localized E3 ubiquitin ligase (OsTT3.1), glycogen synthase kinase 2 (OsGSK2), and the heat shock factor B2c (OsHsfB2c) to modulate thermotolerance. We demonstrate that OsGSK2 directly interacts with and phosphorylates OsHsfB2c both in vitro and in vivo, a modification that promotes its nuclear translocation. Functional analyses revealed that mutations in both OsGSK2 and OsHsfB2c enhance heat tolerance, while their overexpression reduces thermotolerance. Furthermore, we identified OsTT3.1 as an OsGSK2-interacting protein that promotes OsGSK2 degradation through ubiquitin-mediated proteolysis. Double mutant analysis (ostt3.1/osgsk2) showed increased heat tolerance compared to ostt3.1 single mutants, confirming that OsTT3.1 acts upstream of OsGSK2. Transcriptomic profiling of oshsfb2c mutants under normal and heat stress conditions uncovers widespread dysregulation of genes involved in primary metabolism and stress defense, indicating that OsHsfB2c functions as a transcriptional repressor of heat-responsive pathways. Our findings establish a sophisticated regulatory cascade where OsTT3.1-mediated degradation of OsGSK2 relieves the phosphorylation of OsHsfB2c, thereby derepressing heat stress responses and enhancing thermotolerance in rice. This "brake-release" mechanism serves as a critical buffering system that allows rapid attenuation of the OsGSK2-OsHsfB2c repressive module under heat stress, ensuring timely and robust activation of stress responses and optimal metabolic reprogramming.

Diosgenin is an important steroidal sapogenin widely used for the industrial production of steroidal drugs. Phytosulfokine-α (PSK-α) is a sulfated pentapeptide hormone known to regulate plant growth, development, and stress responses. However, its role in the regulation of diosgenin biosynthesis remains unclear. Fenugreek seedlings were treated with PSK-α to investigate its effects on diosgenin biosynthesis through metabolite profiling and comparative transcriptomic analyses. PSK-α induced a dynamic response in diosgenin accumulation, characterized by an initial decrease during the early stage of treatment followed by a gradual increase, ultimately resulting in 61.4% and 72.1% increases in diosgenin accumulation after 10 days and 15 days, respectively, compared with the corresponding random pentapeptide control. Comparative transcriptomic analysis between PSK-α-treated seedlings and the corresponding controls at 5 and 15 days identified key genes or transcript isoforms involved in diosgenin biosynthesis, PSK signaling and MAPK signaling pathways. Meanwhile, five candidate genes involved in diosgenin biosynthesis and PSK signaling exhibited expression patterns consistent with the dynamic accumulation pattern of diosgenin. To further dissect the regulatory network, co-expression network analysis between these 28 candidate genes involved in diosgenin biosynthesis and PSK signaling and 1407 transcription factor-encoding genes identified candidate transcription factors potentially involved in the coordinated regulation of diosgenin biosynthesis and PSK signaling. This study suggests that PSK-α may contribute to enhanced diosgenin accumulation through coordinated transcriptional regulation of diosgenin biosynthesis and PSK signaling pathways, providing new insights into the regulation of plant secondary metabolism by peptide hormones.

KEY MESSAGE: Fine mapping of qSSISFH8.1, a quantitative trait locus (QTL) for reproductive stage salinity tolerance in rice to a 640 kb region, and qRT-PCR analysis of differentially expressed candidate genes in the region. An indel marker was designed and validated for marker-assisted introgression of the trait into elite rice cultivars Rice (Oryza sativa L.) is cultivated globally as a staple food crop for about fifty percent of the human population. However, popular high-yielding rice cultivars are sensitive to soil salinity during seedling and reproductive stages of growth. Most of the reported QTL for salinity tolerance in rice span large chromosomal regions, making it difficult to identify the underlying causal genes or utilize these in marker-assisted breeding. Here we present fine mapping of qSSISFH8.1, a robust QTL for reproductive stage salinity tolerance derived from rice landrace Nona Bokra. A population of 4300 F₂ plants from cross between QTL-NIL (near-isogenic lines) with contrasting alleles was genotyped for the QTL-flanking markers to identify 146 recombinants. Thirteen SNPs were identified by targeted re-sequencing of genes in the QTL region and used to group the recombinants based on the crossing over breakpoints. Phenotyping of these recombinants for salinity tolerance in controlled microplots helped narrow down the QTL interval to 640 kb. Analysis of differential expression of genes in the region in response to salinity stress and allelic sequence variation, identified genes coding for brassinosteroid insensitive 1-associated receptor kinase, prohibitin, tetraspanin, ATPase 1 and an uncharacterized expressed protein as potential candidates for salinity tolerance underlying qSSISFH8.1. We developed and validated an easy-to-use indel marker ‘RSST8.1indel’ for introgression of the QTL into salinity-sensitive elite rice cultivars.

AimsNeonatal hypoxic-ischemic encephalopathy (HIE) causes severe neurodevelopmental impairment, but the upstream immune mechanisms that initiate this process remain unclear. We investigated whether mast cell (MC)-derived tryptase contributes to aberrant microglia-mediated synaptic pruning through PAR-2/MAPK/NF-κB signaling after neonatal hypoxic-ischemic (HI) injury.MethodsA postnatal Day-7 rat HI model was established using the Rice-Vannucci method. MC abundance, c-Kit and tryptase expression, PAR-2/MAPK/NF-κB signaling, and synaptic integrity were assessed using histological, immunofluorescence, biochemical, ultrastructural, and three-dimensional (3D) reconstruction analyses. Microglia-targeted F2rl1 silencing and pharmacological inhibition with FSLLRY-NH2 or APC366 were used to examine PAR-2 and tryptase-related signaling. Complementary oxygen-glucose deprivation/reperfusion (OGD/R) experiments were performed in BV-2 microglia. Cognitive outcomes were assessed using the Morris water maze and Y maze.ResultsHI increased hippocampal MC abundance and tryptase expression, and these changes were associated with acute neurological deficits. HI insult also increased complement associated synaptic labeling, PAR-2/MAPK/NF-κB signaling, CD68 expression, and engulfment of PSD95 positive synaptic material. Microglia-targeted F2rl1 silencing attenuated pathway activation and synaptic engulfment. FSLLRY-NH2 and APC366 similarly reduced HI associated signaling and microglial synaptic engulfment, while APC366 preserved dendritic spine density and synaptic ultrastructure and improved long-term spatial learning and memory. In BV-2 microglial cells, exogenous tryptase enhanced OGD/R associated CD68 expression and MAPK/NF-κB phosphorylation, which were attenuated by F-NH2.ConclusionMC-derived tryptase is an upstream contributor to pathological microglial synaptic pruning and cognitive impairment after neonatal HI, potentially involving the PAR-2/MAPK/NF-κB axis.

HSFA2 is a key regulator of acquired thermotolerance after mild-heat priming, but whether volatile-triggered priming depends on this pathway remains unclear. Here, we examined thermotolerance induced by the reactive green leaf volatile trans-2-hexenal (T2H) in Arabidopsis thaliana Col-0 and an hsfa2 T-DNA insertion mutant. Seedlings were primed with mild heat or a 30-min T2H pulse, recovered for 2 days, and challenged with lethal heat. T2H pretreatment improved survival in both genotypes. Survival analysis and image-based quantification of projected green area showed that T2H-supported recovery was retained in hsfa2, whereas recovery after mild-heat priming was reduced. Total chlorophyll content showed the same genotype-dependent pattern: it was lower in hsfa2 than in Col-0 after mild-heat priming but did not differ between genotypes after T2H. Early reverse-transcription quantitative PCR (RT-qPCR) analysis showed that transcript levels of HSFA1A, HSFA1B, HSFA1D, HSFA1E, and HSP101 did not correlate with the observed survival response. In a separate recovery-period analysis, HSP101 transcript abundance in hsfa2 was significantly lower than in Col-0 after both priming treatments, with the larger reduction after T2H despite strong mutant protection. RNA sequencing (RNA-seq) revealed distinct transcriptional responses between heat and volatile priming: mild heat induced broad transcriptome remodeling, whereas T2H induced a more selective stress-associated response enriched in detoxification- and redox-associated, immune-associated, and hypoxia-like transcript categories. In hsfa2, T2H was also associated with increased transcript abundance of WRKY- and MAPK-associated stress-signaling genes relative to heat priming. These findings suggest that reactive volatile priming may support thermotolerance through HSFA2-independent stress-signaling pathways.

Allyl isothiocyanate (AITC), a sulfur‐containing volatile from the cruciferous glucosinolate‐myrosinase hydrolysis, the core reaction of the “mustard oil bomb”, acts as an antimicrobial, insecticidal, and airborne signaling molecule. In this study, two treatments were established: direct fumigation with AITC (Group D) and plant‐to‐plant aerial chemical communication (Group T) in pakchoi, and RNA‐seq, RT‐qPCR, glucosinolate (GSL)/isothiocyanate (ITC) quantification, and Spodoptera litura feeding assays were used to investigate AITC‐induced insect resistance. Transcriptomic analysis revealed that differentially expressed genes (DEGs) were significantly enriched in plant hormone signal transduction and phenylpropanoid biosynthesis pathways. Metabolically, AITC effectively promoted the conversion of GSL to ITC, resulting in higher total ITC content in all treatment groups compared to the control, following a time‐dependent accumulation pattern. Spodoptera litura larvae displayed reduced feeding preference for AITC‐treated pakchoi, which was negatively correlated with ITC levels. This study reveals the key regulatory mechanism by which AITC enhances secondary metabolism and produces volatile metabolites for insect defense, probably through activating a signaling network centered on jasmonic acid (JA) and synergizing with other hormones. It also confirms AITC's role in airborne early warning and plant‐to‐plant signal transmission, providing theoretical foundations and practical references for developing green pest control technologies based on plant volatile metabolites, such as AITC. However, the specific sensory receptors mediating AITC‐induced plant airborne immunity, the detailed processes of signal transmission between plants, and the interactive networks with other defense pathways still require further in‐depth exploration.

Phytoplasmas are obligate, phloem-limited bacterial pathogens that reprogram host development through secreted effector proteins. The effectors characterized to date, including SAP11, SAP54/PHYL1, SAP05 and SWP1, act principally by binding and destabilizing host transcription factors. Although α/β-hydrolase-fold phytoplasma proteins with catalytic activity toward lipid substrates have recently been described, no phytoplasma effector has been shown to possess enzymatic activity toward a plant hormone. The effector RY378 from rice yellow dwarf (RYD) phytoplasma induces excessive tillering and dwarfing in rice and alters strigolactone- and auxin-responsive gene expression; its predicted α/β-hydrolase domain was proposed to mediate these effects, but the protein was never expressed, purified or biochemically characterized, leaving the mechanism of this effector class unresolved. From the closed genome of 'Ca. P. sacchari' isolate SCGS, associated with sugarcane grassy shoot disease, we identified SGP-7, an orthologue of RY378 that is conserved across at least seven 16Sr phytoplasma groups. SGP-7 adopts a canonical α/β-hydrolase fold with a Ser116-Asp229-His261 catalytic triad, and its predicted substrate pocket is comparable in volume to that of the Arabidopsis strigolactone receptor AtD14. Recombinant SGP-7 is a bona fide esterase with a clear preference for short-chain acyl esters, and hydrolyses both the fluorogenic strigolactone probe Yoshimulactone Green (apparent Km 3.55 uM; kcat 9.38 x 10^-3 s^-1) and the strigolactone analogue rac-GR24. Both activities are abolished by substitution of the catalytic serine (SGP-7S116A). Transient expression of SGP-7 in Nicotiana benthamiana reduced plant height by 46%, increased axillary bud outgrowth approximately fourfold, and lowered transcript levels of the strigolactone-responsive marker NbBRC1, reproducing in a dicot host the architectural phenotype reported for RY378 in rice. Together, these results define the biochemical function of a conserved phytoplasma effector family and identify strigolactone-like molecules as direct enzymatic targets of a bacterial plant pathogen.

PIN-FORMED (PIN) auxin transporters are essential for plant development, relying on asymmetric localization at the plasma membrane. While the central hydrophilic loop (HL) of PINs is known to integrate various regulatory signals, the structural basis of its coordination remains unclear. Here, we demonstrate that the PIN-HL domain undergoes homotypic interaction independent of the transmembrane domains via a conserved β-sheet motif. Deleting this motif in PIN2 reinforced protein clustering and polarity, and impaired intracellular trafficking, leading to defective root gravitropism. These phenotypes were successfully reversed through rapamycin-induced reconstitution of the HL-HL interaction, confirming that this physical association is vital for native PIN2 behavior. Furthermore, we show that phosphorylation of the HL domain inhibits this interaction, which in turn modulates the recruitment of PIN-regulatory factors. Our findings support a model in which the HL-HL interaction serves as a regulatory hub, where phosphorylation and other modulators converge to influence PIN behavior.

Petal size is a critical agronomic trait influencing pollination efficiency and yield in field crops. However, the cellular basis and upstream hormonal interplay governing petal size in Brassica napus remain unclear. In this study, utilizing a natural variation population of "Zhongshuang 11", we investigated biological mechanisms underlying petal development through integrated phenotypic screening, paraffin section-based cytological analysis, targeted hormone metabolomics, and transcriptome. The results revealed that the single-flower area of large petals (BEP; 13.0 mm2) was significantly larger than that of wild type (8.5 mm2) and small petals (SEP; 6.8 mm2), a difference primarily attributable to increased petal width. The formation of BEP was due to a more than two-fold increase in cell number per unit area, rather than cell expansion. Furthermore, salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and cytokinins (CK) were significantly enriched in BEP petals, whereas brassinosteroid (BR) content was lowest. Correlation analysis indicated a significant positive correlation between ABA and JA (r = 0.95). Negative regulators of ABA signaling, BnaC09.ABI1 and BnaA01.PP2CA, were significantly downregulated, while core cell cycle genes were highly expressed in BEP. An "ABA de-repression" model was proposed; a hormonal milieu characterized by high concentrations of SA, JA, and CK, coupled with low BR levels, is associated with the downregulation of negative regulators of ABA, potentially alleviating constraints on cell cycle progression to facilitate cell proliferation. This study elucidates the molecular and physiological basis of petal size in rapeseed and provides a theoretical foundation for enhancing outcrossing and yield through floral trait modification.

The universal stress protein (USP) family is a large but poorly studied group of plant proteins whose roles are considered primarily in the context of stress tolerance. This study demonstrates that the functions of USPs are not limited to the stress response but also influence plant developmental processes, namely, the regulation of seed germination and seedling establishment. Using reverse genetics, we found that disruption of the Arabidopsis thaliana At3g58450 gene, encoding germination-related USP (GRUSP), results in delayed germination and abscisic acid (ABA) hypersensitivity in the mutant the GABI-kat 115C08 (grusp-115) knockout line. This hypersensitivity was only partially rescued by the application of fluridone, an ABA biosynthesis inhibitor. Altered ABA content and expression patterns of genes involved in ABA metabolism and signaling, along with a decrease in the mRNA levels of gibberellin (GA) oxidases in dry and either Murashige and Skoog (MS)- or ABA-imbibed mutant seeds, indicate that GRUSP function is primarily associated with seed imbibition and early germination stages. Furthermore, the grusp-115 mutant showed upregulation of ABI5 transcripts in imbibed seeds and seedlings as well as an accumulation of ABI5 protein level under excess GA conditions, indicating a functional relationship between these proteins. Hence, GRUSP is a novel component of the GA and ABA pathways whose function is initiated during seed germination.

The number of pistils determines the yield of Chinese chestnut (Castanea mollissima Bl.). However, the candidate pathways associated with formation of multi-pistil female flower clusters (MFF) remain largely unclear. This study integrated morphological, anatomical, transcriptomic, and targeted phytohormone metabolomic approaches to compare MFF and normal female flower clusters (NFF). The results showed that MFF exhibited longer basal bracts with fewer basal trichomes. The critical stage for multi-pistil phenotype formation occurred during pistil primordium differentiation, when additional floral meristems continuously expanded and differentiated into new flower primordia. Auxin (IAA), cytokinin (CTK), jasmonic acid (JA), and gibberellin (GA) were identified as potentially associated with this. Candidate genes associated with hormone biosynthesis, signal transduction, and core transcription factors regulating floral organ development at this key developmental stage were also identified. The findings of this study provide insights into the potential regulatory mechanisms underlying the differentiation and development of MFF.

Branching architecture is a key agronomic and ornamental trait in woody plants; however, the molecular mechanisms coordinating hormone-mediated axillary bud formation and outgrowth remain largely unclear. Clerodendrum japonicum (Thunb.) Sweet is an important ornamental woody species with strong branching potential, but the regulatory network underlying its shoot branching has not been characterized. In this study, we investigated the regulatory basis of hormone-induced branching in Clerodendrum japonicum and identified a LATERAL SUPPRESSOR homolog, CjLAS, as a key candidate regulator. Combined application of gibberellic acid (GA3) and 6-benzyladenine (6-BA) significantly promoted axillary bud formation and outgrowth, accompanied by extensive transcriptional reprogramming. Transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) revealed that CjLAS was closely associated with branching development and hormone-responsive pathways. Expression analysis showed that CjLAS expression increased during the progression of hormone-induced axillary bud development and branch formation. Ectopic overexpression of CjLAS in Arabidopsis thaliana enhanced shoot branching, confirming its conserved positive role in regulating plant architecture. Furthermore, overexpression of CjLAS repressed the gibberellin (GA) biosynthetic genes AtGA20ox2 and AtGA3ox2, induced the catabolic gene AtGA2ox6, and reduced the endogenous levels of bioactive GA1 and GA4. These findings suggest that CjLAS promotes shoot branching by integrating axillary meristem regulation with GA homeostasis. This study reveals an additional layer of LAS-mediated regulation linking meristem development and hormone homeostasis during shoot branching in woody plants and provides insights into the molecular regulation of plant architecture in C. japonicum.

Abstract Background Drought stress is one of the major environmental factors limiting citrus growth and productivity. In this study, we systematically analyzed physiological responses and transcriptomic dynamics of Citrus reticulata 'Hong Mei Ren' leaves under varying drought conditions—control group (T0), mild (T1), moderate (T2), and severe (T3) drought conditions. Results Under drought stress, the levels of SOD, CAT, GSH-Px, MDA, soluble proteins and amino acids in the leaves of pomelo increased overall, while POD and soluble sugars decreased; in T2, the increments of SOD, CAT, carotenoids and soluble proteins were the largest, in T3, MDA and amino acids were the highest, and the decrease in soluble sugars was the greatest, and total chlorophyll in T2 increased while in T3 it decreased. Our results demonstrated that citrus leaves employ an integrated adaptive strategy to cope with drought stress through coordinated regulation of antioxidant defense systems, osmotic adjustment metabolism, photosynthetic pathways and plant hormone signal transduction. Gene expression analysis indicated that the plant response to drought exhibits distinct phase-dependent characteristics: under mild to moderate drought, photoprotection genes (e.g., PsbS) were upregulated, whereas genes associated with photosynthetic capacity are downregulated; core components of the abscisic acid (ABA) signaling pathway—including PYR/PYL, SnRK2, and ABF—were significantly induced and act synergistically to suppress growth-related signaling pathways (e.g., AUX1, GID1); concurrently, lipid signaling molecules (such as PLD and PIP5K) and membrane trafficking genes (e.g., Rab11) were upregulated to facilitate plasma membrane remodeling. However, under severe drought stress, the photosynthetic apparatus, hormone signaling networks were broadly suppressed. Conclusions This study provided a theoretical foundation for elucidating the drought tolerance mechanisms in citrus and identifies potential candidate genes for use in drought-resilient breeding programs.

Abstract To reveal the physiological mechanism by which exogenous indole-3-acetic acid (IAA) and 6-benzyladenine (6-BA) regulate sprout number in stumped Pinus yunnanensis seedlings, optimize hormone application schemes, and provide a theoretical basis for its asexual cutting propagation, this study adopted a two-factor three-level completely randomized block design. Different concentrations of IAA (0, 150, 300 mg/L) and 6-BA (0, 100, 200 mg/L) were sprayed on 1-year-old stumped P. yunnanensis seedlings. The contents of endogenous IAA, abscisic acid (ABA), gibberellin (GA₃), and zeatin (ZA) were determined, and the correlations between endogenous hormones and their ratios with sprout number and sprout length were analyzed. The results showed that single application of IAA exerted an overall dose-dependent inhibitory effect on sprout number. Endogenous IAA content increased monotonically with rising exogenous IAA concentration, while GA₃ and ZA showed a non-monotonic response characterized by "promotion at low concentration and inhibition at high concentration". Under single 6-BA application, the responses of sprout number and sprout length were decoupled, and endogenous ZA content decreased continuously with increasing 6-BA concentration. The combined application of IAA and 6-BA exhibited a significant non-additive interaction effect, which generally reduced endogenous IAA levels. The lowest endogenous IAA content was observed under the combination of 150 mg/L IAA + 200 mg/L 6-BA. Path analysis indicated that the GA₃/ZA ratio was the core positive factor regulating sprout number, while sprout length was significantly and positively correlated with the synergistic accumulation of endogenous IAA and GA₃. The relative ratios of hormones had stronger explanatory power for germination phenotypes than the absolute content of a single hormone. In conclusion, the germination and growth of sprouts in stumped P. yunnanensis are co-regulated by the endogenous hormone balance network. The combined application of exogenous IAA and 6-BA can achieve directional induction of sprouts by regulating key ratios such as GA₃/ZA. These results provide quantitative parameters for hormone regulation in the efficient propagation of superior P. yunnanensis clonal scions.

Cotton growth is frequently constrained by drought stress. Previous studies demonstrated that γ-PGA enhances cotton drought tolerance. Transcriptome analysis of γ-PGA-treated cotton revealed that multiple TIFY family members were markedly induced, among which GhJAZ8 showed the most pronounced upregulation. Sequence analysis indicated that GhJAZ8 belongs to the JAZ/TIFY5A subfamily, and its expression was significantly upregulated under drought stress. Virus-induced gene silencing of GhJAZ8 markedly decreased drought tolerance in cotton, evidenced by aggravated leaf wilting, reduced RWC, elevated MDA content, weakened antioxidant enzyme activities, and decreased proline accumulation. Conversely, overexpression of GhJAZ8 in Arabidopsis significantly enhanced drought tolerance. Furthermore, γ-PGA treatment improved drought tolerance in both TRV2 and GhJAZ8-silenced plants, but the effect was more pronounced in TRV2 plants, indicating that GhJAZ8 is required for the full effect of γ-PGA. Exogenous MeJA treatment only partially alleviated the drought-sensitive phenotype of GhJAZ8-silenced plants. Transcriptome analysis revealed that DEGs were predominantly enriched in ABA, JA, and SA signaling pathways, and hormone quantification further confirmed that GhJAZ8 silencing significantly altered endogenous ABA, JA, and SA levels. Taken together, this study reveals that GhJAZ8 functions as a positive regulator in cotton drought response and acts as an important downstream component of the γ-PGA-mediated drought tolerance pathway by modulating multiple phytohormone signaling pathways, providing a candidate gene resource for drought-tolerant cotton breeding.

Epigallocatechin gallate (EGCG) imparts distinctive health benefits and flavor to tea, and its accumulation is modulated by plant growth regulators (PGRs). However, the regulatory mechanisms of Paclobutrazol (PAC) and Lovastatin (LS) underlying EGCG biosynthesis remain uncharacterized. We integrated quantitative analysis, transcriptomics, and metabolomics to investigate the regulatory effects of exogenous PAC and LS on EGCG accumulation in tea plants and systematically elucidate the underlying molecular mechanism governing EGCG biosynthesis. The results demonstrated that exogenous PAC and LS treatments significantly elevated the accumulation of EGCG, as well as endogenous auxin and jasmonic acid (JA) contents. The variation trend of these key metabolites was highly consistent with the accumulation patterns of upstream flavonoid components, including eriodictyol, delphinidin, and epigallocatechin. Transcriptomic profiling further verified the critical involvement of auxin and JA signal transduction pathways in PGR-induced EGCG differential accumulation, and we screened a total of 14 auxin-related and 8 JA-related core signal regulatory factors. Furthermore, integrated bioinformatic analyses and antisense oligonucleotide (AsODN) functional validation experiments revealed that the core hormone signaling genes CsARF2 and CsMYC2 regulate the expression of CsSCPL16 through the transcription factor CsMYB80, and ultimately promote the conversion of epigallocatechin to EGCG and promote the accumulation of EGCG in tea plants. Collectively, the PGRs boost EGCG biosynthesis by mediating endogenous auxin and JA signal transduction. These results support the development of targeted agronomic practices to improve tea quality and lay a theoretical basis for expanding the industrial exploitation of tea bioactive constituents.

BackgroundBasic helix-loop-helix (bHLH) transcription factors constitute the second-largest transcription factor family in plants. They are widely distributed across plant genomes and play vital regulatory roles in plant growth and development, secondary metabolism, and responses to abiotic stress. Although the bHLH gene family has been widely identified and analyzed in many plant species, systematic genome-wide identification and functional analysis of bHLH genes in Pinellia ternata are still lacking.ResultsIn this study, a total of 140 PtbHLH genes were identified, of which 135 were unevenly distributed on 13 chromosomes. Phylogenetic analysis with Arabidopsis thaliana divided the PtbHLH genes into 24 subfamilies, with no members clustered in subfamily VI. Gene structure and conserved motif analysis showed that members of the same subfamily had highly similar structural characteristics. Collinearity analysis identified three paralogous pairs among the 140 PtbHLH genes. Cross-species collinearity analysis revealed eight syntenic pairs between P. ternata and A. thaliana, 76 between P. ternata and Nicotiana tabacum, and 50 between P. ternata and Oryza sativa. Cis-element prediction showed that all PtbHLH promoters contained hormone-responsive elements, stress-responsive elements and transcription factor binding sites. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that PtbHLH genes were mainly involved in hormone signal transduction, environmental signal response and transcriptional regulation. Protein-protein interaction (PPI) analysis suggested that PtbHLH68 was predicted as a putative interaction hub interacting with multiple family members. Tissue expression profiles showed that PtbHLH genes had diverse expression patterns, with most highly expressed in roots and tubers. Under high-temperature and drought stress, PtbHLH9 was most sensitive to high temperature, and PtbHLH85 was most sensitive to drought. Under methyl jasmonate (MeJA), abscisic acid (ABA) and salicylic acid (SA) treatments, PtbHLH73 responded most strongly to MeJA, PtbHLH7 to ABA, and PtbHLH48 responded significantly to all three hormones. These four genes serve as core candidates for exploring PtbHLH functions in abiotic stress tolerance and hormone signal transduction.ConclusionThis study provides a solid foundation for understanding the functional mechanism of PtbHLH transcription factors in Pinellia ternata, and offers a valuable reference for the further screening of PtbHLH genes related to abiotic stress tolerance and hormone signaling pathways.

High‐density rice planting reduces light quality within the canopy, especially the red to far‐red (R: FR) ratio, triggering a physiological shift that enhances elongation growth at the expense of weakened defence mechanisms. This is not a passive consequence but a coordinated regulation controlled by the Phytochrome B (PhyB)–Phytochrome Interacting Factor (PIF) signalling module. Under low R: FR, PhyB becomes inactive, stabilising key PIFs such as OsPIL13 and OsPIF4. These transcription factors promote shade‐avoidance growth by enhancing auxin and gibberellin biosynthesis, which in turn suppresses salicylic acid (SA) and jasmonic acid (JA) signalling. They also directly repress the expression of core defence genes. Together, these changes lower immune readiness in shaded rice plants. Here, we propose a rice‐specific model in which low R: FR light signals directly suppress immunity through PIF‐mediated transcriptional repression, highlighting a monocot‐specific mechanism that integrates light perception with immune downregulation.

Abstract Background Bulbil sprouting is the cornerstone of asexual propagation in yam ( Dioscorea polystachya ). However, gibberellic acid (GA₃), a classic growth-promoting hormone, paradoxically suppresses this process through an unknown mechanism. Results Through an integrated multi‑omics approach encompassing physiological phenotyping, transcriptomic profiling, and metabolomic analysis, we uncovered two diametrically opposed regulatory regimes. GA₃ inhibits sprouting by establishing a "high GA—low GID1 receptor—high DELLA" signal‑desensitization state, which systemically represses growth‑promoting hormone networks (auxin, cytokinin, ethylene, and jasmonates) and dampens primary energy metabolism. In stark contrast, paclobutrazol (PP₃₃₃) promotes sprouting via a "low GA—high GID1 receptor—stable DELLA" signal‑hypersensitization state, which orchestrates the coordinated activation of multiple pro‑sprouting pathways, including those mediated by ethylene, jasmonic acid, cytokinins, and brassinosteroids. Conclusions Our findings delineate the phytohormone crosstalk network governing yam bulbil sprouting, provide a theoretical foundation for precise sprouting management in yam cultivation, and expand the conceptual understanding of GA signaling functional plasticity beyond conventional model systems.

Drought stress is a major abiotic factor that negatively affects plant growth, physiological processes, and yield in pepper cultivation. This study evaluated the ameliorative effects of exogenous glutathione (GSH) at 50 µM and 100 µM in pepper seedlings grown under water-restricted conditions (60% field capacity), using morphological, physiological, and biochemical parameters. Water restriction significantly reduced seedling height, stem diameter, leaf area, and plant and root fresh and dry weights, and decreased chlorophyll a, chlorophyll b, total chlorophyll content, and plant growth hormones. Conversely, water restriction significantly increased hydrogen peroxide (H2O2), malondialdehyde (MDA), proline, sucrose, and abscisic acid (ABA) content. Glutathione application, on the other hand, significantly reduced water-restriction-induced growth damage, increased biomass production and chlorophyll content, and supported the antioxidant defense capacity of plants by decreasing H2O2 and MDA levels, which are indicators of oxidative stress. It also improved osmotic balance by regulating proline and sucrose accumulation and increased seedling adaptation to water restriction. In conclusion, exogenous GSH acted as a potential biostimulant under water-deficit conditions, with its beneficial effects associated with improved osmotic adjustment through the regulation of proline and sucrose accumulation, enhanced antioxidant defense, and modulation of endogenous phytohormone levels.

Plant development is regulated by a complex interplay of multiple signaling pathways driven by small signaling molecules, including plant hormones. A signaling pathway activated by karrikins (KARs), a class of compounds found in smoke that putatively mimic an endogenous plant signaling molecule, "KAI2 ligand" (KL), controls seedling photomorphogenesis in Arabidopsis thaliana. We recently demonstrated that in roots of Lotus japonicus and A. thaliana, the gaseous plant hormone ethylene promotes accumulation of a transcriptional regulator SMAX1 that negatively regulates KAR/KL response and is targeted for degradation by the 26S proteasome after KAR/KL perception. This ethylene-inducible accumulation of SMAX1 suggests convergence of KAR/KL- and ethylene-driven signaling pathways at SMAX1. In this study, we investigated the effects of ethylene signaling on SMAX1-regulated hypocotyl growth in A. thaliana. Chemical inhibition of ethylene biosynthesis reduced hypocotyl elongation in a SMAX1-dependent manner that was independent of KAR/KL signaling. We propose a model in which SMAX1 mediates developmental processes by integrating multiple plant hormone-driven signaling pathways.

Drought stress has a profound impact on yield, with massive repercussions for agricultural production. In addition to ABA, the central hormone controlling the plant response to drought, secondary metabolites also play important functions in drought stress; however, the precise nature of their roles is still obscure. Metabolomics analysis of ABA- and PEG-treated Arabidopsis wild-type (WT) seedlings as well as ABA signal transduction mutants snrk-triple (snrk2.2 snrk2.3 snrk2.6) and pyl-quadruple (pyr1 pyl1 pyl2 pyl4) identified a number of drought-related metabolites. Specifically, we observed differences in the levels of sinapic acid and quercetin. To assess the function of sinapoyl esters in the regulation of drought stress, we characterized the sinapoylglucose accumulator 1 (sng1) mutant, deficient in the conversion of sinapoylglucose (SG) to sinapoylmalate (SM), resulting in high endogenous levels of SG and low SM levels. sng1 mutants showed increased drought resistance compared to WT plants. RNA-sequencing of sng1 and WT rosette leaves before and after dehydration revealed differentially expressed genes related to cuticle synthesis, anthocyanin biosynthesis, leaf senescence and ABA response. Consistently, phenotypic analysis of the sng1 mutant showed increased cuticle synthesis, wax crystal deposition, anthocyanin accumulation, leaf senescence, ABA responsiveness and stomatal closure compared to WT plants. We propose that the deficiency of SM and accumulation of SG in sng1 mutants may affect the metabolic flow of sinapoyl esters to activate multiple biological processes whose combined action enhances drought resistance. Our findings shed light on the secondary metabolites regulatory network in response to drought stress in Arabidopsis.

Gray mold, caused by Botrytis cinerea, severely threatens strawberry production worldwide, yet the molecular mechanisms underlying strawberry resistance remain poorly understood. Here, we show that the transcription factor ETHYLENE INSENSITIVE 3 (FaEIN3), a strawberry EIN3/EIL homolog, positively regulates strawberry resistance to B. cinerea by directly activating transcription of the WRKY transcription factor gene FaWRKY40. FaWRKY40 further induces the cytokinin biosynthesis gene FaLOG1 and the jasmonic acid biosynthesis gene FaAOC4, thereby promoting hormone accumulation and enhancing disease resistance. Moreover, the JAMM-family deubiquitinase FaPSMD14 physically interacts with FaEIN3 in the nucleus and stabilizes FaEIN3, thereby strengthening FaEIN3-mediated activation of FaWRKY40. Our findings reveal a previously uncharacterized defense mechanism in which the FaPSMD14-FaEIN3-FaWRKY40 regulatory cascade promotes strawberry resistance to B. cinerea through coordinated activation of cytokinin and jasmonic acid pathways.

Hulless barley (Hordeum vulgare L. var. nudum) is a climate-resilient crop, but cadmium (Cd) contamination severely threatens its development on the Qinghai-Xizang Plateau. We identified 12 conserved HbPIN gene family members. Physiological tests revealed that 50 μM Cd stress severely suppressed seedling growth and drastically repressed the expression of HbPIN2, HbPIN4, HbPIN9, and HbPIN12. Interestingly, applying exogenous indole-3-acetic acid (IAA) alone also reduced root length, directly supporting the "auxin optimum principle". Crucially, Cd + IAA co-application successfully rescued growth inhibition and fully restored the transcript profiles of HbPIN4, HbPIN9, and HbPIN12. Spatially decoupled LC-MS/MS metabolomics unraveled that this rescue counteracts an auxin transport stalemate under Cd stress, where active auxins paradoxically accumulate in shoots due to transport blockades while roots suffer intensive catabolism. Transcriptome profiling revealed that this bottleneck is initiated by the synchronized transcriptional shutdown of upstream regulatory master switches, including MADS-box, ERF, and WRKY families. Furthermore, downstream qRT-PCR assays demonstrated that the restored HbPIN profiles systematically modulate transmembrane heavy metal routing gatekeepers; upon exogenous IAA administration, the transcript abundance of the xylem-loading facilitator HbHMA2 and the shootward allocation driver HbCAL1 is remarkably recovered under Cd pressure, thereby contributing to the systemic reconfiguration of dynamic ion homeostasis. In summary, our study explains the key regulatory roles of the HbPIN family, providing vital candidate genes and a strong theoretical basis for breeding heavy-metal-tolerant cereal cultivars.

Drought stress imposes severe constraints on cotton yield and fiber quality. Prior research has shown that exogenous melatonin (MT) application effectively enhances drought tolerance in cotton. Through transcriptome sequencing and weighted gene co-expression network analysis (WGCNA), we identified GhMYC5, a basic helix-loop-helix (bHLH) transcription factor, as a melatonin-responsive hub gene governing drought response. To clarify the functional roles and molecular mechanisms of GhMYC5, virus-induced gene silencing (VIGS) was employed to suppress GhMYC5 expression in cotton, alongside the generation of heterologous overexpression lines in Arabidopsis thaliana. Phenotypic and physiological assessments demonstrated that silencing GhMYC5 substantially compromised drought tolerance. Under drought conditions, GhMYC5-silenced plants showed significantly decreased peroxidase (POD) and catalase (CAT) activities, leaf relative water content (RWC), and proline (PRO) accumulation, while malondialdehyde (MDA) levels were markedly increased. In contrast, transgenic Arabidopsis overexpressing GhMYC5 exhibited improved drought resistance. Transcriptomic profiling identified 9,525 differentially expressed genes (DEGs) in GhMYC5-silenced plants under drought stress. Gene Ontology analysis indicated that these DEGs were primarily linked to protein kinase-related biological processes. Importantly, key genes and kinases associated with Ca2+ signaling, abscisic acid (ABA) and jasmonic acid (JA) crosstalk, and the mitogen-activated protein kinase (MAPK) cascade pathway exhibited substantial alterations. Overall, these findings suggest that GhMYC5 serves as a positive regulator in cotton drought stress response, with its regulatory mechanism closely associated with ABA, JA, and MAPK signaling pathways. This study offers novel genetic resources and a theoretical foundation for elucidating molecular mechanisms underlying cotton drought tolerance, thereby facilitating the development of drought-resistant cotton cultivars.

Karrikins (KARs) are a class of butenolide molecules discovered in smoke hypothesized to mimic an undiscovered plant hormone, KAI2 ligand (KL). KAR/KL signaling regulates germination, seedling development, stress tolerance, and symbiotic interactions with soil microbes, among other traits. KAR/KL signaling is initiated by KARRIKIN INSENSITIVE2 (KAI2), an ɑ/β-hydrolase related to the strigolactone enzyme-receptor DWARF14 (D14). Activated KAI2 forms protein-protein interactions that trigger proteasomal degradation of a transcriptional regulator, SUPPRESSOR OF MAX2 1 (SMAX1), initiating changes in gene expression. D14-LIKE2 (DLK2), an ancient paralog of KAI2 and D14, is a prominent transcriptional marker of KAR/KL signaling in many plants that has uncertain function. We find that DLK2 forms a negative feedback loop that attenuates KAR/KL signaling in Arabidopsis thaliana. This mechanism complements that of KARRIKIN UPREGULATED F-BOX1 (KUF1), which putatively restricts KAR/KL metabolism through targeted protein degradation. Loss-of-function mutations of DLK2 show little effect alone, but synthetically enhance the constitutive KAR/KL responses of kuf1 seedlings. Overexpression of DLK2 proteins from several plants increases the abundance of a SMAX1 ratiometric reporter. DLK2 does not require nuclear localization to protect SMAX1, suggesting its function is independent of interactions with SMAX1 or its transcriptional regulator partners. DLK2 hydrolyzes a profluorescent, desmethyl butenolide reporter molecule that is putatively analogous to KL. We hypothesize that DLK2 catabolizes KAI2 ligand(s) without participating in KAR/KL signaling directly. This functional antagonism could have evolved after KAI2 gene duplication through subfunctionalizing mutations that disrupted protein-protein interactions while preserving enzymatic activity.

Bermudagrass (Cynodon dactylon) is widely used in ecological restoration, soil conservation, industrial landscaping, and potentially bioenergy, ethanol, and biochar production. Shading critically influences bermudagrass growth and survival. However, current knowledge on bermudagrass responses to shade from a hormonal perspective, particularly regarding growth and defense mechanisms, remains limited. We investigated shade responses in bermudagrass with two accessions, shade-tolerant (C792) and shade-sensitive (GZ), through morphological, physiological (hormone profiling by UPLC-MS), and transcriptomic (|log2FC| > 1, adjusted P < 0.05) analyses. Under shading, C792 decreased less on shoot density, coverage, and clipping dry weight than GZ, while maintaining leaf thickness and increasing turf height. Shading significantly increased indole-3-acetic acid (IAA) and gibberellin 1 (GA1) concentrations in C792 but not GZ, with corresponding upregulation of GA biosynthesis genes, changes correlated with its shade tolerance; furthermore, the expression of GA signaling-related gene GID1 was down-regulated to a lesser extent in C792 than GZ, while the up-regulation extent of DELLA was also less in C792 than GZ. Although cis-12-Oxo-Phytodienoic Acid (cis-OPDA) increased under shading, JA concentration remained stable in both bermudagrass under shading. C792 exhibited upregulated jasmonate ZIM domain 1 (JAZ1) and JAZ2 but downregulated salicylic acid (SA) concentrations, whereas GZ exhibited opposite trends. Notably, the SA pathway gene NPR1 in C792 downregulated significantly more pronounced than that in GZ. These coordinated hormonal adjustments in C792, associated with increased growth-promoting hormones (IAA/GA1) contents while showed adjustments in defense responses (reduced JA response), consistent with its shade adaptation through minimal growth trade-offs and strategic defense modulation.

Main conclusionCytokinins and abscisic acid act as auxin antagonists during adventitious root induction, thereby limiting AR development depending on plant genotype and the distance to the wounding site. This study investigated the role of plant hormones in the lower rooting capacity of cuttings of the Hydrangea macrophylla cultivar 'Clarissa' compared to 'Caipirinha', further considering interrelations to carbohydrates. At first, the rooting response to 1 min pulse treatments of the stem base with indole-3-acetic acid (IAA) was analyzed. Without IAA, rooting of 'Clarissa' lagged behind that of 'Caipirinha'. Increasing IAA doses up to 50 mM particularly enhanced rooting in 'Clarissa' to the same level as 'Caipirinha'. The number of adventitious roots was positively correlated with the length of the rooting zone, which was greater in 'Caipirinha' and was further enlarged by IAA application. Carbohydrate analysis indicated higher carbohydrate utilization in the 0-1 cm basal stem section compared to the 1-2.5 cm section above, which was further stimulated by IAA. Phytohormone profiling by LC-MS/MS in both stem base sections revealed similar dynamics for jasmonic acid, jasmonoyl-isoleucine, and IAA in both cultivars, but significantly higher cytokinin and abscisic acid (ABA) levels in 'Clarissa' compared to 'Caipirinha'. 'Clarissa' revealed lower IAA/cytokinin and IAA/(cytokinin + ABA) ratios during the first 48 h after cutting excision compared to 'Caipirinha', while these ratios were higher in the basal 0-1 cm compared to the 1-2.5 cm stem section above. These findings provide new perspectives on hormonal crosstalk in adventitious rooting and support the conclusion that the lower rooting capacity and shorter rooting zone of 'Clarissa' is likely based on locally higher cytokinin and ABA levels that antagonize IAA during the induction phase.

Abstract Arundo donax L. is a highly promising bioenergy and forage crop; however, its seed set is frequently constrained by abnormalities in gametophyte development, and the mechanisms underlying pollen abortion remain unclear. In this study, we integrated morphological observation, scanning electron microscopy, paraffin sectioning, pollen germination assays, meiotic cytology, and transcriptome analysis to investigate the developmental and molecular basis of male gametophyte abortion. Mature anthers were shriveled and did not undergo normal dehiscence, leading to reduced pollen production and fertility. Cytological analysis revealed spindle defects during meiosis, leading to unequal chromosome segregation. Premature tapetal degeneration disrupted nutrient supply to developing microspores, resulting in pollen wall collapse and loss of viability at the tricellular stage. Transcriptome profiling showed that upregulated genes in the male gametophyte were enriched in membrane components, redox processes, and protein ubiquitination, accompanied by activation of ethylene (ET) and jasmonate signaling, indicating their potential involvement in the disruption of normal gametophyte development. Conversely, downregulated genes were primarily associated with microtubule binding, mitotic cell cycle progression, and DNA-binding activities. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis further indicated significant downregulation of genes involved in base excision repair, nucleotide excision repair, and homologous recombination, implying impaired DNA double-strand break repair during meiosis. These defects likely lead to abnormal chromosome behavior, defective microspore formation, and ultimately male gametophyte abortion. Collectively, these findings provide novel insights into the molecular basis of pollen abortion in A. donax .

IntroductionThe early stage of cold acclimation (CA) is a critical period during which plants gradually transition from cold sensing to the establishment of acquired freezing tolerance. Winter rye (Secale cereale L.) is a cereal crop with superior cold hardiness. However, the temporal transcriptional dynamics during early CA and the tissue-specific expression patterns between leaves and roots remain poorly understood.MethodsWe evaluated the growth and freezing survival of 'White BK-1' during long-term CA and profiled leaf and root transcriptomes at multiple time points during the first 48 h of CA.ResultsFollowing CA, enhanced freezing tolerance in rye was associated with growth inhibition. Time-course transcriptome analysis of leaves and roots during early CA (0-48 h) revealed distinct transcriptional reprogramming patterns. At 2 h, 2,585 and 137 DEGs (Differentially expressed genes) were identified in roots and leaves, respectively. DEG numbers peaked at 48 h in both tissues, reaching 8,795 in leaves and 8,984 in roots. The leaf responses were initially dominated by photosynthesis-related pathways, followed by the upregulation of protective metabolism, such as trehalose biosynthesis, glutathione metabolism, and phenylpropanoid biosynthesis. The response in roots was characterized by rapid transcriptional reprogramming, followed by plant hormone signal transduction and damage clearance mechanisms, including ABA/ethylene responses, autophagy, and linoleic acid metabolism, several genes associated with ABA signaling pathways, as well as a gene encoding the DELLA inhibitor RHT1 (SECCE4Rv1G0219050), were significantly upregulated. In total, 764 DETFs (Differentially Expressed Transcription Factors) were identified, mainly from the ERF, MYB, WRKY, bHLH, and NAC families. Moreover, SECCE5Rv1G0340400 (DREB1H, ERF family), SECCE5Rv1G0304800 (CPRF2, bZIP family), and SECCE3Rv1G0171840 (CDF2, Dof family) were upregulated in both tissues. WGCNA (Weighted gene co-expression network analysis) identified modules closely associated with 48 h cold treatment in 'White BK-1' leaves and roots. Within the ICE-CBF-COR signaling pathway, SECCE7Rv1G0464580 (COR27) and SECCE5Rv1G0354870 (COR413PM1) exhibited the highest connectivity in the leaf and root subnetworks, respectively.DiscussionOur study provides insight into tissue-specific transcriptional responses in rye leaves and roots during early CA, identifying candidate genes and co-expression modules potentially relevant to cereal cold tolerance.

This study aimed to investigate the effect of welan gum on camptothecin (CPT) accumulation in Camptotheca acuminata leaves. We evaluated the impact of welan gum treatment on CPT biosynthesis by measuring CPT accumulation and the expression of key CPT biosynthetic genes in C. acuminata leaves. Transcriptome analysis was performed to elucidate the underlying molecular mechanisms driving welan gum-induced CPT biosynthesis. Welan gum application significantly enhanced CPT accumulation by upregulating the expression of CPT biosynthesis genes. Transcriptome data revealed strong activation of the salicylic acid (SA) signaling pathway through increased SA production, which appears to play a central role in mediating CPT biosynthesis in response to welan gum. In addition to SA signaling, jasmonic acid (JA), abscisic acid (ABA), and gibberellin (GA) signaling pathways also responded to welan gum, highlighting a complex regulatory network underlying the plant’s adaptation to microbial polysaccharide signals. Welan gum treatment robustly activates multiple signaling pathways, particularly SA signaling, thereby promoting CPT accumulation in C. acuminata leaves. The coordinated modulation of these pathways reflects the intricate transcriptional reprogramming that balances growth and defense responses, enabling the plant to dynamically adjust to environmental cues triggered by microbial polysaccharides.

Leaf rust disease caused by Melampsora larici-populina severely impacts the photosynthesis and growth of Populus cathayana, causing substantial productivity losses in forest trees. Abscisic acid (ABA) and nitric oxide (NO) are known to play key roles in plant defense. However, their synergistic interplay in woody plant rust resistance remains unresolved. Here, we investigated the roles of ABA and NO in P. cathayana defense against the biotrophic rust fungus M. larici-populina using physiological, transcriptomic, and transgenic approaches. Exogenous ABA and the NO donor sodium nitroprusside synergistically enhanced rust resistance by inducing stomatal closure, modulating reactive oxygen species homeostasis, promoting lignin deposition, and increasing chitinase activity. Transcriptomic profiling revealed that ABA modulated phenylpropanoid and amino sugar/nucleotide sugar metabolism pathways, with DEGs in these pathways tightly correlating with lignin biosynthesis and chitinase activity. Among these, the chitinase gene PcCHIB1 was identified and functionally validated as a pivotal positive regulator of rust resistance, as its overexpression significantly reduced fungal biomass, whereas knockout compromised resistance. Notably, ABA exerted cross-kingdom regulatory effects on M. larici-populina, downregulating genes involved in the spliceosome pathway and two small secreted protein effector genes (MLP105684 and MLP124543) that are critical for fungal virulence. Collectively, these findings establish a bipartite defense paradigm in which ABA and NO synergistically reinforce host basal defense while ABA suppresses fungal virulence, providing a mechanistic foundation for engineering rust resistance in poplar.

Aphid feeding begins with brief exploratory probing and transitions to prolonged phloem ingestion, yet whether these distinct phases trigger different plant immune programs remains unknown. We show that probing and phloem feeding are associated with distinct plant immune cascades. Under probing-enriched conditions, we observed selective activation of jasmonate (JA) signaling, accompanied by significant ROS bursts and vascular callose deposition, whereas phloem feeding elicits only marginal salicylic acid accumulation. Probing-enriched conditions imposes severe fitness penalties on aphids, a phenotype recapitulated by exogenous methyl jasmonate. Consistent with this, aphids exhibit high feeding‑site fidelity, which likely minimizes probing events and helps evade these penalties. Transcriptome analysis identified the squash‑type trypsin protease inhibitor CsTrypPI1, characterized by six conserved cysteines, as a JA-dependent component upregulated under probing-enriched conditions. Recombinant CsTrypPI1 directly impairs aphid survival and fecundity in artificial diet assays. Transgenic cucumber lines constitutively over‑expressing CsTrypPI1 displayed broad‑spectrum resistance against the aphid Aphis gossypii, the whitefly Bemisia tabaci, and the lepidopteran Spodoptera litura. Our findings indicate that the probing versus phloem feeding comparison defines two distinct immunological trajectories, which helps reconcile conflicting evidence on JA and SA roles in plant and phloem‑feeder interactions. The identification of CsTrypPI1 as a broad‑spectrum resistance candidate highlights this behavioral-immunological interface as a promising intervention point for sustainable pest management.

Potato spindle tuber viroid (PSTVd) infection can induce tuber cracking and knob-like protuberances at tuber eyes, but the molecular differences between these two localized symptoms remain unclear. In this study, smooth surface regions and normal eye tissues were collected from mock-inoculated tubers of the potato cultivar 'Kexin 18', whereas crack regions and regions of knob-like protuberances at tuber eyes were collected from PSTVd-infected tubers. RNA sequencing and targeted phytohormone quantification were combined with weighted gene co-expression network analysis, infection × region interaction analysis, and gene-hormone association analysis. PSTVd infection markedly inhibited plant growth and tuber development, and 2972 genes with significant infection × region interaction effects were identified. Crack regions were characterized primarily by enhanced defense and oxidative stress related transcriptional responses, remodeling of cell wall and surface barrier related processes, and accumulation of several cytokinin (CK) metabolites, salicylic acid/salicylic acid glucoside (SA/SAG), and 12-oxo-phytodienoic acid (OPDA); CRK2, RBOHA, and CSLG2 were among the representative candidate genes. Regions of knob-like protuberances at tuber eyes showed pronounced reprogramming of processes related to DNA replication, the cell cycle, chromosome maintenance, and chromatin organization, together with decreases in several CK metabolites, a downward trend in jasmonate-related oxylipins, and an increase in indole-3-butyric acid; ACL5-like, PILS7-like, CYCA3;1, and MCM4 were identified as candidate genes. Gene-hormone association analysis further revealed distinct molecular association patterns in the two regions. Collectively, the two symptomatic regions exhibited distinct region-dependent transcriptomic and phytohormone metabolic states after symptom development.

Main conclusionThis review highlights the importance of the CYTOKININ RESPONSE FACTOR 6 transcription factor as a plant regulatory nexus integrating responses to multiple abiotic stresses with metabolism, growth and development. Cytokinin Response Factors constitute a subfamily of plant transcription factors that have been identified as cytokinin-responsive APETALA2/ethylene-responsive element binding proteins and further characterised as components of cytokinin signalling. Besides being involved in the canonical cytokinin signalling pathway, they are part of complex networks of interactive regulation. Their mode of action, their interactions and their functions however remain to be fully understood. Among cytokinin response factors that are currently identified and characterised in Arabidopsis thaliana, CYTOKININ RESPONSE FACTOR 6 has been extensively studied as it is related to numerous developmental, nutritional, hormonal, abiotic stress and biotic stress processes. CYTOKININ RESPONSE FACTOR 6 was thus recently shown to be part of a SNF1-related kinase1/Jumonji C domain-containing JMJ15 regulatory axis involved in energy, oxidative stress and growth co-regulation. This review focusses on involvement of CYTOKININ RESPONSE FACTOR 6 in the integration of abiotic stress responses with metabolism, growth and development, and on the underlying mechanisms of this integration. The characterisation of CYTOKININ RESPONSE FACTOR 6 as a stress-growth nexus will be highlighted in terms of regulatory processes and mechanisms. The essential roles of this stress-growth nexus will be discussed in the context of multiple stresses and global changes with potential implications for plant biodiversity dynamics and agricultural sustainability.

Waterlogging is a serious abiotic stress caused by climate change that negatively affects plant growth and productivity. In response to waterlogging, plants activate a coordinated series of physiological, metabolic and molecular adjustments that maintain cellular homoeostasis and support survival. This review synthesises recent advances in the mechanisms by which waterlogging-induced oxygen deprivation is sensed and transduced into coordinated molecular programmes that determine plant survival and stress recovery. Particular attention is given to oxygen-sensing hubs centred on ethylene response factor group VII, which are regulated by the N-end rule/N-degron pathway and activate downstream stress-response programmes. Additionally, metabolic reprogramming under hypoxia, including the shift from aerobic respiration to anaerobic pathways (e.g., lactate and ethanolic fermentation) that sustain ATP production, is further examined. Furthermore, redox regulation during hypoxia-reoxygenation is discussed, highlighting the dynamics of reactive oxygen species, antioxidant defences, heat shock protein networks, and proteostasis mechanisms that constrain metabolic flux. Moreover, hormone-mediated growth-survival decisions, contrasting adaptive strategies, such as gibberellic acid-DELLA-mediated growth restraint and ethylene-driven gibberellic acid-abscisic acid signalling, are discussed. Building on this foundation, we present an integrative framework linking these regulatory layers into a coordinated system and compare its deployment across species to explain divergent tolerance outcomes. Finally, we translate these mechanisms into applied crop improvement, addressing breeding targets, ideotype design and field management, and identify key regulatory nodes and priority directions for advancing waterlogging tolerance.

Low temperature constrains nitrogen (N) acquisition and assimilation in aquatic plants, yet how different inorganic N forms regulate growth and stress physiology under cold conditions remains poorly understood. Here, Hippuris vulgaris was exposed for 40 days to ammonium (NH4+-N) or nitrate (NO3--N) supplied at 0, 2, 5, 10, 20 and 50 mg L-1 N under 2-10°C conditions. A mixed-N experiment with different NH4+-N/NO3--N ratios was further conducted at the same total N concentration to evaluate growth-based N-form preference. Growth traits, tissue N accumulation, N-assimilation enzymes, antioxidant responses, nitric oxide (NO) signal, and phytohormone profiles were examined to identify N-form response. Results showed that H. vulgaris had clear preference for NO3--N over NH4+-N. NH4+-N at ≥10 mg N L-1 significantly inhibited root development and biomass accumulation, whereas NO3--N up to 50 mg N L-1 did not cause comparable growth suppression. After 40 days, weight growth rates under NO3--N treatments were 38.34-141.83% higher than 29.64-63.48% under corresponding NH4+-N treatments, and increasing the NO3--N proportion in mixed-N treatments further promoted fresh weight accumulation. The NO3--N advantage was associated with higher NR, NiR, GS and GOGAT activities and greater tissue N accumulation. Compared with NH4+-N, NO3--N reduced malondialdehyde accumulation, maintained higher antioxidant enzyme activities and enhanced root NO signal. High NH4+-N induced abscisic acid accumulation and was associated with weaker jasmonate-related responses, indicating a shift toward stress-dominated hormonal regulation. The above findings suggest that coordinated N assimilation, oxidative balance and phytohormone reprogramming underlie the contrasting responses of H. vulgaris to NO3--N and NH4+-N, providing physiological evidence for its use in nitrate-dominated cold-season aquatic environments.

Soil salinization poses a significant challenge to soybean production, yet the regulatory mechanisms underlying root responses to salt stress remain incompletely understood. In this study, we analyzed the expression patterns, subcellular localization, and functions of overexpressed GmJAZ5 in soybean roots. Under salt stress, GmJAZ5 expression was consistently downregulated in leaves, whereas roots exhibited initial suppression followed by a recovery phase. The GmJAZ5 protein was localized to the nucleus. GmJAZ5 overexpression markedly attenuated salt-induced growth inhibition and enhanced the activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT). This manipulation also reduced malondialdehyde (MDA) accumulation and decreased both Na+ content and the Na+/K+ ratio in leaves. Additionally, it exerted tissue-specific effects on jasmonic acid (JA) and abscisic acid (ABA) levels. These results indicate that overexpression of GmJAZ5 enhances salt tolerance in soybean by strengthening antioxidant defenses, limiting Na+ translocation, and modulating phytohormone homeostasis, highlighting its potential as a candidate gene for breeding salt-tolerant soybean varieties.

Strigolactones are phytohormones that regulate shoot branching and facilitate communication with neighbouring plants, bacteria and arbuscular mycorrhizal fungi. In seed plants, strigolactone perception begins with the enzyme-receptor DWARF14 (D14), an α/β-hydrolase likely to have evolved via gene duplication within a larger gene family that includes the karrikin receptor, KARRIKIN INSENSITIVE 2 (KAI2) and other D14-like (DLK) proteins of uncertain function. However, it is unclear when the functional characteristics that define a bona fide strigolactone receptor evolved. Here, we apply ancestral sequence reconstruction to generate a D14 protein representative of seed plants to study the evolution of ligand specificity and the conservation of signalling mechanisms and to explore desirable traits for protein engineering. Ancestrally reconstructed D14 is structurally and functionally comparable to D14 from Arabidopsis thaliana. The ligand specificity of ancestral D14 does not meaningfully differ, despite having unusual active site geometry and altered enzyme kinetics. We show that the catalytic aspartic acid residue of ancestral D14 is not required for strigolactone signalling when expressed in A. thaliana, suggesting that this mechanistic feature is conserved throughout seed plants. We find that ancestral D14 exhibits higher recombinant yields, greatly increased thermostability and enhanced catalytic activity relative to D14 from A. thaliana. This work provides insight into the evolution of phytohormone signalling and presents a robust scaffold for the application of D14-type proteins in diverse applications.

Abstract The plant hormone jasmonoyl-isoleucine (JA-Ile) reallocates resources from growth towards induced defenses upon microbial or herbivore attacks or other environmental threats. JA-Ile and its precursor jasmonic acid (JA) undergo complex enzymatic turnover that shape hormonal dynamics for optimal signaling. JA-Ile is inactivated through gradual oxidation by CYP94 monooxygenases, followed by 12-OH-JA-Ile deconjugation towards 12-OH-JA. 12-OH-JA was believed to originate also from direct JA hydroxylation by JAO/JOX dioxygenases. Here we investigate the nature and origins of hydroxy-JAs and their derivatives upon stress responses in Arabidopsis. Using a set of analytical methods to explore enzyme assays and multiple pathway-impaired mutants, we show that JAO enzymes produce exclusively 11-OH-JA rather than 12-OH-JA and define a separate branch in JA catabolism. Upon leaf wounding, the CYP94/AH and, to a lesser extent, the JAO pathways direct the respective accumulation of 12- and 11-OH-JAs and their 11/12-glucosylated and 12-sulfated derivatives with isoform-specific occurrences. In contrast, Botrytis infection triggers the exclusive accumulation of 11-OH-JA and 11-Glc- O -JA with complex patterns of HSO 4 -JA. Finally, treatment of jar1 seedlings with 11-OH-JA does not trigger transcriptional changes, reinforcing the notion that the JAO/11-OH-JA pathway represents a metabolic sink modulating JA-Ile formation. Our findings elucidate the 11-OH-JA biosynthetic pathway and highlight stress-specific hydroxylation signatures.

Phytohormone jasmonic acid (JA) plays a central role in plant defense against herbivores by activating transcriptional and metabolic reprogramming. However, how JA-responsive transcription factors coordinate defense signaling and metabolic outputs remains incompletely understood. Here, we identify the WRKY transcription factor OsWRKY10 as an early JA-responsive regulator that enhances rice resistance to brown planthopper (BPH). OsWRKY10 expression is rapidly induced by BPH infestation in a MYC2-dependent manner. Overexpression of OsWRKY10 suppresses BPH feeding and oviposition, whereas wrky10 mutants exhibit increased susceptibility. Mechanistically, OsWRKY10 directly binds to the promoters of JA biosynthetic genes, including OsLOX6, which contributes to BPH-induced JA biosynthesis, thereby promoting JA accumulation and activating downstream defense responses. This MYC2-WRKY10-LOX6 module forms a positive feedback loop that amplifies JA signaling during herbivore attack. We further identify OsDIP1 as a negative regulator that interacts with OsWRKY10 and promotes its degradation, thereby attenuating OsWRKY10-dependent JA signaling. Consistently, dip1 mutants show enhanced resistance to BPH. Together, these findings reveal a JA-amplifying regulatory circuit centered on OsWRKY10 and fine-tuned by OsDIP1 to maintain defense homeostasis, providing new insights into how plants balance defense signal amplification and attenuation during herbivore defense.

Abstract The plant hormone abscisic acid (ABA) can regulate developmental processes like the flower time under various abiotic stress conditions. However, the regulatory roles of genes encoding for receptors in floral transition remain underexplored. Here we demonstrate a previously uncharacterized connection that ABA receptor-encoding-genes PYL8 and PYL9 redundantly repress floral transition by cooperating with the AGL16-SVP-SOC1 regulatory module. The pyl8-1 pyl9 double mutant exhibited accelerated flowering compared to single mutants and wild-type under long-day conditions, independent of exogenous ABA or drought stress. Genetic analysis revealed that, despite of the direct binding within PYL8 promoter, AGL16, as well as its partner SVP, acts epistatic to PYL8 and PYL9 in flowering time regulation. Loss-of-function of these two receptor genes systematically upregulated the SOC1 expression, while they could independently form heterologous complexes with AGL16, thereafter enhanced the AGL16 binding to the SOC1 promoter to transcriptionally repress this flowering integrator in Arabidopsis thaliana. AGL16 likely acts as a molecular glue to tie the interactions between PYL8/PYL9 and SVP. Our findings establish a molecular framework wherein PYL8 and PYL9 collaborate with flowering repressors to delay floral transition through transcriptional regulation of SOC1, thus uncover an important crosstalk between the ABA receptors and the flowering time regulation.

IntroductionCitrus huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is a globally devastating plant disease characterized by severe disruption of host physiology. Although alterations in plant hormone homeostasis, including salicylic acid and abscisic acid (ABA), are hallmarks of HLB, the contribution of plant-associated microbiota to these hormonal changes remains poorly understood. Here, we investigated whether ABA depletion in HLB-affected citrus is associated with microbial metabolism.MethodsABA and its metabolites were quantified in HLB-affected citrus tissues by liquid chromatography-mass spectrometry. The capacity of CLas strain Ishi-1 and Flavobacterium spp. strains CGRL1 and CGRL2 to metabolize ABA was assessed using in vitro culture assays. The effects of exogenous ABA on bacterial growth were also evaluated.ResultsABA concentrations were significantly decreased in symptomatic HLB-affected tissues, concomitant with the accumulation of the ABA catabolite dehydrovomifoliol. Furthermore, CLas strain Ishi-1 and Flavobacterium strains CGRL1 and CGRL2 degraded ABA and produced dehydrovomifoliol, whereas no such activity was detected in non-inoculated controls. Notably, exogenous ABA promoted bacterial growth, suggesting that ABA may serve as a metabolic substrate or growth-enhancing factor for these bacteria.DiscussionThese findings identify microbial ABA metabolism as a potential driver of hormone depletion in HLB-affected citrus. Our results further suggest that CLas and its associated bacteria may actively reshape host hormone homeostasis through ABA metabolism, providing a mechanistic link between pathogen-associated microbial communities and hormonal imbalance during HLB development.

Nitric oxide (NO) and gibberellin (GA) antagonistically regulate plant cell elongation. Here, we report an unexpected observation that NO confers partial GA-insensitivity on hypocotyl elongation in Arabidopsis thaliana. We identify GASA13 and GASA14 as essential downstream specificity filters underlying this phenomenon. Loss-of-function of GASA13 or GASA14 converted the NO-mediated inhibition from a partially GA-insensitive state to a fully GA-reversible state, as revealed by hypocotyl phenotyping analyses. Both GASA proteins showed increased protein accumulation upon NO treatment and decreased accumulation upon GA treatment, while their transcript levels were also regulated by DELLA. Hormone profiling showed that NO modulated bioactive GA levels in a GASA-dependent manner. Transcriptomic analysis revealed that wild-type plants exhibited a focused NO response, whereas gasa13 and gasa14 mutants displayed broad, diffuse transcriptional signatures, indicating a loss of signal specificity. Further analysis of GA metabolic and cell wall-related genes supported the model that GASA13 and GASA14 restrict NO-induced transcriptional changes to a precise growth-suppressive program. We propose that GASA13 and GASA14 act as downstream transcriptional specificity filters that channel NO signals into a focused output, conferring partial GA-insensitivity. This work uncovers a novel regulatory layer of NO-GA crosstalk beyond DELLA stabilization, and provides a mechanistic framework for understanding how plants achieve signal specificity during growth regulation.

Abiotic stress frequency and intensity are increasing, severely impacting plants' health, hence leading to significant crop yield losses (~20%–40% globally). In addition to modifying their genetic and physiological traits to increase stress tolerance, growing research revealed that plant–microbiome interaction plays a remarkable role in determining stress resilience. This review integrates physiological, ecological, and multi‐omics data suggesting holobiont plasticity is an unifying paradigm for mechanistic understanding of stress‐induced plant–microbe system reorganization. Abiotic stress causes rapid changes in plants' root metabolism and root exudate composition, which alter the release of organic acids, phenolics, osmolytes, and signaling compounds, which selectively change the microbial community's structure in the rhizosphere and endosphere. Microbial taxonomic diversity usually declines under stress conditions. Meanwhile, functional redundancy within the microbial communities is generally maintained or can increase. However, network connectivity may often remain stable or become stronger under stress, and the centrality of keystone taxa usually increases. These keystone microbes play a critical role in sustaining microbial community structure and function. Microbial regulation of phytohormones (such as auxin, ethylene, and abscisic acid), along with control of redox balance, osmotic adjustment, and nutrient cycling, improves plant water use, nutrient uptake, and root development. This often makes them more tolerant to stress by 15%–60% without increasing their biomass. Holobiont plasticity emerges as a quantifiable and potentially predictive characteristic of plant stress responses by integrating microbial network structure, functional gene profiles, metabolomic responsiveness, and host regulatory mechanisms. These responses function on ecological timescales (days to weeks), preceding the more gradual process of host genetic adaptation. This halobiont plasticity‐based framework shows promising potential but requires validation under field conditions to prove its robustness and applicability. This opens new avenues for microbiome‐assisted plant growth and development of a climate‐resilient agricultural system.

BackgroundMethyl jasmonate (MeJA) is a potent elicitor of secondary metabolism in plants, but its regulatory role in flavonoid biosynthesis in Scutellaria barbata remains unclear.MethodsHere, we performed an integrated transcriptomic and metabolomic analysis of S. barbata seedlings following exogenous MeJA treatment, combining differential expression and correlation network approaches to identify candidate genes, metabolites, and transcription factors (TFs) associated with flavonoid accumulation.ResultsMeJA treatment significantly promoted the accumulation of multiple flavonoids and upregulated key biosynthetic genes, including Sbar2C282T9_CHS2, Sbar5A47T51_CHI, and Sbar9A209T130_C4H. A total of 2,453 differentially expressed genes (DEGs) were identified, which were significantly enriched in flavonoid biosynthesis and plant hormone signal transduction pathways. Among these, 180 differentially expressed TFs were detected, with the ERF, MYB, bHLH, NAC, and bZIP families being markedly enriched. Integrative correlation analysis revealed 40 TFs whose expression patterns were strongly correlated with 19 flavonoids. TF binding site prediction further identified seven candidate TFs predicted to target flavonoid structural genes, including Sbar2C282T9_CHS2 and Sbar9A209T130_C4H. Protein‑protein interaction prediction suggested that several of these TFs, particularly bHLH members, may interact with JAZ repressors and form intra‑family interaction clusters.DiscussionCollectively, these findings suggest a potential JA-responsive transcriptional regulatory network and point to a conserved JAZ-TF module that may modulate flavonoid biosynthesis in S. barbata. This study provides valuable insights into the regulatory mechanisms of secondary metabolism in medicinal plants and offers candidate gene resources for metabolic engineering and molecular breeding of high‑flavonoid varieties.

Cadmium (Cd) phytoextraction requires efficient metal acquisition and translocation without compromising plant growth and detoxification. We investigated the effects of silicon nanoparticles (Si-NPs) and jasmonic acid (JA), applied alone or together, on Cd phytoextraction by Sedum alfredii. The combined treatment increased shoot biomass and total Cd accumulation by 32.6% and 67.4%, respectively. Si-NPs-containing treatments increased rhizosphere available Cd by 5.4-5.9%, promoted Cd uptake and root-to-shoot translocation, and induced Cd- and Si-transport-related genes. JA strengthened antioxidant enzyme activities. The combined treatment further increased phytochelatin and glutathione levels by 152% and 48.7%, respectively, while reducing malondialdehyde by 38.5%. These responses indicate enhanced thiol-mediated detoxification and oxidative protection. Metabolomic and microbiome analyses further revealed compartment-specific responses. The rhizosphere was associated with Cd-mobilization-related metabolites and enrichment of potentially beneficial genera, including Polycyclovorans and Ramlibacter. In contrast, leaves showed enhanced sulfur-thiol metabolism, redox regulation, and secondary metabolism. Multiblock integration linked Cd phytoavailability and accumulation with detoxification and biomass production. Overall, Si-NPs and JA exhibited asymmetric functional complementarity, integrating enhanced Cd acquisition and transport with thiol-centered detoxification and growth maintenance. This accumulation-compatible tolerance strategy provides a mechanistic framework for improving assisted Cd phytoextraction by coordinating Cd flux with whole-plant detoxification capacity.

Chloroplasts drive photosynthesis and regulate cellular signaling and phytohormone biosynthesis processes that greatly influence crop yield and quality traits. In this study, we identified a recessive chlorophyll-deficient mutant, reduced chlorophyll mutant 1t (rcm1t), from an advanced-generation tomato inbred line. Genetic and phenotypic characterization confirmed that chloroplast degradation accelerates rapidly and chlorosis occurs during late leaf development in the rcm1t mutant. Subsequently, we fine-mapped rcm1t to an approximately 60 kb region on chromosome 8. This interval encompasses Lutescent (L1)/SlRCM1, a well-characterized gene essential for chloroplast biogenesis and developmental maintenance. An allelism test between rcm1t and the previously characterized ethyl methanesulfonate (EMS)-induced rcm1 mutant revealed non-complementation. Moreover, transgenic complementation with the wild-type SlRCM1 coding sequence rescued the chloroplast premature senescence phenotype in rcm1t, providing definitive genetic evidence that rcm1t is a loss-of-function allele of SlRCM1. Resequencing identified a ~4.8 kb retrotransposon insertion in the first exon of the gene, which disrupts normal transcription and results in a premature stop codon. Quantification of abscisic acid (ABA) revealed significantly higher ABA levels in the rcm1t mutant than in the wild type. Protein-protein interaction analyses demonstrate that SlRCM1 interacts with SlNCED2. Osmotic stress assays showed that the rcm1t near-isogenic line exhibited enhanced tolerance under osmotic stress. This study successfully mapped the natural allele rcm1t of SlRCM1, preliminarily explored its potential application in enhancing plant tolerance to osmotic stress, and provided valuable insights for further elucidation of SlRCM1 function.

Plants are rarely exposed to ultraviolet C (UV-C) radiation because it is absorbed by the ozone layer. Nevertheless, low doses of UV-C can induce physiological and developmental responses, although the underlying hormonal mechanisms are not well understood. We show that a single one-minute UV-C pulse (20 W/m2) is associated with temporally distinct differences in gibberellin (GA) homeostasis in Arabidopsis. At early sampling times, levels of GA12, a precursor of bioactive GAs, are lower in UV-C-treated plants compared with controls. This is accompanied by lower transcript levels of KS and KAO2. Similarly, bioactive GA4 levels are lower in UV-C-treated plants. These results suggest that UV-C suppresses the early stages of GA-biosynthesis. At later time points, however, the GA4 levels are higher in UV-C-treated plants than in controls, coinciding with earlier flowering, enhanced growth, and increased fertility. The GA-signalling mutant gdella and the early GA-biosynthesis mutants kao1 and kao2 do not show wild-type developmental responses to UV-C, indicating that GA biosynthesis and signalling contribute to this response. In contrast, the C19-GA 2-oxidase quintuple mutant (C19-2oxq) retained wild-type-like responses, suggesting a limited role for C19-GA 2-oxidation. These results highlight the importance of GA-homeostasis in UV-C-mediated regulation of plant development.

Summary Cereal grain is a global food staple. Filial tissues in grain, such as the embryo and endosperm, grow embedded in the maternal ovule and surrounding ovary wall. Maternal tissues provide nutrition and protection but are progressively reduced and degraded as filial tissues expand, driving grain expansion between floret hulls. While we know some enzymatic players, regulation of this process remains less understood. However, our previous work in barley, Hordeum vulgare spp. vulgare suggested a role for the phytohormone jasmonate (JA). We edited the barley ALLENE OXIDASE CYCLASE ( HvAOC ) gene encoding a JA biosynthetic enzyme. We examined hvaoc mutants for developmental phenotypes, genetic interactions and effects on the transcriptome. Defective HvAOC alleles reduced JA levels, and caused enhanced vegetative growth, faster flowering and lengthened grain, all phenotypes rescued by MeJA treatment. HvAOC control of grain length appears parentally‐derived, consistent with enriched HvAOC expression in maternal compared to filial tissues. Grain dimension changes were uncoupled from hull size and correlated with larger endosperm cavities, more persistent maternal tissues and higher expression of genes linked to sucrose transport. This work provides the first genetic dissection of JA in barley and highlights roles in coordinating maternal tissues within grain.

AtATG8h and AtATG8i belong to a unique sub‐group of the nine ATG8 proteins encoded in the Arabidopsis genome. Unlike other ATG8s that need ATG4 protease for cleavage to expose their C‐terminal Gly residue for attachment of phosphatidylethanolamine (PE) and thus subsequent recruitment to the autophagosomal membranes, AtATG8h and AtATG8i directly carry a Gly residue at their C‐termini and can be lipidated without the action of ATG4. We previously showed that CLATHRIN LIGHT CHAIN 2 (CLC2) participates in the autophagy process via interacting with AtATG8h and AtATG8i. Simultaneously knocking out AtATG8h and AtATG8i by CRISPR/CAS9 technology compromised autophagy, and as a consequence, enhanced the resistance to a biotrophic fungal pathogen. In this study, we took a gain‐of‐function approach to further investigate the roles of AtATG8h in disease resistance. Our results showed that overexpression of AtATG8h enhanced the resistance to biotrophic bacterial and fungal pathogens but compromised the resistance to a toxin secreted from a necrotrophic fungal pathogen. The enhanced resistance to the biotrophic pathogens was correlated with the increased expression of Pathogenesis‐related (PR) gene, enhanced callose deposition and levels of both salicylic acid (SA) and H₂O₂, whereas the compromised resistance to the necrotrophic fungal toxin was correlated with the significantly reduced expression of the genes in the jasmonic acid (JA) pathway. These results indicated that either knocking out or overexpressing AtATG8h resulted in a similar outcome in Arabidopsis disease resistance. The underpinning molecular mechanism is discussed.

We previously found that inoculation with rhizobia tends to increase both biomass production and glycyrrhizic acid (GL) production in the medicinal plants Glycyrrhiza uralensis and G. glabra. In this study, we investigated the mechanism by which rhizobial inoculation promotes GL production. Transcriptome analysis of plants grown for 3, 6, 9, and 13 weeks after rhizobial inoculation revealed significant enrichment of GO terms related to root tissue differentiation and reorganization in inoculated plants. In addition, jasmonic acid (JA)-mediated signaling pathway, fatty acid biosynthetic process, and isoprenoid biosynthetic process were specifically enriched in inoculated plants, and these terms included Jasmonate ZIM-domain protein (JAZ)-like, MYC-related transcription factor 2 (MYC2)-like, Allene oxide cyclase (AOC)-like, Allene oxide synthase (AOS)-like, and Squalene synthase 2 (SQS2)-like genes. Time-course analysis of the expression patterns of these genes showed that JAZ-like genes were upregulated at the early growth stages in inoculated plants, whereas at 13 weeks after inoculation, expression of JAZ-like genes decreased while MYC2b and CYP88D6 expression increased. These results suggest that CYP88D6 expression may be regulated by MYC2. These results suggest that activation of JA biosynthesis and JA signaling is involved in the promotion of GL production by rhizobial inoculation, thereby supporting our previously proposed hypothesis at the transcriptome level. Further studies, including JA quantification, elucidation of transcriptional regulatory mechanisms, and functional analyses, will be necessary.

Efficient hybrid seed production requires controllable male sterility systems, yet how developmental processes can be selectively manipulated to disrupt male fertility while preserving vegetative growth remains poorly understood. Whether filament elongation represents a distinct hormonal sensitivity checkpoint and how its regulation can be exploited to uncouple male fertility from vegetative growth remain largely unknown. Here, we identify a series of gibberellin-sensitive genic male sterililty (GGMS) mutants carrying allelic variations in the maize GA biosynthetic gene ZmKAO. Unlike previously characterized GA-deficient mutants with broad defects in reproductive development, ggms mutants exhibit male sterility primarily due to defective filament elongation, while anther development, pollen maturation, and female fertility remain largely unaffected even under severe GA deficiency. Notably, filament elongation in GGMS mutants can be restored by a single GA application after tasseling. Genetic, biochemical, and molecular analyses support a model in which reduced D9 accumulation relieves D9-mediated repression of the filament-preferential transcription factor ZmMYB53, thereby enhancing ZmBXL7 expression and promoting filament cell elongation. Quantification of endogenous GA levels across a series of ZmKAO alleles with different functional strengths reveals a graded relationship between GA reduction and developmental outputs. While strong alleles (ggms1/2) cause severe growth defects, weak alleles (ggms3/4) uncouple vegetative growth from male fertility by selectively disrupting filament elongation. Based on these findings, we propose a three-threshold model in which different developmental processes exhibit distinct sensitivities to GA reduction. This framework enables rational selection and engineering of GGMS materials for maize hybrid seed production. Together, our study reveals a hierarchical GA sensitivity mechanism underlying maize reproductive development and provides both conceptual insights into hormone-regulated fertility and practical strategies for engineering controllable GGMS systems in maize.

Gibberellins (GAs) are ubiquitous phytohormones that regulate plant growth and are widely used in agriculture. Among these, GA3 and GA4+7 are the only commercially available products, yet GA4+7 commands a much higher price than GA3, primarily due to its low titer in the industrial fungus Fusarium fujikuroi. To engineer a high-yielding GA4+7 producer, we first deleted p450-3 to block the conversion of GA4 and GA7 to GA1 and GA3. This led to the accumulation of GA4+7 at 0.934 g/L, a 37-fold increase over the wild-type strain, albeit with over half reduction in total GA accumulation. Using this Δp450-3 mutant as a platform, we combined metabolic engineering (overexpressing rate-limiting enzymes) with process optimization (pH, medium composition and fermentation duration). These combinatorial interventions synergistically boosted GA4+7 production. Under optimized conditions, the engineered strain achieved a final titer of 6.08 g/L (a 6.51-fold increase over the Δp450-3 parent), comprising 2.13 g/L GA4 and 3.96 g/L GA7, representing 5.30- and 7.44-fold increases, respectively. Preliminary optimization in a 10 L fermenter yielded 3.51 g/L of GA4+7. Finally, a solid-state fermentation system was developed on wheat bran, yielding 17.34 g GA4+7 per kg and enabling green, in-house, in-situ gibberellin production. In addition to substantially increasing the GA4+7 titer and total GA accumulation, this study demonstrated that the GA4/GA7 ratio can be modulated through both molecular and fermentation strategies.

Progression of leaf senescence is regulated by internal factors like the plant hormone cytokinin (CK) that acts to delay senescence and retain photosynthesis by maintaining chloroplast-functionality. While initial CK-signaling components controlling senescence have been described, downstream targets regulating physiological performance have remained less characterized. Here we present the CHLOROPLAST IMPORT APARATUS 2 (CIA2) and CIA2-like (CIL) transcription factors (TFs) as mediators of CK senescence activity, enabling chloroplast-function. Utilizing loss-of-function mutants, we show CIA2 and CIL negatively regulate senescence progression and alter physiological performance in Arabidopsis thaliana . Integrated transcriptomic-metabolomic analysis shows that CIA2 and CIL action is distinct from other known chloroplast-related TFs (like GLKs/GATAs) and can boost antioxidant response over senescence. We find that CIA2 and CIL are essential in relaying CK activity during chloroplast biogenesis, photosynthesis, rubisco function, and metabolite accumulation. In turn, ARR10/12 CK signaling components were needed for CK induction of CIA2 and CIL themselves - a cascade that induced chloroplast- and photosynthesis-associated nuclear genes, to boost chloroplast-function and metabolism. We also show that CK acts to repress senescence through accumulation of the polyamine, spermidine, in a CIA2/CIL dependent manner. This work presents a CIA2/CIL-cascade as a new anterograde signal for CK antisenescence effects, indicating a mechanism through which CK prolongs leaf function. This fundamental understanding of CK action has implications towards engineering plants with elevated photosynthesis performance and delayed senescence.

Clubroot, caused by Plasmodiophora brassicae, poses a serious threat to the rapeseed (Brassica napus) industry. Due to B. napus being an allopolyploid with a complex genome and the current scarcity of available resistance gene resources, the molecular basis of rapeseed resistance to P. brassicae remains poorly understood. Here, we performed a functional characterization of BnEIN2 (ethylene-insensitive protein) to explore the role of ethylene signaling in rapeseed resistance to P. brassicae. The Bnein2 mutants generated through CRISPR/Cas9 technology exhibited enhanced resistance to P. brassicae, along with reduced 1-aminocyclopropane-1-carboxylic acid (ACC)/S-adenosyl-L-methionine (SAM) accumulation and ethylene insensitivity. Pharmacological assays demonstrated that inhibitors of ethylene biosynthesis or signaling improved the resistance of Bnein2 mutant plants to P. brassicae. Transcriptome analysis revealed that loss-of-function of BnEIN2 affected the expression of ethylene-, auxin-, and cytokinin-related genes. Moreover, the increased resistance of Bnein2 mutants to P. brassicae was accompanied by a reduction in auxin (indole-3-acetic acid, IAA) biosynthesis and degradation of cytokinin (trans-zeatin, TZ). Collectively, these findings establish the negative regulatory role of ethylene signaling in rapeseed resistance to P. brassicae. This study represents the first effort to elucidate rapeseed resistance to P. brassicae by directly obtaining rapeseed genetic material and offer novel insights into the hormonal regulatory network underlying disease resistance and valuable resources for breeding clubroot-resistant varieties.

Cyclocarya paliurus leaves are rich in bioactive triterpenoids, yet their regulatory mechanisms, particularly in response to methyl jasmonate (MeJA), remain unclear. This study aimed to characterize the spatial and temporal effects of MeJA on triterpenoid accumulation, identify MeJA-responsive genes involved in triterpenoid biosynthesis, and functionally evaluate CpSQS and its promoter regulatory regions. Upper and lower leaves were sampled at multiple time points after MeJA treatment. Six triterpenoid monomers were quantified by HPLC, and transcriptomes were analyzed at 5 and 10 days. MeJA altered triterpenoid accumulation in a time- and leaf-position-dependent manner, with stronger responses in upper leaves. Differentially expressed genes were enriched in terpenoid backbone biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, plant hormone signaling, and MAPK pathways, while HMGR, FPPS, SQS, and SE were generally upregulated. CpSQS was induced by MeJA and showed a spatial expression pattern associated with triterpenoid accumulation. Heterologous overexpression of CpSQS in tobacco calli significantly increased CpSQS transcript abundance and total triterpenoid content, supporting its positive role in triterpenoid biosynthesis. Promoter deletion analysis further identified a 188 bp region from -961 to -773 bp that contributed substantially to promoter activity, with its deletion reducing GUS activity by 56%. These findings reveal the spatiotemporal regulation of triterpenoid biosynthesis by MeJA and identify CpSQS and its transcriptionally active promoter region as potential targets for further functional studies and metabolic improvement in C. paliurus.

Pectobacterium carotovorum is a gram-negative phytopathogenic bacterium that causes soft rot disease on diverse plant species. It encodes the type III secretion system effector protein, DspE, and its chaperone, DspF. The DspE family proteins form water and solute channels in plant cells, flooding the apoplast to aid bacterial multiplication. In Pseudomonas syringae, the DspE ortholog, AvrE, upregulates abscisic acid (ABA) expression, leading to stomatal closure. In this study, a P. carotovorum dspEF mutant did not cause leaf cell death in tobacco leaves. This observation is supported by the lower expression of plant cell wall–degrading enzymes such as pelB, pelI, celV, prtW, and the quorum-sensing system transcript expI in tobacco plants prior to visual symptoms (5 h postinoculation). Interestingly, neither dspE/F nor hrpL mutation affected synthesis of quorum-sensing signaling molecule acyl-homoserine lactone under microbiological settings. However, maceration symptoms occurred if leaves infiltrated with the dspEF mutant were kept under high humidity or detached postinfiltration. These leaves showed elevated transcription of ABA synthesis genes compared with infiltrated leaves maintained on the plant under ambient conditions. To validate this involvement, coinfiltration of ABA with the dspEF mutant restored its ability to cause maceration in attached leaves under ambient conditions. Overall, our data suggest that DspE/F facilitates host susceptibility by creating an aqueous apoplast, promoting ABA accumulation and stomata closure.

The urgent demand for precision agriculture has intensified the need for reliable sensors capable of monitoring phytohormones. To address this, we constructed a novel organic-inorganic intercalation hybrid, Cu-SDA, by embedding aromatic stilbene-4, 4'-dicarboxylate (SDA) linkers into layered copper hydroxide. This design moves beyond conventional carbon-based electrodes, leveraging the synergistic effect of π-π stacking for structural robustness and exposed copper centers for specific electrocatalytic activity. Comprehensive physicochemical characterization, including X-ray diffraction (XRD), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS), confirmed the successful intercalation and structural integrity of the framework. Furthermore, density functional theory (DFT) calculations revealed enhanced adsorption toward salicylic acid (SA) (-1.01 eV) and indole-3-acetic acid (IAA) (-1.14 eV), accompanied by reduced bonding distances. Consequently, the Cu-SDA sensor exhibits a high electroactive surface area (0.229 cm2) and superior charge transfer efficiency compared to pristine Cu(OH)2. The platform achieves low detection limits (2.43 μM for SA, 1.15 μM for IAA) with excellent selectivity against common interferents. These detection limits are suitable for monitoring stress-induced accumulation of SA and IAA, rather than their basal physiological concentrations. Finally, the practical applicability of the proposed sensing platform was demonstrated through an on-leaf standard addition assay for SA and IAA in living peanut seedling leaves, highlighting its potential for monitoring stress-induced phytohormone variations in precision agriculture.

Drought stress severely limits grapevine productivity and compromises fruit quality, with its adverse effects expected to intensify under ongoing climate change. This review synthesizes recent advances in elucidating the molecular and physiological mechanisms underlying drought tolerance in Vitis vinifera. Key physiological responses include abscisic acid (ABA)-mediated amplification of stomatal closure, osmotic adjustment, activation of antioxidant defense systems, and hydraulic vulnerability segmentation-a protective strategy involving the targeted sacrifice of petioles and basal leaves to maintain hydraulic integrity in stems and roots. These coordinated responses are regulated by a complex transcriptional network centered on NAC, WRKY, MYB, and bZIP transcription factors, and further modulated by hormone metabolism, potassium channel activity, aquaporin-facilitated water transport, and both enzymatic and non-enzymatic antioxidant pathways. We also identify critical knowledge gaps requiring urgent attention: (First) field-scale validation of transgenic or CRISPR-edited grapevine lines; (Second) the molecular basis of rootstock-scion signaling during drought stress; and (Third) the integration of multi-omics data with high-resolution phenotyping to accelerate precision breeding. Collectively, this review provides an integrative conceptual framework to support sustainable viticulture in water-limited environments.

Jasmonates are essential phytohormones that coordinate defense responses and developmental programs across land plants. In angiosperms, the active jasmonate ligand jasmonoyl-L-isoleucine (JA-Ile), is produced through GH3-mediated conjugation of jasmonic acid to isoleucine and JA-Ile homeostasis is further shaped by ILR1/ILL-family amidohydrolases. In contrast, the primary bioactive jasmonate ligand in bryophytes, dinor-12-oxo-phytodienoic acid (dn- iso -OPDA), is inactivated through conjugation with amino acids, raising the question of whether these conjugates constitute a reversible hormone reservoir or an irreversible catabolic end point. Although the ILR1-like family has been characterized extensively for its role in auxin and jasmonate homeostasis in angiosperms, its function in bryophytes remains basically unexplored. Here we show that MpILR1, the sole Marchantia ortholog of the ILR1/ILL family, hydrolyzes a specific subset of dn- iso -OPDA-amino acid conjugates in vivo . Loss-of-function Mp ilr1 mutants exhibit enhanced accumulation of dn- iso -OPDA conjugated to hydrophobic amino acids (Val, Leu and Ile) but not to hydrophilic residues (His, Glu and Gln), demonstrating substrate-selective hydrolysis. MpILR1 hydrolytic activity is required for full dn- iso -OPDA-mediated responses, including transcriptional activation and defense against gastropod herbivory. These findings establish MpILR1 as a key positive regulator of jasmonate signaling in Marchantia polymorpha and suggest that hormone conjugation/deconjugation is an ancient regulatory mechanism evolved during plant terrestrialization.

Main conclusionNO and H2 synergistically promote cucumber adventitious rooting by regulating hormone, photosynthesis and antioxidant pathways. Adventitious root formation is essential for plant propagation and is modulated by nitric oxide (NO) and hydrogen gas (H2). This study used transcriptome sequencing to investigate cucumber explants treated with sodium nitroprusside (SNP, an NO donor) and hydrogen-rich water (HRW, an H2 donor). In total, 2716 differentially expressed genes (DEGs) were identified under SNP treatment and 1443 under HRW treatment, with 570 genes co-regulated by both signals. KEGG enrichment analysis revealed significant enrichment in pathways involved in phytohormone signaling, photosynthesis-antenna proteins, and glutathione metabolism. Functional characterization of the 570 common genes showed enrichment of protein kinases, cytochrome P450, and transcription factor families (AP2/ERF, MYB, bHLH, NAC), and protein-protein interaction network analysis identified five hub genes (CsAUX1, CsCYCD3-3, CsGSTU8, CsXTH3, CsERFC3). Physiological assays verified that SNP and HRW coordinately regulated phytohormone homeostasis: they decreased zeatin content while increasing brassinosteroids and ethylene levels. Additionally, both treatments improved photosynthetic parameters and enhanced glutathione antioxidant activity. Time-course qPCR revealed that HRW induced faster gene expression changes (peaks at 12-24 h) than SNP (peaks at 24-48 h), and NO scavenger (cPTIO) experiments confirmed that SNP effects are strictly NO-dependent while HRW acts through both NO-dependent and NO-independent pathways. Oxidative stress markers (MDA, H2O2, O2-) were reduced, and antioxidant enzymes (SOD, POD, CAT, APX, GR, GPX, GSTs) were elevated, indicating enhanced redox buffering capacity rather than oxidative stress. Cis-regulatory analysis further detected transcription factor binding sites related to light, hormone, and stress responses. Collectively, these findings systematically reveal a synergistic molecular network by which NO and H2 promote adventitious rooting. This study enhances our understanding of the regulatory mechanisms underlying adventitious root development and offers a theoretical foundation for optimizing propagation techniques in horticultural crops.

Brassica napus L. is a member of the family Cruciferae. This species is also referred to as "rape," “oilseed rape," "rapa," "rappi," and "rapeseed." It is the third most important source of edible oil globally after soybean and palm oil. Quantitative trait loci (QTLs) analysis has proven to be a powerful tool to reveal the genetic mechanisms of seed weight regulation in this species. We identified and compiled a comprehensive list of 1,905 candidate genes associated with seed weight regulation in Brassica napus. Five candidate genomic regions with 204 seed weight–related genes were identified, including 21 differentially expressed genes from transcriptomic analysis. A CRISPR/Cas mutant library targeting silique- and seed size-related genes was generated in rapeseed, indicating a potential role of BnaHRDs in their development. DEGs were involved in developmental processes, cell division, and nutrient storage, indicating their role in regulating seed size in Brassica napus. HB1 and HAIKU2 also regulate seed weight by controlling endosperm proliferation through the IKU pathway. A total of 1,643 SNP markers were aligned to the pseudochromosomes of Brassica napus using BLAST to identify significant hits. Furthermore, BRASSINAZOLE-RESISTANT1 (BZR1) is an important regulator of seed growth acting through maternal tissues. TDZ treatment enhanced both cell size and number. Plant hormones, including auxin, gibberellin (GA), and brassinosteroid (BR) signaling pathways, have been reported to regulate seed size. Transcription factors including LEC2, WRI1, FUS3, MYB30, and ABI3 were active during early seed development, while LEC1, LEC2, ABI3, and FUS3 remained active during seed maturation. Physiological processes play a key role in yield variation and the selection of high-yielding genotypes. Currently, marker-assisted selection and transgenic approaches are commonly used in rapeseed breeding. Temperature and light are key environmental factors affecting oil production in rapeseed. The integration of genetic engineering, molecular marker technologies, and conventional selection is expected to be a key approach for improving high-oil-content rapeseed. These limitations highlight the need for further studies on rapeseed applications.

Platycladus orientalis (P. orientalis) is a monoecious and evergreen tree. It is not only one of the most common ornamental species, but also has some pratical value. Cutting propagation is a common technique used to preserve excellent P. orientalis resources. But the rooting rate is influenced by both internal and external factors. In this study, we used our new cultivar “Liye” as the material to design experiment. We detected the determination of physiological indicators including soluble protein, peroxidase (POD), polyphenol oxidase (PPO), and indole acetic acid oxidase (IAAO). Meanwhile, the content of indole‐3‐acetic acid (IAA), zeatin riboside (ZR), abscisic acid (ABA), and gibberellin 3 (GA₃) were detected after indole‐3‐butyric acid (IBA) treatment. According to the data on phenotypic changes, we analyzed some proteins related with hormones by isobaric tag for relative absolute quantitation (iTRAQ). We found IAA is a key endogenous hormone that induced the formation of adventitious root in young branches of P. orientalis. Besides, the content of ZR, ABA, and GA₃ showed the different trends of change. We found 136 differential expression proteins related to hormones, and selected two proteins related to cytokinin, three proteins related to auxin, and one protein related to abscisic acid based on the changing trends of hormone content at different stages. The results provided a more holistic view of cellular processes and laid a theoretical foundation for the production of uniform and high quality seedlings in P. orientalis.

Salicylic acid (SA) and jasmonic acid (JA) are key plant defense hormones that typically exhibit antagonistic interactions, with JA levels peaking during the day and SA at night. However, how plants sustain SA-mediated immunity during daylight when JA defenses dominate remains unclear. Here, we identify the rice oscillator gene OsRVE6aas a central regulator enabling simultaneous activation of SA and JA pathways during daytime biotic stresses. Daytime feeding by the small brown planthopper (SBPH) induces OsRVE6a expression and alters circadian rhythms in rice. OsRVE6a maintains phased JA and SA peaks, activates JA signaling via the receptor OsCOI1a to align JA signaling with JA content peaks, and alleviates their antagonism by suppressing OsNPR1. Concurrently, OsRVE6a directly activates OsNPR1-responsive defense genes such as OsWRKY20 and OsPR1 ensuring SA-responsive gene expression despite OsNPR1 reduction. Genetic analysis confirms that OsWRKY20 is essential for OsRVE6a-mediated SBPH resistance. These findings establish OsRVE6a as a molecular orchestrator linking circadian timing and immune coordination, enhancing rice resilience to biotic stresses.

Pathogenesis-related (PR) proteins are central to plant defense; however, their roles in regulating secondary metabolism remain largely unexplored. In Panax ginseng, the molecular characteristics and functional links of PR1 and PR10 gene families to ginsenoside biosynthesis remain unclear. Here, we conducted genome and transcriptome identification, evolutionary analysis, co-expression network construction, and methyl jasmonate (MeJA) induction assays to investigate their roles. We identified 34 PR1/PR10 family members (11 PgPR1s and 23 PgPR10s) unevenly distributed across 15 chromosomes, with gene duplication driving family expansion. Functional divergence was evident: PgPR1 members are evolutionarily conserved and root-preferential, associated with antibacterial defense, whereas PgPR10 underwent species-specific expansion, showed broad tissue expression, and possessed promoters enriched with hormone- and stress-responsive cis-elements. Co-expression analysis (|r| ≥ 0.3, p PgPR1-5 and PgPR10-8 as the only PR members significantly correlated with key ginsenoside biosynthetic enzymes. Under MeJA, the ginsenoside pathway displayed bidirectional temporal regulation, with core PR genes showing synchronized expression with the negatively regulated enzyme gene CAS_14. Our study characterizes the molecular and evolutionary features of PgPR1/PgPR10 families, identifies core PR genes associated with ginsenoside metabolism, and reveals their coordinated response to MeJA, providing key insights and candidate targets for exploring the potential link between PR proteins and ginsenoside biosynthesis and promising candidate genes for further research on P. ginseng.

Histone acetyltransferases (HATs) regulate transcription epigenetically through histone acetylation and play crucial roles in plant development. In this study, we identified a cotton HAT gene, GhHAT11, associated with plant height and investigated its regulatory functions and underlying mechanisms. Tissue-specific expression analysis showed that GhHAT11 was predominantly expressed in stems. Virus-induced gene silencing (VIGS) of GhHAT11 significantly reduced cotton plant height and internode cell length, whereas heterologous overexpression of GhHAT11 in Arabidopsis thaliana increased plant height, confirming its positive role in plant height regulation. Metabolomic and transcriptomic analyses revealed that silencing GhHAT11 decreased auxin and gibberellin (GA) levels, altered the expression of 3233 genes, downregulated genes involved in auxin and GA biosynthesis/signaling, and upregulated genes related to cytokinin metabolism and signaling. Furthermore, GhHAT11 positively regulates the expression of GA biosynthetic genes by maintaining H3K9ac levels at their promoters. Collectively, GhHAT11 regulates cotton plant height by coordinating gibberellin, auxin, and cytokinin pathways through modulation of hormone biosynthesis/signaling gene expression and H3K9ac-mediated control of GA biosynthetic genes.

Auxin is a fundamental phytohormone that orchestrates developmental processes in plants. Flavin monooxygenase (YUCCA) serves as the rate-limiting enzyme in the indole-3-pyruvic acid pathway (IPA) pathway, and its main function is to convert IPA into IAA. In this study, 12 members of the MiYUCCA gene family were mined and identified from the genomic data of mango (Mangifera indica). Phylogenetic tree analysis classified mango YUCCA into five categories. Cis-elements analysis of the promoter indicated that the promoter region contained a large number of hormone response elements and abiotic elements. Expression analysis revealed that the majority of MiYUCCA gene family members displayed high expression abundance at the early developmental stages of fruits, and their expression decreased or even remained unexpressed with the fruit growth and development. MiYUCCA10A and MiYUCCA10B were selected for functional verification. Overexpression of MiYUCCA10A and MiYUCCA10B promoted the early flowering in transgenic Arabidopsis and tomato, enhanced the salt and drought resistance, and also increased the content of IAA in transgenic plants. In addition, MiYUCCA10A and MiYUCCA10B significantly reduced the seed number of transgenic Arabidopsis and tomato plants, exerted no effect on the pod length of Arabidopsis, but markedly decreased the fruit size of tomato. The interaction of MiYUCCA10A/B with the SHORT VEGETATIVE PHASE 1/2/3/4/5 (MiSVP1/2/3/4/5) and FLOWERING LOCUS C (MiFLC) was revealed using both yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) analyses. These results indicate that the MiYUCCA10A/B were not only involved in flowering, but also related to stress tolerance and fruit development.

Key messageAltered ABA-related regulation is associated with transgressive salinity tolerance in a synthetic tetraploid rice carrying extensive post-polyploidy homoeologous exchanges. Plant polyploids often show higher environment resilience than their congeneric diploid progenitors. However, it remains unclear whether these traits arise directly from whole-genome duplication (WGD) or evolve through post-WGD changes. Here, we show that transgressive salt tolerance occurs in ca. 1.71% of an early-generation synthetic rice tetraploid from japonica-indica subspecies hybridization. Genome resequencing unravels extensive homoeologous exchanges (HEs) but without genomic features distinguishing the tolerant vs. sensitive plants. In contrast, RNA-seq-based transcriptome-profiling enables distinct separation of the tolerant and sensitive plants. A KEGG pathway analysis indicates that the downregulated genes in tolerant plants are enriched in the abscisic acid (ABA)-degradation pathway. Expression and coding-sequence analyses identified the ABA catabolic gene OsABA8ox3 as a candidate associated with the salt-tolerant phenotype. Endogenous ABA measurements and exogenous ABA and Na2WO4 treatments in selected tetraploid recombinant inbred lines further supported an association between ABA homeostasis and salinity tolerance. Our results suggest post-WGD regulatory diversification contributes to adaptive phenotypic variation in polyploids.

Stem spines are key adaptive organs in desert plants, playing a critical role in resisting drought stress and maintaining plant survival. However, the molecular mechanisms governing their responses to drought remain insufficiently characterized. We investigated the stem spines of two representative halophytic shrubs from the Ebinur Lake Wetland desert region, Alhagi sparsifolia and Nitraria tangutorum, under a natural drought gradient: mild drought (Mi), 15.51% ± 0.61%; moderate drought (Mo), 8.87% ± 0.77%; severe drought (S), 3.34% ± 0.26%), and integrated transcriptomic and metabolomic analyses were performed to systematically reveal the molecular basis of stem spine responses to drought stress. The results showed that, compared with Mi, chlorophyll content in N. tangutorum stem spines decreased by 1.22- and 1.67-fold under Mo and S, respectively, whereas superoxide dismutase activity increased by 1.10-fold under Mo. Integrated transcriptome and metabolome analyses revealed that drought-responsive genes and metabolites under Mo and S were predominantly enriched in the isoflavonoid biosynthesis, arachidonic acid metabolism, steroid/brassinosteroid biosynthesis, and tryptophan metabolism pathways. In the tryptophan metabolism pathway, both metabolites and genes in N. tangutorum changed significantly, and tryptamine, auxin (IAA), and serotonin accumulated markedly under both Mo and S. In the isoflavonoid biosynthesis pathway, N. tangutorum exhibited increased genistein under Mo and elevated isoformononetin under S, whereas A. sparsifolia showed sustained accumulation of coumestrol and glyceollin II, with particularly strong increases in glyceollin II under both drought levels. This study revealed the molecular basis of drought responses in desert plant stem spines and offers insights into desert plant adaptation and ecosystem conservation strategies.

The OVATE family proteins (OFPs) are plant-specific regulators that modulate organ morphogenesis and grain development. To elucidate their roles in sorghum (Sorghum bicolor L.) grain development, we performed a pan-genome-informed comparative analysis of the OFP family using 17 published sorghum genomes and identified 498 SbOFP genes. These 498 genes were classified into 35 SbOFP orthologous gene families, which were grouped into 5 Core, 9 Softcore, and 21 Shell families based on presence/absence variation (PAV). Collinearity and Ka/Ks analyses showed that 2 Softcore (OFP7, OFP13) and 3 Shell (OFP25, OFP26, OFP30) members exhibited elevated pairwise Ka/Ks ratios in subsets of inter-accession comparisons, whereas the majority of SbOFPs evolved under purifying selection during sorghum evolution. Core OFP genes were consistently retained in all 17 accessions (at least one copy per genome) and exhibited higher sequence stability than Shell members. SbOFP genes were predominantly expressed during seed and inflorescence development. In addition, SbOFP promoters contain abundant hormone-responsive and grain development-related cis-acting elements. Exogenous hormone treatment further showed that SbOFP genes exhibited expression changes in response to ABA, BR, MeJA, and IAA, and that the response patterns were broadly consistent with the presence of corresponding hormone-responsive cis-elements in their promoters. Grain transcriptome analysis identified SbOFP14 and SbOFP15 as dominantly expressed members throughout grain development. Weighted gene co-expression network analysis revealed hub SbOFP genes co-expressed with NAC, MYB, MADS, and bHLH transcription factors, among which SbOFP32 and SbOFP13 showed the highest connectivity. These transcription factors are candidate partners whose expression covaries with that of SbOFP hub genes during grain development. These findings provide the theoretical basis for further elucidating the molecular mechanism of SbOFP genes in sorghum grain development and hormone signal regulation, and lay the foundation for genetic improvement of sorghum grain traits.

Gall induction by hymenopteran gall wasps is one of the most sophisticated forms of plant-insect interaction, in which insects manipulate plant development to produce specialized, nutrient-rich structures that support their growth and reproduction. Despite substantial advances, the biochemical and molecular mechanisms underlying gall formation remain incompletely understood. This review synthesizes current knowledge on gall induction, focusing insect-derived signals, phytohormonal regulation, host developmental reprogramming, nutrient manipulation, and defense modulation. Evidence from physiological, transcriptomic, proteomic, and metabolomic studies indicates that gall development involves extensive reprogramming of host cellular, developmental, metabolic, and defense-related pathways. Altered auxin and cytokinin dynamics are associated with abnormal cell proliferation, tissue differentiation, vascular reorganization, and nutrient sink establishment, while gall-inducing wasps modulate host defenses through changes in oxidative signaling, secondary metabolism, and gene expression. Host genotype influences gall outcomes, with resistant plants exhibiting stronger defenses and restricted gall development. Collectively, available evidence suggests that gall formation results from the coordinated interaction of developmental, hormonal, metabolic, and defense-related processes. However, candidate insect-derived effectors may trigger phytohormonal alterations, nutrient sink formation, and defense modulation, collectively contributing to the reprogramming of host tissues into specialized organs that support insect development. Future multi-omics and functional studies are needed to identify key drivers of gall induction and host manipulation.

Red radish (Raphanus sativus L. var. carminatus) is a characteristic economic crop in China, with flower bud differentiation efficiency directly determining its yield and quality. However, how salicylic acid (SA) affects this process and tuber quality remains unexplored. Here, we investigated different SA concentrations' effects on endogenous hormones (GA3, IAA, CTK), flower bud differentiation, flowering, and pollen traits, alongside leaf soluble sugar, starch, and protein levels. We also applied response surface methodology (RSM) to build a regression model for optimizing SA application parameters, and conducted real-time PCR to detect the expression of flowering-related genes. SA and GA₃ most strongly affected apical buds (F = 45.2, p Raphanus sativus L. var. carminatus), and highlight a feasible strategy for manipulating flowering and improving economic traits via SA application. These findings provide a theoretical basis for shortening red radish's flowering cycle and improving its economic traits.

Seed vigor is a critical determinant of crop establishment and yield, yet the epigenetic mechanisms underlying this trait remain largely unexplored. Here, we identified a histone methyltransferase, OsSET37, and characterized its essential role in regulating seed vigor in rice (Oryza sativa). Consistent with OsSET37 being a member of the SDG (SET domain group) family, our results demonstrate that the loss of OsSET37 leads to a marked decrease in H3K36me3 in vivo. Phenotypic analysis showed that osset37 mutants displayed normal vegetative and reproductive growth, but exhibited delayed germination and enhanced sensitivity to salt, drought, and ABA stresses during seed germination relative to the wild type. Y2H (Yeast two-hybrid) assays demonstrated that OsSET37 interacts with stress-responsive proteins OsSUB1C and OsDIP1. Furthermore, transcriptome analysis and qRT-PCR indicated that OsSET37 modulates the expression of key genes involved in the GA (gibberellin) and ABA (abscisic acid) signaling pathways. Collectively, our findings suggest that OsSET37 positively regulates seed vigor by modulating H3K36me3 deposition and fine-tuning the hormone signaling network, highlighting its potential as a valuable target for rice breeding programs aimed at improving stress resilience.

Coffee leaf rust, caused by the obligate biotrophic fungus Hemileia vastatrix, remains the most destructive disease of coffee worldwide. Although genomic and transcriptomic studies have identified a large number of candidate effectors, experimental evidence supporting their biological roles during infection remains limited. Here, we integrated functional assays, temporal expression profiling during coffee infection and subcellular localization analyses to investigate the biological properties of 44 H. vastatrix effector candidates (HvECs). Using the Pseudomonas fluorescens EtHAn effector delivery system in Nicotiana benthamiana, 15 HvECs consistently suppressed pattern-triggered immunity (PTI), indicating that immune suppression is a widespread property among the H. vastatrix effector repertoire, as assessed in this heterologous system. Five HvECs also attenuated AvrB-triggered effector-triggered immunity (ETI), and three suppressed both PTI and ETI, suggesting that a subset of HvECs targets conserved regulatory nodes shared by these interconnected immune pathways. Temporal expression profiling revealed sequential deployment of HvECs throughout infection, with distinct subsets predominating during pre-biotrophic development, host penetration or biotrophic colonization, consistent with stage-specific functions during fungal pathogenesis. Subcellular localization analyses further showed that HvECs preferentially accumulated in the nucleus or chloroplasts, compartments known as central hubs of plant immune regulation. This study provides the most comprehensive functional characterization of H. vastatrix effector candidates to date, establishes a biologically informed framework for prioritizing candidates for future identification of avirulence determinants recognized by SH resistance genes, and advances our understanding of how the coffee rust fungus orchestrates immune suppression across time and cellular space during pathogenesis.

Light quality is a key environmental signal that integrates plant immune pathways, yet how specific wavelengths regulate the execution, timing, and efficiency of the hypersensitive response (HR) remains incompletely resolved. This study investigated how light spectral composition modulates HR dynamics and resistance outcomes during a non-host interaction. Tobacco leaves were inoculated with Pseudomonas syringae pv. tomato DC3000 (PstDC3000) and incubated under red (RL), green (GL), blue (BL), white light (WL), or darkness. Monochromatic light treatments distinctly reshaped HR progression. BL and WL induced rapid HR characterized by early accumulation of reactive oxygen species (ROS), accelerated electrolyte leakage, and pronounced callose deposition. RL and GL delayed visible HR while effectively restricting bacterial proliferation by moderating ROS production, delaying membrane disruption, and sustaining cell wall reinforcement. In darkness, ROS accumulation was dysregulated, tissue collapse was uncontrolled, and pathogen growth was not effectively constrained. These results demonstrate that light wavelength fine-tunes HR kinetics and qualitatively distinct defense outcomes, with RL and GL favoring controlled immune responses over rapid cell death. By identifying GL as a key modulator of regulated immunity, this study provides a conceptual framework for managing light environments to optimize plant disease resistance.

Transcription activator-like effectors (TALEs) are major virulence factors of Xanthomonas oryzae, the causal agent of bacterial blight in rice. The BED-domain nucleotide-binding leucine-rich repeat receptor Xa1 confers resistance by recognizing TALEs, but the mechanisms underlying Xa1 activation remain unclear. Here we show that structurally conserved canonical TALEs differ in their ability to activate Xa1, revealing unexpected specificity in effector recognition. Using transient expression assays, endogenous epitope tagging, and prime editing of the native Xa1 locus, we demonstrate that nuclear localization is essential for Xa1-mediated immunity. We identified three functional nuclear localization signals (NLSs) within the Xa1 N-terminal region. Prime-edited rice lines carrying mutations in these motifs exhibited compromised resistance, with mutation of the first NLS causing the strongest susceptibility phenotype. Disruption of the NLS motifs in either Xa1 or TALEs did not prevent their physical interaction, indicating that effector binding can occur independently of nuclear localization. However, only nuclear-localized Xa1 triggered cytosolic Ca2+ influx, a hallmark of immune activation. Together, our results establish nuclear localization as a prerequisite for Xa1 immune function and reveal a nuclear surveillance mechanism for BED-NLR-mediated immunity in plants.

Plasmodiophora brassicae, an intracellular biotrophic protist that causes clubroot in crucifers, extensively alters the host endomembrane system for colonization. However, the molecular mechanisms and functions of effectors to achieve this remain poorly understood. Using fluorescent and transmission electron microscopy of P. brassicae-infected Arabidopsis, we show that host endomembranes and organelles become closely associated with P. brassicae secondary plasmodial structures and are subsequently phagocytosed by the pathogen. PbALE is a secreted endomembrane-targeting effector that binds a range of phospholipids. Overexpression of the effector results in plants with glossy leaves that progressively become chlorotic. Fluorescent protein-tagged PbALE is observed in large condensates that cluster with plant organelles, disrupting endocytic recycling of plant membrane receptors and immune regulatory proteins, thereby impairing penetration resistance to nonadapted barley powdery mildew infection. PbALE's association with the P. brassicae plasmodial surface and the extrahaustorial membrane during adaptive powdery mildew infection, along with the recruitment of PI(4,5)P2 to these membrane contacts, supports a model in which the effector nucleates lipid-rich compartments and enriches organelles at the host-pathogen interface. Our findings reveal a lipid-binding effector that remodels host endomembranes, disrupts immune trafficking, and aids pathogen colonization, offering insights into strategies for durable disease resistance.

ABSTRACT Zoospores are unicellular, wall-less and flagellated cells produced by a number of eukaryotic microorganisms. They allow microbial dispersion, enable the search and location for new sources of nutrients where they aggregate, initiate pathogen-host interactions and communication within microbiota. Little is known about their ability to release bioactive extracellular vesicles (EVs) supporting these adaptations. Here we used electron microscopy to establish that in the biflagellate zoospores of the heterokont and phytopathogenic species Phytophthora parasitica, EV biogenesis occurs from vesicles budding either at cell body plasma membrane or at the front flagellum, in particular from the tubular mastigonemes. Zoospore-conditioned water supernatant was fractionated and characterized by means of morphological, immunochemical, proteomic and lipidomic analyses. Three fractions enriched in cell body, flagella and EVs were obtained by differential ultracentrifugation at 3,000g, 31,000g and 100,000g, respectively, with the EV fraction consisting of small vesicles (100-150 nm). Using mass spectrometry, label-free proteomic analysis of the EV fraction (1,470 proteins) revealed a collection of proteins involved in lipid transport, vesicle membrane and cell wall organization, and tubular mastigoneme architecture, but not in virulence. Label-free quantitative lipidomic analysis (191 lipids) revealed enrichment of EVs in sphingolipids, particularly ceramides compared with the cell body. These findings provide a first hallmark of features characteristic of zoosporic EVs, with ceramides, mastigoneme proteins and the EV-related protein PPTG_13069 containing two tetraspanin MARVELous domains among molecular markers. They define the molecular and cellular principles in understanding zoosporic EV biogenesis within Stramenopiles and intercellular communication between self-aggregating zoospores, with host plant cells or microbiota. GRAPHICAL ABSTRACT

Fusarium sacchari is one of the major pathogenic fungi that cause sugarcane Pokkah Boeng disease (PBD). Effectors play pivotal roles in F. sacchari-sugarcane interaction; thus, characterizing these effectors is essential for elucidating the molecular mechanisms underlying F. sacchari pathogenicity and for developing effective strategies to control PBD. However, only a limited number of effectors have been functionally validated to date. Here, we report FsRGAE1, a candidate effector protein from F. sacchari predicted to encode a rhamnogalacturonan acetylesterase (RGAE). FsRGAE1 exhibits high expression during the early stages of infection and maintains relatively elevated expression levels throughout the F. sacchari-sugarcane interaction. Targeted deletion of the FsRGAE1 gene in F. sacchari had no discernible impact on mycelial growth, conidiation, or carbon-source utilization, yet it significantly attenuated fungal virulence. FsRGAE1 possesses both a signal peptide conferring secretory capacity and a transit peptide enabling its translocation into the host cytoplasm and nucleus. Using the Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana, FsRGAE1 was confirmed to suppress cell death induced by Bcl-2-associated X protein (BAX), as well as ROS accumulation and callose deposition, and its nuclear localization is indispensable for this immunosuppressive activity. Collectively, these findings indicate that FsRGAE1 promotes F. sacchari virulence by suppressing host immune responses in a nuclear localization-dependent manner, providing new insights into effector-mediated F. sacchari pathogenesis and potential target for resistance breeding in sugarcane.

- The ascomycete fungal pathogen Blumeria hordei (Bh) is predicted to secrete over 530 effector proteins to infect barley and cause powdery mildew disease, yet their individual and collective functions are largely unexplored. We used yeast two-hybrid next-generation interaction screening (Y2H-NGIS) to probe the host targets of 48 Bh candidate secreted effectors (CSEPs), including multiple AVR A effectors that are recognized by MLA nucleotide-binding leucine-rich repeat (NLR) receptors. - Y2H-NGIS and subsequent binary retesting identified 356 high-confidence protein-protein interactions (PPI) among Bh CSEPs, MLA NLR fragments, and barley proteins. We uncovered unique and shared host targets, including numerous proteins involved in ubiquitin-associated processes, gene expression, immune and kinase signaling, metabolism, and vesicle trafficking. - We identified a host protein (HvCAP2) that interacts with MLA and at least five Bh effectors, as well as additional effector-host PPI that correlate with loss of recognition of AVR A13 and AVR A6 by MLA13 and MLA6, respectively. - Results were integrated with an interolog-based barley interactome (HvInt) to assemble a host-pathogen network of 1248 proteins and 1701 interactions. This PPI resource can serve as a platform for functional investigations into the molecular basis of powdery mildew pathogenesis and host recognition.

Effector-triggered immunity (ETI) safeguards intestinal epithelial cells (IECs) by surveilling bacterial virulence effectors or the damage they cause, such as pore-forming toxins (PFTs) that breach plasma membrane integrity (PMI). However, how metazoan ETI regulators guard IEC homeostasis compromised by PFT in vivo remains elusive. Moreover, while the molecular architectures of ETI are well-defined in plants, functionally analogous systems in animals are only beginning to emerge. Here, we unveil a conserved ETI guard system - the intestinal ETI regulator methyl-HLH-30/TFEB upregulates a novel intrinsic cellular defense (INCED) orchestrator Y54G11A.4/TTC38 - in Caenorhabditis elegans and human IECs. This conserved ETI system coordinates the restoration of PMI, microvilli architecture, and nutrient absorption concurrently by promoting phase condensation of the PTRN-1/CAMSAP-dependent guard program through an HSPA/HSP70-HSP90 chaperone system. Together, our findings reveal an intestinal ETI guard system that couples PFT-damage sensing to comprehensive epithelial recuperation, underscoring its physiological relevance for mucosal homeostasis across metazoans.Abbreviations and acronyms B. thuringiensis (Bt): Bacillus thuringiensis; CAMSAP: calmodulin regulated spectrin associated protein; C. elegans: Caenorhabditis elegans; Cry toxins: crystal toxins; ETI: effector-triggered immunity; FRAP: fluorescence recovery after photobleaching; FRET: Förster/fluorescence resonance energy transfer; GFP: green fluorescent protein; hlh/HLH: helix loop helix; HSP: heat shock protein; IDRs: intrinsically disordered regions; IECs: intestinal epithelial cells; INCED: intrinsic cellular defense; IPTG: isopropyl-β-D-1-thiogalactopyranoside; lgg/LGG: LC3 and GABARAP family; ncMTOC: non-centrosomal microtubule-organizing center; LLPS: liquid-liquid phase separation; PFTs: pore-forming toxins; PI: propidium iodide; PMI: plasma membrane integrity; PTMs: post-translational modifications; qRT-PCR: quantitative real-time PCR; RNAi: ribonucleic acid interference; SEM: scanning electron microscopy; SLO: streptolysin O; SQST-1/SQSTM1/p62: sequestosome related 1; TFEB: transcription factor EB; TPR: tetratricopeptide repeat motifs; TTC38: tetratricopeptide repeat domain 38.

Although α-1,3-glucan represents a major polymer in cell walls of filamentous plant pathogenic fungi, its role in vegetative and pathogenic development is poorly understood. The maize anthracnose fungus Colletotrichum graminicola differentiates elaborate appressoria on the leaf surface and exhibits a hemibiotrophic lifestyle after host invasion. Promoter-eGFP-fusion analyses of all three α-1,3-glucan synthase (AGS) genes, designated as AGS1, AGS2, and AGS3, suggest that AGS3 is expressed in vegetative and pathogenic hyphae, whereas expression of AGS1 and AGS2 was not, or barely, detectable. Targeted gene deletions yielded single and double deletion mutants, as well as triple mutants, which lacked the entire repertoire of α-1,3-glucan synthase genes. Conidia of mutants lacking AGS3 developed multiple germ tubes, and vegetative hyphae had cell walls containing strongly reduced amounts of alkali-soluble glucose, showed hyperbranching, reduced growth rates on all substrata tested, and hypersensitivity to the cell wall-perturbing agents Caspofungin and Nikkomycin Z. Moreover, mutants lacking AGS3 exhibited strong virulence defects and, intriguingly, biotrophic hyphae of these mutants that differentiated in planta were severely misshapen. Increased cell wall pore diameters, increased mobility of uncharged dextran-FITC conjugates through cell walls of AGS3-deficient mutants and lack of PAMP-triggered immune responses in maize leaves inoculated with these mutants suggest that α-1,3-glucan does not play a role in masking of fungal cell walls and immune evasion, but rather controls mobility of fungal cell wall-modifying enzymes and cell wall integrity.

The green alga Chara corallina (Charophyceae) is a long-standing model for plant electrophysiology and, owing to its phylogenetic proximity to the ancestors of land plants, an informative system for probing the evolutionary depth of plant–microbe interactions. Bacterial lipopolysaccharide (LPS) is a potent microbe-associated molecular pattern (MAMP) that triggers innate immune signaling in both animals and land plants, but nothing was known about how a charophyte alga responds to this elicitor at the level of the plasma membrane. Here we measured resting membrane potential (V m ) changes in C. corallina internodal cells exposed to purified LPS from three Gram-negative bacteria: Escherichia coli, Pseudomonas aeruginosa , and Pectobacterium carotovorum subsp. carotovorum . All three LPS preparations triggered hyperpolarization of the plasma membrane, but with strikingly different kinetics: E. coli LPS produced a transient hyperpolarization that decayed within minutes, whereas LPS from P. aeruginosa and P. carotovorum produced a sustained, non-transient hyperpolarization that persisted for the duration of recording. Pretreatment with vanadate, a specific inhibitor of P-type H + -ATPases, abolished the electrogenic component of the resting potential and strongly reduced the LPS-induced hyperpolarization, suggesting that the plasma-membrane proton pump participates in this response. Strikingly, membrane-potential recordings performed in Arabidopsis thaliana cell-suspension cultures reproduced chemotype-specific kinetic dichotomy: LPS from the non-phytopathogen E. coli and P. aeruginosa , both induced a transient hyperpolarization, whereas LPS from the phytopathogen P. carotovorum subsp. carotovorum induced a non-transient, sustained hyperpolarization of A. thaliana cells. These results demonstrate that Chara possesses an electrophysiologically detectable LPS-sensitive perception system that pre-dates the divergence of land plants, that this system can discriminate between LPS chemotypes with distinct kinetic signatures, and that the same transient/non-transient signature is conserved in A. thaliana . These findings argue that the transient/non-transient dichotomy documented here may reflect an ancestral, broadly conserved capacity to decode structural variation in bacterial glycolipids at the level of the plasma membrane.

To cause rice blast disease, the filamentous fungus Magnaporthe oryzae develops a specialised infection cell called an appressorium, which generates enormous turgor to breach the rice leaf cuticle. Although key regulators of appressorium development have been identified, it is not known how they drive the extensive transcriptional reprogramming required for infection-related morphogenesis. Here, we show that the Pmk1 MAP kinase orchestrates plant infection by regulating a transcriptional network controlled by the Mst12 and Bip1 transcription factors. Bip1 is regulated both by Pmk1-dependent phosphorylation and at the transcriptional level, while Mst12 binds to a cis -acting element upstream of BIP1 essential for pathogenesis. Bip1 and Mst12 are both necessary for regulating a set of Pmk1-dependent appressorium-specific genes, including transcriptional regulators, cell wall-degrading enzymes, and effector proteins. In addition, Mst12 specifically regulates functions required for appressorium-mediated penetration, while Bip1 controls a distinct sub-set of effector genes deployed during invasion of plant tissue. When considered together, these findings define a Pmk1-dependent transcriptional network required for plant infection by the blast fungus.

Summary The extracellular space in plant tissues, known as the apoplast, remains one of the least characterized cellular compartments. The apoplastic fluid, akin to mammalian extracellular fluid, serves as the primary interface between pathogens and their host. It allows the exchange of signalling molecules, coordinates host cell responses and enables the circulation of pathogen effectors that modulate the immune response. We describe here a comprehensive multi‐omics analysis of the apoplastic washing fluids content just 6 h after the onset of Arabidopsis thaliana infection with Botrytis cinerea , a fast‐killing necrotrophic fungus. By varying plant nitrogen nutrition, known to affect both plant defenses and pathogen virulence, we identify candidates that do not stand out under optimal conditions. Our analysis uncovers novel nitrogen‐dependent mechanisms that regulate both basal and early induced apoplastic immunity, revealing the presence of previously unidentified, potentially protective apoplastic metabolites as well as RAN GTPase, a nuclear protein, thereby offering new insights into early apoplastic immune responses.

Medicinal and aromatic plants are increasingly important in modern society, as approximately 70% of global populations rely on plant‐based natural products for their primary healthcare, according to the World Health Organisation (WHO). Catharanthus roseus is a medicinal plant that produces two key terpenoid indole alkaloids (TIAs), namely vinblastine and vincristine, which are used in anticancer therapies. C. roseus has shown susceptibility towards pathogenic fungi such as Alternaria alternata, which can significantly reduce plant growth. In this study, 17 different biotic elicitors were evaluated to determine alterations in innate immunity and production of TIAs in C. roseus. Among tested elicitors, LMW_CS‐0.01% showed immunity‐boosting capacity, significantly increasing peroxidase (2.73‐fold), polyphenol oxidase (3.93‐fold), phenylalanine ammonia‐lyase (1.57‐fold), β‐1,3 glucanase (1.51‐fold), phenolic compounds (2.13‐fold) and flavonoid (1.67‐fold) accumulations on the ninth day after treatment compared to the control. Increases in vinblastine (3.15‐fold) and vincristine (2.76‐fold) were also quantified through LC–MS analysis. These biochemical responses show the activation of effector‐triggered immunity, which strengthens the plant cell against pathogenic microbes and improves resistance against leaf blight disease caused by A. alternata. Our results showed that chitosan not only induced activity of defence‐related enzymes, but also modified NO‐mediated signalling pathways. In addition, it reduced pathogen‐induced oxidative stress by lowering reactive oxygen species and minimising cellular damage during infection.

Juniperus species are a rich source of pharmacologically important secondary metabolites, including terpenoids, lignans, flavonoids, and phenolic compounds. However, the commercial utilization of these metabolites is limited by the plant's slow growth, poor natural regeneration, and environmental variability affecting metabolite accumulation. Integrating plant tissue culture with elicitation strategies is a promising approach for sustainably producing these valuable compounds. This review summarizes the diversity, biological activities, and biotechnological significance of secondary metabolites in Juniperus, focusing on tissue culture systems and enhancement of metabolite biosynthesis through elicitors. It critically evaluates the current knowledge of the effects of signaling compounds such as methyl jasmonate (MeJA), jasmonic acid (JA), and salicylic acid (SA), as well as other abiotic and biotic elicitors including silver nanoparticles (AgNPs), chitosan, and chito-oligosaccharides. Particular attention is given to their effects on podophyllotoxin, phenolic, flavonoid, and other bioactive metabolite accumulation. Elicitation responses appear to depend strongly on species, culture system, elicitor type, and treatment conditions. This review also highlights major knowledge gaps, particularly the limited understanding of the regulatory mechanisms controlling secondary-metabolite biosynthesis in Juniperus species. Overall, the available evidence suggests that integrating optimized elicitation strategies with transcriptomics, metabolomics, and metabolic engineering could improve our understanding of secondary metabolite regulation and facilitate the development of sustainable Juniperus tissue culture platforms for producing high-value natural products.

ABSTRACT Transcriptional memory enables organisms to respond more rapidly to recurrent stress, yet the underlying features of chromatin that contribute to this transcriptional recalibration remain poorly defined. Here we identify the genes displaying transcriptional memory in response to the bacterial immune elicitor, flg22, in Arabidopsis thaliana. In comparison to non-memory response genes, these memory genes show a preference for tissue-specific over uniform spatial expression patterning. The chromatin architecture of these genes in the resting state displays depletion of H3K4me3, elevation H3K27me3 and a subset are marked by H3K27me3-H3K4me3 bivalency. The H3K4me3 demethylase, JMJ14, is required for transcriptional memory, with JMJ14 occupancy enriched over memory gene loci. Upon priming, chromatin is reconfigured, with H3K4me3 levels increasing in a sustained manner at memory gene loci. To assess the function of this H3K4me3 accrual, we employ epigenome-engineering, observing that its targeted deposition at memory gene loci, including the WRKY29 locus, is sufficient to drive transcriptional memory and can endow plants with enhanced resistance to the bacterial pathogen, Pseudomonas syringae. Together, the findings demonstrate a causal role for H3K4me3 in transcriptional memory, under the regulation of JMJ14, and open the door for rational rewriting of chromatin to enhance organismal resilience.

We investigated the roles of the cognate, redundantly acting transcription factors SARD1 and CPB60g in the metabolic reprogramming of leaves of Arabidopsis thaliana suffering from bacterial infection or UV light exposure. Comprehensive metabolite analyses reveal that SARD1/CBP60g exert distinct influences on major stress-inducible metabolic pathways. The SARD1/CBP60g transcriptional node decisively boosts the biosynthesis of the systemic acquired resistance (SAR)-inducing metabolites N-hydroxypipecolic acid (NHP) and salicylic acid (SA), as well as their metabolism to major glucose conjugates. Additionally, it significantly promotes the biotic stress-induced accumulation of aromatic and branched-chain amino acids, and the generation of the antioxidant γ-tocopherol. By contrast, SARD1/CBP60g does not impact or yet exerts negative influence on the stress-induced biosynthesis of indolic defense compounds, including the phytoalexin camalexin, accumulation of Lys, Ser and Gly, and sterol desaturation. Significantly, we show that SARD1/CBP60g also acts downstream of NHP and SA in immune signaling, indicating a critical function of this transcriptional node in an NHP- and SA-driven immune amplification relay operating in SAR. RNA-sequencing analysis highlight that the transcriptional response to NHP is separable into two gene groups with different regulatory characteristics. One group consists of strongly SARD1/CBP60g-promoted genes that invariably contain NHP and SA-pathway genes and is enriched in SA-inducible genes. Genes of the other group, which overrepresents H2O2-inducible genes and concentrates genes of indolic metabolism, are not promoted by SARD1/CBP60g. Interestingly, SARD1/CBP60g oppositely affect the NHP-mediated priming of pathogen-induced SA and camalexin biosynthesis as well, illustrating that plants realize priming of distinct defenses by different mechanisms.

Plant NLR immune receptors can undergo dynamic changes in subcellular localization upon effector recognition. The Arabidopsis ADR1 helper NLRs mainly act at the plasma membrane (PM) in a phospholipid-dependent manner. Whether ADR1s display nuclear translocation and how ADR1s subcellular partitioning influences their immune function remains unknown. Here, we show that ADR1s relocate from the PM to the nucleus after TNL activation and identified TRANSPORTIN1 (TRN1) as a specific nuclear transport receptor that mediates nuclear import of ADR1s through direct binding, thus enabling the formation of TNL-induced EDS1-PAD4-ADR1s complex in the nucleus. Interestingly, TRN1 suppresses the autoactivity of ADR1s through compromising their PM localization and oligomerization. Nucleus-localized ADR1s restrict their autoactivation capacity but the nuclear pool of ADR1s is required for TNL-activated transcriptional reprogramming and disease resistance. Thus, TRN1-mediated nuclear import of ADR1s coordinates the PM and as-yet-undiscovered nuclear activities of ADR1s, thus enabling plants to mount a complete immune response.

The plant defence hormone salicylic acid (SA) triggers systemic acquired resistance by signalling through its receptor, NON-EXPRESSOR OF PATHOGENESIS-RELATED GENE 1 (NPR1), leading to widespread transcriptional reprogramming1,2. However, the molecular mechanism by which NPR1 senses SA and activates transcription remains unresolved. Here we show that SA stabilizes the SA-binding domain (SBD) of NPR1 and allosterically promotes its interaction with MED15A, a subunit of the Mediator complex. Leveraging the NPR1 proxiome and AlphaFold structural predictions, we identify a direct interaction between NPR1 and the kinase-inducible domain interacting (KIX) domain of MED15A. We show that recombinant NPR1 recruits MED15A in an SA-dependent manner, and that MED15A binding reciprocally enhances the affinity of NPR1 for SA. Cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry reveal that SA and the MED15A KIX domain cooperatively potentiate NPR1-SBD for ternary complex formation by stabilizing its SA-binding core via separate interfaces. We further demonstrate that NIMIN1, a repressor of the SA-NPR1 pathway, antagonizes SA signalling by competing with MED15A for the same NPR1-SBD docking site and allosterically blocking hormone binding. In line with previous genetic evidence establishing a critical role of MED15A in SA signalling, our findings provide a mechanistic resolution to the longstanding question of how SA promotes transcriptional activation during plant immune responses.

Arsenic (As) pollution poses serious threats to soil ecosystems and plant growth. Arbuscular mycorrhizal fungi (AMF) have been confirmed to enhance As tolerance in host plants, making mycorrhizal-assisted phytoremediation a promising and practical strategy for remediating As-contaminated soils. Populus tomentosa Carr. (Chinese white poplar) is a native fast-growing woody species suitable for phytoremediation in East Asia. However, comprehensive transcriptomic analyses focusing on the molecular mechanisms by which AMF improve As tolerance in this species remain limited. In this study, a pot-based experiment was performed on P. tomentosa seedlings using a two-factor experimental design with four treatments: non-inoculated seedlings under non-As stress (CK0), non-inoculated P. tomentosa seedlings under As stress (CK100), Rhizophagus irregularis-inoculated seedlings under non-As stress (Ri0), and R. irregularis-inoculated seedlings under As stress (Ri100). Plant-growth measurements, root morphological assessment, and Illumina RNA-seq transcriptomic analysis were applied to characterize seedling responses. Our results revealed that As stress significantly inhibited AMF colonization rate, suppressed seedling growth, and disrupted root morphological architecture. Nevertheless, R. irregularis inoculation substantially alleviated As-induced growth repression, increasing plant height, shoot and root dry biomass, as well as key root morphological parameters under As exposure. Transcriptome profiling identified large sets of differentially expressed genes (DEGs) triggered by AMF symbiosis and As stress. Functional enrichment indicated that signal transduction of jasmonate (JA) biosynthesis and metabolism represented the dominant response pathways. AMF symbiosis dynamically rewrote the transcriptional patterns of core JA biosynthesis and metabolism genes in P. tomentosa seedlings under As stress. Weighted gene co-expression network analysis further highlighted hub transcription factors, including GATA5 and WRKY57, which were tightly co-expressed with JA-synthesis-related genes and potentially bridged mycorrhizal symbiotic signals and downstream defense responses. These findings illustrated that AMF enhanced As tolerance in P. tomentosa seedlings by reprogramming JA-associated transcriptional regulatory networks. This study provided novel mechanistic insights for understanding AMF-wood plant-As interactions, and offered theoretical support for developing AMF-assisted poplar phytoremediation technology in As-contaminated soils.

The apoplast is a primary location for interactions between plants and invasive pathogens and is the site where many pathogen-secreted effectors are perceived by cell surface immune receptors to activate defence responses. However, our understanding of apoplastic interactions remains limited because protein interactions are difficult to investigate in this harsh extracellular environment. TurboID-based proximity labelling (PL) has emerged as a powerful approach for studying protein interactions in plants, though its application has to date been restricted to intracellular proteins. Here, we designed and validated a TurboID-based PL strategy for investigating protein interactions in the leaf apoplast using the well-characterised interaction between the Phytophthora infestans elicitor INF1 and the receptor-like protein (RLP) REL in Nicotiana benthamiana . Transient expression of SP-INF1-TurboID (INF1-T) induced a cell death (CD) response comparable to that triggered by native INF1, demonstrating that fusion of the TurboID tag did not impair INF1 recognition by REL. Apoplastic localisation of both INF1-T and the control construct SP-eGFP-TurboID (eGFP-T) was confirmed, validating their suitability for PL experiments. Efficient TurboID-mediated biotinylation was achieved in the apoplast using co-infiltration of biotin, ATP and magnesium acetate. Streptavidin-HRP immunoblotting revealed distinct biotinylation profiles for INF1-T and eGFP-T. Furthermore, co-immunoprecipitation demonstrated specific biotinylation of REL by INF1-T, but not by eGFP-T, in wild-type, bak1 and sobir1 / sobir1 -like N. benthamiana . These findings demonstrate that TurboID-based PL is functional in the apoplast and provides a proof-of-concept for investigating elicitor-receptor interactions in this compartment.

Pyrroloquinoline quinone (PQQ) is a redox cofactor derived from prokaryotes that participates in various biological processes involving dehydrogenase enzymes. Previous field trials identified a PQQ‐producing endophyte, Burkholderia seminalis 869T2, which enhances banana growth and reduces Fusarium wilt incidence from 24.5% to 3.4%. While more recent studies have confirmed its agricultural benefits across multiple plant species, the underlying molecular mechanisms remain unclear. Here, integrated omics and imaging mass spectrometry were employed to investigate the role of PQQ in planta. Our results indicate that PQQ achieves these outcomes by modulating key aspects of plant energy metabolism, including the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and NAD/NADP pathways. In addition, PQQ appears to influence phytohormone signalling pathways and trigger systemic plant resistance. Consistent with these molecular responses, exogenous PQQ enhanced root and shoot development and improved resistance to Fusarium infection. Collectively, these findings indicate that the endophyte functions as a biostimulant through PQQ production, coordinating plant metabolism and defence to counter pathogen invasion. This study provides mechanistic insight into plant–endophyte mutualism and highlights the potential of both PQQ and PQQ‐producing endophytes as biostimulants for sustainable agricultural applications.

Ziziphora clinopodioides Lam., a valuable medicinal plant, exhibits significant potential for flavonoid production, which significantly contributes to its therapeutic properties. In the initial phase of this ongoing study, phenolic derivatives were analyzed in 13 populations during two vegetative growth stages under greenhouse conditions. In the next phase, the effects of precursors [phenylalanine (Phe) and naringenin (Nar)], biotic elicitor [yeast extract (YE)], and abiotic elicitors [salicylic acid (SA) and methyl jasmonate (MeJA)] were evaluated on the accumulation of specific flavonoids and phenolic acids in a selected population. Younger plants (2‐MO growth stage) exhibited higher phenolic content than older ones (5‐MO growth stage). Selected populations (P1, P4, P5, and P10 for shoots; P4, P8, P11, and P13 for roots) were subjected to further LC–MS/MS analysis, revealing that P5 shoots had the highest potential for producing quercetin (QUE), rutin (RUT), and apigenin (API). Treatment with 5 mM Phe resulted in its highest level in 96 h post‐treatment, reaching 3.49‐ and 6.66‐fold higher levels than the control, respectively. Nar at concentrations of 0.4‐ and 0.8‐mM enhanced API and QUE production by 1.91‐ and 3.28‐fold, respectively. YE notably increased API and ACA (acacetin) levels, while SA resulted in the highest accumulation of QUE and RUT, highlighting its crucial role in flavonol biosynthesis. YE and Nar significantly enhanced FNSII gene expression, with Nar being the most effective. This makes Nar an effective candidate for increasing flavonoid production in Z. clinopodioides. This finding may have important implications for industries seeking to elevate flavonoid content, particularly the medicinal compound ACA, in plant‐based products.

Cassava (Manihot esculenta Crantz) is a vital food and energy crop in tropical regions, yet its yield is significantly threatened by cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xpm). Mildew resistance locus O (MLO) genes are known negative regulators of plant immunity, but their roles in cassava disease susceptibility remain unclear. In this study, we identified the MeMLO12 gene from cassava and investigated its function in response to Xpm infection. Subcellular localization revealed that MeMLO12 is specifically localized in the nucleus. Functional analysis via transient overexpression and virus-induced gene silencing (VIGS) demonstrated that MeMLO12 suppresses disease resistance: overexpression enhanced susceptibility to Xpm, while silencing conferred resistance. Furthermore, MeMLO12 modulated the expression of genes involved in jasmonic acid and salicylic acid signaling pathways, as well as reactive oxygen species production and callose deposition, which are key components of plant innate immunity. These findings indicate that MeMLO12 acts as a susceptibility factor in cassava and may serve as a potential target for breeding bacterial blight-resistant varieties.

IntroductionSeveral Trichoderma species are recognized as biocontrol agents that can enhance plant defenses against a wide range of biotic and abiotic stresses. However, few studies have investigated Trichoderma activity against bacterial pathogens, and the Xanthomonas axonopodis pv. passiflorae (Xap)-Passiflora edulis pathosystem remains largely unexplored.MethodsWe analyzed the effects of cell-free filtrates from Trichoderma cultures on the growth of Xap. We also investigated the potential of T. asperellum T25 and T. harzianum T146, when applied to the plant growth substrate, to control bacterial blight caused by Xap in P. edulis, as well as the mechanisms underlying systemic defense responses in the host plant. Fifty-day-old plants grown in substrate treated or untreated with a Trichoderma strain were inoculated or not with Xap, and changes in the expression of marker genes representative of jasmonic acid (JA) biosynthesis (LOX2), salicylic acid (SA)-dependent (PR1), and ethylene (ET)-dependent (PDF1.2) defense pathways were analyzed at 48 h post-inoculation (hpi) and 14 days post-inoculation (dpi). At 14 dpi, H₂O₂ content and the activities of three antioxidant enzymes -superoxide dismutase, peroxidase, and catalase- were also evaluated.Results and discussionTrichoderma filtrates showed antimicrobial activity against Xap. In the absence of the pathogen, strain T25 promoted the growth of P. edulis plants. Both Trichoderma strains controlled disease symptoms with similar efficacy. However, at the early stage of the P. edulis-Xap interaction, differential expression of SA- and JA-related genes was detected between T25-pretreated and T146-pretreated plants. Furthermore, at the later stage of the interaction (14 dpi), pretreatment with strain T146 increased ROS levels (measured as H₂O₂ content) in response to Xap, whereas the lowest oxidative levels were observed in plants inoculated with the pathogen alone. Our results demonstrate that T. asperellum and T. harzianum effectively control bacterial blight caused by a virulent Xap strain in P. edulis.

Abstract Bacterial effectors target diverse components of plant immunity, yet how they exploit nuclear regulatory proteins remains poorly understood. Here, we identify the Pseudomonas syringae effector HopT1-1 as a nucleo-cytoplasmic virulence factor that targets LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), a chromatin-associated regulator. Despite its canonical association with transcriptional repression, LHP1 positively contributes to immune competence: loss of LHP1 in Arabidopsis thaliana and tomato compromises PAMP-triggered immunity and increases bacterial susceptibility. HopT1-1 interacts with the C-terminal chromo-shadow domain of LHP1 and interferes with LHP1 self-association without detectably altering its abundance or nuclear localization. Transcriptome profiling further shows that Arabidopsis LHP1 supports both basal and PAMP-responsive defense-associated transcription. These findings reveal how a bacterial effector exploits an interaction-dependent property of a chromatin regulator to compromise plant immune competence.

Global anthropogenic climate change is restructuring the thermohygric parameters that govern Aspergillus flavus and Aspergillus parasiticus ecology, expanding the geographic range and contamination intensity of aflatoxin in staple food crops worldwide. Aflatoxin B1, a Group 1 IARC carcinogen and the most potent naturally occurring hepatocarcinogen, undergoes CYP3A4/CYP1A2-mediated bioactivation to the exo-8,9-epoxide, which alkylates N7-guanine residues in DNA, generating AFB1-N7-guanine adducts that produce G → T transversions culminating in the TP53 R249S gain-of-function mutation, a molecular fingerprint of aflatoxin-driven hepatocellular carcinoma. Beyond direct genotoxicity, AFB1 drives mitochondrial dysfunction through oxidative phosphorylation impairment, glutathione depletion, cardiolipin peroxidation, and mitochondrial membrane potential collapse. It activates NF-κB, STAT3, and TGF-β inflammatory signaling; and engages hepatic stellate cells in fibrogenesis culminating in cirrhosis. Critically, AFB1 also disrupts the gut microbiome, depleting butyrateproducing Ruminococcaceae, Faecalibacterium prausnitzii, and Akkermansia muciniphila while expanding pro-inflammatory Proteobacteria and Enterobacteriaceae. These dysbiotic changes compromise tight junction integrity, increase intestinal permeability, elevate portal LPS, and activate hepatic TLR4/MyD88/NF-κB and NLRP3 inflammasome cascades that amplify the carcinogenic consequences of direct AFB1 genotoxicity. This review integrates mechanistic evidence from molecular toxicology, microbiome biology, immunometabolism, and systems oncology to establish a unified framework positioning gut dysbiosis as a modifiable intermediate pathway in climate-sensitive aflatoxin-driven carcinogenesis.

Crop diseases caused by fungal and oomycete pathogens threaten sustainable agricultural production, and the efficient identification of pathogen effectors is essential for understanding virulence mechanisms and developing disease-resistant crops. However, the rapid evolutionary diversification of effectors often obscures conserved motifs, limiting the effectiveness of conventional computational approaches and making experimental prioritization challenging. To address this, we developed DeepEffector, a high-precision predictor for fungal and oomycete effector identification. DeepEffector leverages protein language models (ProtT5 for fungi and esm1b for oomycetes) to generate functional semantic embeddings, integrates G-SMOTE to mitigate class imbalance in the latent feature space, and employs a hybrid multi-head self-attention and convolutional neural network architecture to capture both global contextual dependencies and local functional patterns. Benchmarking on independent test sets showed that DeepEffector outperformed state-of-the-art tools, providing a reliable high-confidence candidate list to reduce downstream experimental screening. Its practical utility was further evaluated in Fusarium oxysporum f. sp. cubense TR4, the causal agent of destructive banana Fusarium wilt. Four prioritized candidates were experimentally confirmed to be secreted proteins, localized to host-relevant subcellular compartments, and significantly suppressed Bax-induced programmed cell death, supporting their potential roles as immune-suppressing effector candidates. DeepEffector is available as a user-friendly web server (https://deepeffector.hainanu.edu.cn/), offering an accessible pipeline to accelerate effector discovery and support crop protection research.

Hemibiotrophic fungi of the genus Colletotrichum provide valuable systems for studying plant immunity and pathogen lifestyle transitions; however, compatible and incompatible interactions have rarely been compared within the same host species. Here, we show that Arabidopsis thaliana exhibits contrasting responses to two phylogenetically distinct hemibiotrophic fungi, Colletotrichum coccodes KACC40803 and Colletotrichum scovillei KC05. Multilocus phylogenetic analyses using ITS, ACT, GAPDH, and TUB2 sequences confirmed their taxonomic identities. The two fungi also differed morphologically, particularly in colony characteristics and appressorial structures. Inoculation of Arabidopsis leaves with Colletotrichum coccodes caused progressive chlorosis, necrotic lesions, extensive fungal colonization, acervulus formation, and increasing fungal biomass, indicating a compatible susceptible interaction. Host defense-related genes associated with salicylic acid- and jasmonic acid-mediated pathways were suppressed during infection. By contrast, Colletotrichum scovillei induced restricted lesion development, rapid accumulation of callose and reactive oxygen species, localized plasma membrane disruption, and strong induction of defense-related genes in Arabidopsis leaves, consistent with an incompatible hypersensitive response (HR)-like cell death. Fungal biomass remained low throughout infection, demonstrating effective restriction of colonization. Together, these findings establish a dual Arabidopsis-Colletotrichum pathosystem that enables comparative analysis of susceptibility and HR-like resistance within a single host, providing a tractable model for dissecting host specificity, immune signaling, and regulated cell death during hemibiotrophic infection.

Escalating crop losses caused by insect pests, together with pesticide resistance, environmental persistence, and increasing regulatory scrutiny of conventional agrochemicals, have intensified interest in biologically based pest-management strategies. Endophytic bacteria and fungi represent a promising avenue for crop protection because they can influence plant defense, pest performance, and multitrophic interactions within plant tissues. This review critically synthesizes the 'landscape' of endophyte-mediated pest resistance-the 'journey' of microbes from colonization to field protection-through direct antagonism by insecticidal metabolites and enzymes, immune priming, signaling crosstalk (i.e., jasmonic acid, salicylic acid, and ethylene pathways), volatile organic compound-mediated indirect defense, and microbiome restructuring. We further evaluate the strength of evidence across laboratory, greenhouse, and field studies, distinguishing well-supported mechanisms from responses that remain context-dependent or insufficiently validated under agronomic conditions. This implies that endophyte-based pest resistance will remain difficult to translate unless colonization stability, host specificity, ecological trade-offs, formulation performance, biosafety, and regulatory requirements are addressed together. Future progress will require standardized efficacy testing, multiomics-based mechanism validation, improved delivery systems, predictive strain-selection pipelines, and transparent biosafety frameworks. By linking mechanism, evidence strength, and deployment barriers, this review provides a translational framework for moving endophyte-mediated pest resistance from experimental promise toward field-ready crop protection.

Plant intracellular immune receptors are widely deployed in breeding to protect crops from disease. In addition to nucleotide-binding leucine-rich repeat receptors (NLRs), tandem kinase proteins (TKPs) have recently emerged as an important family of immune receptors within staple cereal food crops, but how TKPs recognize effectors and whether they are amenable to engineering is essentially unknown. Here, we show that the barley and wheat TKPs Rmo2 and Rwt7 recognize different blast fungus effectors via their integrated HMA domains using different protein interfaces with nanomolar binding affinity. Structural analysis pinpointed interface residues that dictate effector recognition and enabled engineering of dual-specificity TKPs. These results establish integrated HMA domains as programmable modules within TKPs for designing new specificities in plant immunity for diseases relevant to global agriculture.

SUMMARY The ubiquitin proteasome system (UPS) and autophagy cooperatively orchestrate plant immune homeostasis, while diverse phytopathogens deploy secreted effectors to disrupt their crosstalk for successful colonization. Proteaphagy, the autophagic turnover of intact 26S proteasome holoenzymes, is a conserved stress response co‐opted by Pseudomonas syringae effector HopM1 to cripple host proteolysis. However, how the Huanglongbing (HLB) pathogen ‘ Candidatus Liberibacter asiaticus’ ( C Las) simultaneously manipulates UPS and autophagy to subvert citrus immunity remains largely uncharacterized. Here, we functionally characterize the secreted C Las effector SDE5640, which exacerbates HLB susceptibility in sweet orange ( Citrus sinensis ). SDE5640 directly binds the 19S proteasome chaperone CsPSMD10, whose homodimerization via Cys163/Cys190 disulfide bonds is required for immune suppression. CsPSMD10 overexpression enhances citrus susceptibility to C Las, whereas the dimerization‐deficient CsPSMD10‐C190S mutant confers robust HLB resistance. Mechanistically, CsPSMD10 targets CsATG8 isoforms for UPS‐dependent degradation to restrain autophagic flux; SDE5640 competitively occupies the Ank2/Ank3 domains of CsPSMD10 to disrupt CsPSMD10‐CsATG8c complexes, thereby stabilizing CsATG8c and triggering autophagy dependent on CsPSMD10. Moreover, SDE5640‐induced autophagy drives selective autophagic degradation of the 20S proteasome catalytic subunit CsPBA1, which ultimately disrupts cellular proteasome activity and attenuates salicylic acid‐mediated citrus immunity. Collectively, our findings identify a novel SDE5640‐CsPSMD10‐CsATG8c‐CsPBA1 regulatory cascade, illustrating how a single C Las effector synchronously rewires the UPS‐autophagy crosstalk to facilitate pathogen proliferation.

Potato late blight caused by Phytophthora infestans threatens global food security. Unlike the fact that most race-specific resistance to P. infestans (Rpi) genes is rapidly overcome by evolving pathogen populations, the R8 gene from Solanum demissum has conferred durable, quantitative resistance for nearly a century despite encoding a canonical coiled-coil-type nucleotide-binding, leucine-rich repeat (NLR) protein. We identified specific R8 gene analogs that act as negative modulators of R8-mediated immunity. These R8 gene analogs lack independent resistance functions but perturb the recognition of avirulence effector Avr8 by R8 and interfere with oligomerization and plasma membrane association of NRC2, a helper NLR essential for the R8 signaling pathway. Stable overexpression of such RGAs effectively compromises R8-mediated resistance in both potato and Nicotiana benthamiana. We propose that this endogenous regulation balances the intensity of R8-mediated immunity, likely reducing selection pressure on the pathogen population and prolonging resistance durability. Our findings reveal a regulatory layer where genetically linked RGAs control resistance (R) protein-mediated immunity, providing a conceptual framework for durable resistance breeding.

Antiviral defence systems frequently utilise cyclic nucleotide second messengers. A prominent example is the type III CRISPR-Cas system, which generates cyclic oligoadenylates (cOA) on detecting viral RNA. cOA molecules can bind and activate a wide range of effectors to provide antiviral defence. In both prokaryotes and higher plants, activation of a catalytic Toll/Interleukin Receptor (TIR) domain by multimerization results in degradation of NAD+, limiting cell metabolism and thus viral replication. Here, we describe a CRISPR-associated TIR-containing effector that includes a SAVED (SMODS-associated and fused to various effector domains) domain for nucleotide sensing and a cOA-degrading ring nuclease Crn4 domain. We demonstrate that the TIR-SAVED-Ring nuclease (TSR1) effector binds cA3, resulting in activation of the TIR NADase activity. The Crn4 domain, which imposes an unusual dimeric quaternary structure on the effector, degrades cA3, providing a mechanism to auto-deactivate the effector. TSR1 is thus a highly unusual example of a dimeric and self-limiting TIR effector in antiviral immunity.

The characterization of the seasonal dynamics of endophytic bacteria in beech leaves can be hindered by co‐amplification of chloroplast and mitochondrial plant DNA. This study applies established peptide nucleic acid (PNA) clamps to suppress host‐derived amplification while resolving bacterial succession across the vegetative season. Chloroplast‐ and mitochondrion‐specific PNAs inverted the proportion of host to bacterial reads, enabled the recovery of bacterial sequence variants, and increased alpha diversity accordingly. Beta‐diversity analyses showed that, once host contamination was removed, samples displayed a clear seasonal trajectory. Early‐season leaves contained high abundances of Pseudomonas together with taxa likely introduced through plant–insect–microbe interactions. As leaves matured, the microbiome shifted toward a more stable composition dominated by well‐established genera. The transition from early transient taxa to the later enrichment of phyllosphere‐adapted and nutrient‐cycling genera demonstrates that beech leaves host a temporally structured microbiome shaped by leaf development and seasonal environmental stress.

Phytic acid (IP6) serves as the primary phosphorus reservoir in plant seeds and functions as a potent antinutritional defense to inhibit herbivore growth. Despite this formidable barrier, the bean bug Riptortus pedestris preferentially feeds on IP6-rich leguminous seeds, yet the molecular mechanisms underlying this dietary adaptation have remained elusive. Here, we demonstrate that R. pedestris overcomes this nutritional defense by co-opting the ancient, highly conserved multiple inositol polyphosphate phosphatase 1 (MINPP1) family. We identified four RPMINPP1 paralogs abundantly expressed in both the salivary glands and the midgut. Using 3 1P NMR spectroscopy, we showed that these enzymes completely dephosphorylate IP6 to inorganic phosphate. Functional analyses revealed a sophisticated dual-action detoxification strategy: salivary RPMINPP1 is secreted into host tissues to degrade IP6 in planta, whereas midgut-localized RPMINPP1Ls mediate IP6 hydrolysis in vivo. RNA interference experiments confirmed that RPMINPP1Ls are essential for insect feeding and survival on soybean hosts. Remarkably, RPMINPP1 also functions as a broad-spectrum suppressor of pattern-triggered immunity (PTI) triggered by diverse elicitors, acting independently of its catalytic activity. Collectively, our findings uncover a dual-pronged strategy in which an herbivore repurposes MINPP1 family effectors to simultaneously neutralize phytic acid-based antinutritional defense and suppress host plant immunity.

Elevated atmospheric carbon dioxide (ECO2) is a key climatic factor influencing the resilience of plant-microbial symbiotic systems against heavy metal contamination. Robinia pseudoacacia-rhizobia symbiosis shows great potential for cadmium (Cd) remediation. However, the mechanism by which ECO2 regulates Cd phytostabilization in symbiosis remains unclear. This study conducted a 90-day experiment in growth chambers to investigate the effects of ECO2 on the growth, Cd accumulation and chemical forms, as well as nutrient uptake and antioxidant system in Robinia pseudoacacia-rhizobia symbiosis. Results indicated that ECO2 significantly increased plant biomass and photosynthetic efficiency while significantly raising Cd content in roots (34.5%, p p 2 markedly increased Cd accumulation in roots (81.2%, p 2 promoted the content of nutrients and stimulated the antioxidant system. The random forest model indicated that root weight, Cd and Mn contents are the core factors for ECO2-driven Cd phytostabilization. This study demonstrates that ECO2 enhanced Cd phytostabilization by optimizing the resistance of symbiosis to Cd, offering a novel perspective for predicting plant-microbe joint restoration of heavy metal pollution under global climate change scenarios.

Pistachio (Pistacia vera L.) is among the most economically important nut crops worldwide. They are increasingly exposed to the environmental constraints associated with climate change, including drought, salinity, nutritional imbalances, and heightened disease pressure. These stressors compromise plant growth, physiological performance, nutrient acquisition, and orchard productivity, highlighting the need for sustainable strategies to enhance crop resilience. This review critically examines the current knowledge on the functional roles of arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) in pistachio production. Evidence indicates that AMF and PGPR contribute to plant performance through multiple complementary mechanisms, including improved nutrient mobilization and uptake, maintenance of ionic homeostasis, enhancement of water-use efficiency, stimulation of antioxidant defenses, modulation of stress-related signaling pathways, and suppression of phytopathogens. AMF primarily enhance phosphorus acquisition, water relations, and soil structural stability, whereas PGPR contribute to nutrient solubilization, biological control, and induction of plant defense responses. Despite promising experimental results, most studies have been conducted under controlled conditions, limiting the translation of microbial inoculation strategies to commercial orchards in the field. We identified the key knowledge gaps and research priorities required to improve the consistency, scalability, and field validation of microbiome-based approaches for sustainable pistachio production under increasingly challenging environmental conditions.

As sessile organisms, plants continuously perceive and adapt to dynamic light environments and complex microbial communities. Light serves not only as the energy source for photosynthesis, but also a key signal that regulates plant growth, development, and plant-microbe interactions. Through photoreceptor-mediated perception and downstream signaling networks, light modulates plant immunity and orchestrates beneficial microbial associations. This review summarizes how light signaling regulates plant-microbe interactions through three interconnected layers. First, photoreceptors and downstream factors directly modulate immune and hormonal defense pathways. Second, light regulates physical barriers, including cuticular wax deposition, cell wall lignification, and stomatal immunity. Third, light-driven reprogramming of carbon allocation and secondary metabolism reshapes rhizosphere chemistry and microbiome assembly. These findings highlight light signaling as a central coordinator of plant immunity, physical defense, and microbiome assembly, providing a basis for precisely manipulating plant-microbe interactions through light management to advance sustainable agriculture.

As sessile organisms, plants rely on intricate molecular regulatory networks to navigate complex, fluctuating environments and diverse stress conditions. Nitrogen Limitation Adaptation (NLA), a RING-type ubiquitin ligase harboring an SPX domain, has emerged as a central hub integrating plant responses to nutrient availability, pathogen attack, and cold stress. This review provides a systematic overview of the structural features of NLA, with a particular focus on the regulatory role of inositol pyrophosphates (PP-InsPs) in plant phosphorus signaling. We highlight the core functions of NLA in maintaining nitrogen-phosphorus homeostasis and mediating the crosstalk between nutrient status and immune defense. A key mechanism we discuss is the role of maize ZmNLA in "cold-phosphorus" trade-off, whereby ZmNLA activates jasmonate signaling to enhance cold tolerance by mediating the ubiquitination and degradation of the signaling repressor ZmJAZ11, while simultaneously restricting phosphate uptake via targeting the phosphate transporter ZmPT4. Notably, the ZmNLAΔ12 variant, which has an impaired PP-InsPs binding site, functionally uncouples this trade-off through structure-guided protein design. Finally, we propose future research directions and discuss the potential for breeding new crop varieties with enhanced stress adaptation and improved nutrient use efficiency.

Arbuscular mycorrhizal (AM) symbiosis is a widespread mutualism between plant roots and Glomeromycotina fungi that enables nutrient exchange through arbuscules. Although many transcriptional regulators of AM symbiosis have been identified, the role of the INDETERMINATE DOMAIN (IDD) proteins remain unknown. We show that rice IDD7 expression is strongly induced in arbuscule-containing cells, and its loss markedly reduces fungal colonization and disrupts arbuscule development. IDD7 is required for induction of genes associated with fatty acid biosynthesis, nutrient transport, and symbiotic signaling during AM symbiosis. IDD7 interacts with the transcription factors SLENDER RICE 1 (SLR1) and PHOSPHATE STARVATION RESPONSE 2 (PHR2) through its conserved TQDFLG domain. Electrophoretic mobility shift assays show that IDD7 binds multiple promoter motifs. Together with SLR1 and PHR2, IDD7 synergistically activates the promoters of PHOSPHATE TRANSPORTER 11 (PT11) and the AM-associated transcription factors PHR2, REQUIRED FOR ARBUSCULAR MYCORRHIZATION 1 (RAM1), WRINKLED 5a (WRI5a), and CYCLOPS. Moreover, IDD7 is required for PHR2 expression in arbuscule-containing cells. Here, we show that IDD7 is a central transcriptional regulator in arbuscule-containing cells.

Plant colonizing mutualistic symbionts confer beneficial effects to their hosts, which often includes increased growth, and biotic and abiotic stress resilience. How these benefits are activated on a molecular level is mostly unknown. Here, we describe effector candidates of the fungal symbiont Serendipita indica (Si), which modulate plant stress signalling pathways. By analyzing the Si effector interactome, we reveal frequent targeting of stress related host proteins, which are linked to the identified effector signalling functions. Moreover, functional data indicate that Si effectors modulate abiotic stress response of Arabidopsis, as well as resistance to pathogen infection. Analysis of symbiont effectors might not only uncover previously unreported molecular mechanisms that increase plant fitness but might also be used to identify potential genetic traits for crop improvement under changing climates.

Plants continuously encounter a wide range of biotic and abiotic stresses that adversely affect their growth, development, and productivity. Because they are sessile, plants cannot escape these unfavorable conditions and therefore rely on a diverse array of morpho-physiological, biochemical, and molecular adaptations to survive. Among these adaptive strategies, symbiotic associations with beneficial microorganisms have emerged as a crucial mechanism for enhancing stress tolerance. These plant-microbe interactions are mediated by intricate chemical signaling networks that regulate nutrient exchange, defense responses, and stress adaptation. Despite their immense potential for sustainable agriculture, the large-scale application of beneficial microbes remains limited owing to poor microbial establishment under field conditions and an incomplete understanding of the complex mechanisms governing plant-microbe mutualism. Deciphering these interactions is particularly challenging because they are highly dynamic and involve continuous communication between plants and diverse microbial communities. Recent advances in omics technologies, synthetic biology, and nanotechnology provide unprecedented opportunities to unravel these complex relationships at the molecular and systems levels. This review summarizes plant adaptive strategies under biotic and abiotic stresses, examines the role of microbial symbiosis in stress alleviation, and highlights emerging approaches, including multi-omics integration, synthetic microbial consortia, engineered quorum-sensing circuits, holobiont-level analyses, and nanoparticle-mediated modulation of the rhizosphere microbiome, for understanding and engineering beneficial plant-microbe interactions. Collectively, these advances offer new insights into symbiotic crosstalk and provide a foundation for developing resilient and sustainable agricultural systems.

The ubiquitin-proteasome system (UPS) constitutes a highly conserved regulatory hub governing protein turnover and signal transduction in eukaryotes, which precisely determines the fate of substrate proteins via dynamic and reversible ubiquitination. During long-term coevolution between plants and viruses, the UPS has evolved into a critical battlefield for host-virus arms races. Plants exploit the substrate recognition specificity and proteolytic activity of the UPS to selectively eliminate essential viral proteins required for infection, thereby establishing multilayered antiviral immune barriers. In contrast, viruses have evolved diverse effector proteins to antagonize or hijack this pathway to facilitate their replication and spread. Competitive exploitation of this shared regulatory machinery underlies the fundamental logic of bidirectional regulation in plant-virus interactions. This review systematically summarizes the molecular basis of UPS-mediated plant antiviral immunity, as well as convergent pathogenic strategies adopted by diverse viruses to perturb ubiquitin signaling, suppress host immune responses, and reprogram the intracellular environment. The work aims to provide theoretical insights for deciphering viral pathogenesis and breeding crops with durable virus resistance.

BackgroundSeed endophytes are the earliest microbial companions of plants. They play a beneficial role in seed germination, seedling development, and plant growth. Endophytes help plants to adapt to changing environments; however, their role in the aspect of toxic metal-polluted environments is poorly known. We investigated the impact of metal toxicity on the structure of seed-associated microbiota in Arabidopsis arenosa and explored the role of these microbes in host plant adaptation to metal-rich, post-mining environments.ResultsToxic metals markedly altered the alpha and beta diversity of bacterial seed endophytes, whereas fungal endophyte diversity was largely unaffected. Similar patterns were observed in a vertical transmission experiment, indicating that metal exposure can influence seed-associated microbiota across generations. To explore the functional relevance of these changes, we assembled synthetic microbial communities from culturable bacterial and fungal isolates recovered from seeds of plants originating from non-metalliferous and metalliferous sites. A synthetic community composed of isolates from non-metalliferous populations enhanced plant growth under metal stress, whereas the community assembled from metalliferous populations showed no growth-promoting effect. Crude seed extracts also did not reproduce the growth-promoting effect of the defined synthetic community. Further fractionation indicated that the beneficial effect was mainly associated with the fungal component, while bacterial fractions did not enhance plant growth under the tested metal-stress conditions. Fungal isolates from non-metalliferous populations promoted plant biomass under metal stress, whereas this effect was not observed for isolates from metalliferous populations under the tested conditions.ConclusionsLong-term exposure to toxic metals exerts strong selective pressure on seed-associated microbiota, markedly reducing bacterial diversity and reshaping community composition while leaving fungal diversity largely unaffected. Metal toxicity causes losses in the abundance of bacteria. High metal tolerance of these microorganisms indicates rather that metal exposure affects the interaction between the microbe and its host and the ability of the microbe to be retained in the seeds. Fungal endophytes from metal-free populations proved more effective in promoting plant growth under metal stress than those from metalliferous populations, suggesting that intrinsic strain-level traits, rather than prior environmental adaptation, are key to enhancing host tolerance. This challenges the assumption that endophytes from contaminated sites are inherently better suited for phytoremediation and highlights the potential of "naive" microbial sources. Together, these results expand our understanding of vertical transmission, microbial adaptation, and host-microbe interactions under heavy metal stress, and underscore the value of broadening the search for effective endophytes beyond contaminated habitats.

In a recent issue of Science, Gao et al.1 discover a link between pattern-triggered immunity (PTI) initiated by plant cell surface receptors and effector-triggered immunity (ETI) initiated upon detection of pathogen effectors by intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors. PTI activates alternative splicing that results in mRNAs for the potato NLR Rpi-vnt1.1 that lack a repressive N-terminal prodomain.

Arbuscular mycorrhizal fungi (AMF) colonization influences plant-insect interactions, but the mechanisms underlying JA-mediated AMF-induced resistance remain unclear. Using rose as the experimental material, Rhizophagus intraradices inoculation combined with the jasmonic acid (JA) biosynthesis inhibitor SHAM was applied, integrating physiological measurements, hormone quantification, metabolomics, and insect bioassays to elucidate the role of JA-mediated secondary metabolites in AMF-induced resistance. The results showed that AMF colonization significantly increased JA levels and promoted the accumulation of flavonoid (37.21%) and total phenolic (15.38%), whereas SHAM treatment attenuated these responses. Feeding assays showed that larvae of Lymantria dispar exhibited reduced growth on mycorrhizal plants, while SHAM weakened this suppressive effect, suggesting that JA signaling may be involved in AMF-induced resistance. Metabolomic analysis revealed significant enrichment of flavonoid biosynthesis across treatments. Flavonoids, including naringenin, (-)-epicatechin, and kaempferide, were upregulated by AMF but reduced by SHAM. Correlation analysis showed that the contents of these metabolites were significantly positively correlated with JA levels, and negatively correlated with the growth of L. dispar larvae. Partial least squares structural equation modeling (PLS-SEM) provided statistical support for the hypothesized regulatory pathway, suggesting that AMF-induced resistance of rose against L. dispar may be associated with enhanced flavonoid accumulation in leaves, with JA potentially playing a regulatory role in this process (Goodness-of-fit = 0.79). This study provides a theoretical basis for sustainable pest management through AMF-plant symbiosis.

Abstract Spodoptera frugiperda severely damages maize and causes massive agricultural economic losses. Combining endophytic entomopathogenic fungi with natural enemies is a sustainable pest management approach. However, how these fungi modify plant physiology to affect S. frugiperda and regulate parasitoid performance remains unclear. Two endophytic entomopathogenic fungal strains (GL-5 and H8) were used. We detected fungal colonization effects on maize growth, leaf antioxidant enzyme activity, phyllosphere microbiota, and metabolome. We further investigated fungal-mediated changes in the development, reproduction, and detoxification metabolism of S. frugiperda and analyzed the tritrophic interaction with the parasitoid Microplitis manilae . Both strains significantly promoted maize seedling growth and disrupted S. frugiperda pupal development, prolonging development and suppressing pest population growth. GL-5 increased the parasitism rate and cocoon weight of M. manilae . Fungal colonization inhibited maize superoxide dismutase (SOD) and peroxidase (POD) activities. GL-5 activated larval glutathione S-transferase (GST), carboxylesterase (CarE), and cytochrome P450 (CYP450) detoxification pathways, while H8 enhanced acetylcholinesterase (AChE) activity and suppressed core detoxification systems. GL-5 enriched Enterobacter and induced flavonoid and phenolic acid accumulation; H8 recruited Chryseobacterium and regulated carbohydrate metabolism and alkaloid biosynthesis. Endophytic entomopathogenic fungi remodel maize phyllosphere microecology and metabolome, impairing pest detoxification and development and synergizing with natural enemies to suppress S. frugiperda . This study provides mechanistic support for eco-friendly fall armyworm management.

Soybean is a major source of plant protein and oil, yet its production is severely constrained by diverse pathogen infections. Here, we identify eugenol as a pathogen-induced defense metabolite in soybean with broad-spectrum antimicrobial activity against fungal, oomycete, and bacterial pathogens. Pathogen-associated molecular patterns (PAMPs), including Flg22, Chitin, and Elicitin, strongly induced soybean eugenol synthase (GmEGS) genes and promoted eugenol accumulation. Phylogenetic and expression analyses identified five GmEGS genes, among which GmEGS1a, GmEGS1c, and GmEGS2a were strongly induced by PAMPs and pathogen infection. Eugenol inhibited mycelial growth, spore germination, and zoospore release, while transcriptome analyses of Phytophthora sojae and Fusarium graminearum showed disruption of central carbon, amino acid, and membrane-associated metabolic pathways. In soybean, exogenous eugenol induced antioxidant metabolism and protein homeostasis without activating canonical immune responses, consistent with a protective effect associated with its antimicrobial activity rather than canonical immune activation. Overexpression of GmEGS1a or GmEGS1c increased endogenous eugenol accumulation and conferred broad-spectrum disease resistance without detectable growth penalties. Moreover, exogenous eugenol reduced disease severity in soybean, rice, maize, and tomato. Together, our findings establish GmEGS-mediated eugenol biosynthesis as an inducible chemical defense pathway and highlight eugenol as a promising natural antimicrobial compound for sustainable crop protection.

Arbuscular mycorrhizal (AM) symbiosis is often presented as a linear sequence of fungal recognition, root colonization, arbuscule formation, and nutrient exchange. This view underrepresents the repeated regulatory transitions through which plants influence symbiotic establishment, function, and persistence. Here, we propose a checkpoint framework organized around invitation, admission, accommodation, investment, maintenance, and termination and renewal. We examine how nutrient status, carbon availability, hormonal and immune signaling, systemic root-shoot communication, and environmental context regulate fungal recruitment, intracellular entry, interface construction, resource exchange, and arbuscule turnover. We distinguish strong mechanistic evidence from correlative observations and emphasize that gene expression, colonization abundance, or arbuscule degeneration alone do not demonstrate resource flux, performance-sensitive evaluation, partner-level sanction, or selective interface termination. Evidence is strongest for plant control of presymbiotic signaling, cellular accommodation, interface construction and resourcing, nutrient acquisition, and regulated arbuscule turnover. By contrast, direct performance-sensitive evaluation of individual fungal interfaces remains insufficiently demonstrated. AM fungi also providing regulatory inputs: fungal signals, secreted molecules, and small RNAs can modify host processes, while fungal physiology and extraradical networks influence nutrient acquisition and allocation. Plant control is therefore substantial but not absolute, operating through asymmetrical, reciprocal regulation across interface, whole-plant, and fungal-network scales. We conclude by outlining experiments that link fungal nutrient contribution, plant response or allocation, and subsequent fate at the same interface, with fungal fitness additionally required for claims of partner-level sanction. Such experiments provide tests for distinguishing developmental and physiological regulation from performance-sensitive interface maintenance.

Fusarium pseudograminearum, the dominant causal agent of Fusarium crown rot (FCR), secretes numerous effectors to modulate host immunity during infection, but the functions of most remain unknown. In this study, we identified FpSP5, an effector from F. pseudograminearum, as a pathogen-associated molecular pattern (PAMP) that triggers plant immunity. FpSP5 induces cell death, oxidative burst, and PTI in Nicotiana benthamiana, conferring resistance against multiple pathogens. Furthermore, its perception specifically depends on the coreceptor BAK1 in N. benthamiana. Interestingly, FpSP5 is not only conserved among fungi and capable of activating defense responses in different plants but also essential for the full virulence of F. pseudograminearum on wheat. Collectively, these findings enhance our understanding of how F. pseudograminearum effectors modulate host immunity and contribute to pathogenicity, laying a foundation for developing targeted strategies to control FCR.

Abstract Rice bacterial blight, caused by Xanthomonas oryzae pv. oryzae ( Xoo ), to investigate the genomic diversity and evolutionary characteristics of Xoo , whole-genome sequencing was performed on ten representative strains collected from different geographic regions, followed by comparative genomic analyses. Average nucleotide identity (ANI) analysis confirmed that all ten isolates belonged to Xoo and supported their phylogenetic classification. Phylogenomic analysis classified the strains into three distinct lineages, which were highly consistent with gene family composition, CRISPR distribution, and TAL effector (TALE) repertoires. Comparative genomic analyses revealed that the core genome, virulence genes, and antimicrobial resistance genes were highly conserved among the strains, whereas plasmid distribution, gene family composition, and TALE repertoires exhibited substantial variation. Thirty-three nonredundant core TALEs were identified, displaying extensive diversity in repeat-variable diresidue (RVD) composition, repeat number, and chromosomal organization. TALE copy numbers ranged from 12 to 18 among the genomes and showed lineage-specific distribution patterns that closely reflected phylogenetic relationships. These findings indicate that the diversification of TALE repertoires, rather than variation in conserved pathogenic systems, is a major contributor to genomic differentiation and host adaptation in Xoo populations. This study provides valuable genomic resources for understanding the evolution of Xoo pathogenicity and offers a foundation for the discovery of novel resistance genes and the development of durable strategies for bacterial blight resistance breeding

Pathogens target and degrade the extracellular matrix surrounding plant cells. A central question is how cell wall-derived damage-associated molecular patterns (DAMPs) are recognized and integrated to trigger immune responses. We address this question by determining the structure of the extracellular multidomain of the IGP1 receptor in both apo form and bound to the cellulose-derived DAMP cellotriose. Structural analyses reveal that constitutive interactions of Leucine Rich Repeat (LRR)-malectin domains preconfigure IGP1 for ligand recognition and that the receptor features a highly specific sugar-binding pocket in the LRR domain capable of distinguishing fine variations in glycan structures. By directly sensing cello-oligomers, IGP1 operates as a cell wall surveillance receptor that links pathogen-induced wall degradation to immune alerting, equipping plants to mount rapid and robust defense responses.

The symbiosome, a temporary plant organelle enabling nitrogen fixation in legume-rhizobia symbiosis, consists of a plant-derived symbiosome membrane (SM), symbiosome space (SS), and enclosed bacteroid. Here, we isolate and purify symbiosomes from Medicago truncatula-Sinorhizobium meliloti root nodules and perform label-free quantitative mass spectrometry to profile protein abundances in the symbiosomes. We identify 1,018 M. truncatula proteins, including 829 in the SM and 457 in the SS. Combined with transport assays, our data reveal multiple dicarboxylate transporters in the SM that potentially deliver carbon sources to bacteroids. The SM is enriched in membrane trafficking proteins, lipid raft-associated components, and receptor-like proteins, together with numerous cell wall-associated proteins, highlighting the extracellular properties of the symbiosome. Proteomic and metabolomic analyses reveal the SS as a metabolically active compartment enriched in both plant and rhizobial proteins involved in carbon and amino acid metabolism. These findings offer insights into the molecular basis of symbiotic nitrogen fixation.

The coordination of intracellular carbon and nitrogen levels is essential for optimal bacterial growth and, in rhizobia, for survival in the soil, root colonization, and symbiotic interactions with their host plants. Here, we show that the phosphotransferase system PTSNtr, a global regulator of carbon and nitrogen metabolism, modulates the tricarboxylic acid (TCA) cycle activity and the accumulation of three major carbon storage polymers: glycogen, polyhydroxybutyrate, and exopolysaccharide in Rhizobium leguminosarum. The unphosphorylated form of the effector protein ManX is sufficient for the full activation of the TCA cycle dehydrogenase enzymes. Accordingly, loss of manX reduced dehydrogenase activity and redirected overflow carbon into storage polymers, phenocopying wild-type cells grown under nitrogen starvation. We further demonstrate that carbon metabolism in R. leguminosarum is also tightly regulated by the HPr kinase (HprK) protein. In an hprK mutant, loss of NPr Ser48 phosphorylation favors phosphotransfer through the histidyl arm and is predicted to increase phosphorylation of downstream PTSNtr components. When grown on glucose, the hprK mutant accumulates elevated intracellular pyrimidine levels and exhibits increased malate dehydrogenase and TCA cycle activity, consistent with a misregulated shift toward gluconeogenesis. This mutant also overproduces exopolysaccharide, an effect potentially mediated by crosstalk between phosphorylated PtsN and the ChvI/ChvG two-component regulatory system. Together, these data suggest that HprK exerts pleiotropic control over processes regulated by PTSNtr, influencing central metabolism while repressing exopolysaccharide production, likely by promoting the unphosphorylated state of PtsN.IMPORTANCEBacteria must continuously balance growth, nutrient availability, and storage to ensure fitness and survival in fluctuating environments. However, how regulatory networks integrate central metabolism with carbon storage remains poorly understood. This study reinforces the role of the PTSNtr system as a central coordinator of carbon routing and polymer accumulation. By revealing how ManX and HprK-dependent phosphorylation influence central metabolism and the production of major carbon storage polymers in Rhizobium leguminosarum, we show that disruption of PTSNtr signaling reprograms carbon allocation, uncoupling growth from carbon storage and mimicking nitrogen starvation even under nitrogen-replete conditions. These findings highlight a regulatory link between carbon-nitrogen signaling and bacterial storage strategies, with implications for cellular survival and plant symbiosis. More broadly, our work illustrates how global metabolic regulators shape bacterial physiological states, providing new insights into mechanisms that support microbial adaptation in complex environments.

Photosynthesis impairment and the accompanying leaf yellowing represent a prominent and physiologically critical symptom of phytoplasma infection. However, the underlying molecular mechanisms remain poorly understood. In this study, we identified the Jujube Witches' Broom pathogenic effector PHYL1JWB, which was previously reported to induce phyllody in jujube, as a key inducer of photosynthesis impairment. Overexpression of PHYL1JWB in sour jujube and Arabidopsis thaliana disrupted photosynthesis by causing severe disorganization of chloroplast ultrastructure. PHYL1JWB directly interacts with the integral thylakoid membrane protein ZjCURT1A and promotes its 26S proteasome-dependent degradation independently of lysine ubiquitination, leading to disruption of thylakoid structure and photosynthetic dysfunction. The conserved two α-helices of PHYL1JWB and the C-terminal segment of ZjCURT1A containing the thylakoid membrane curvature-related CAAD motif are essential for their interaction. Together, these findings reveal that PHYL1JWB impairs photosynthesis by disrupting chloroplast ultrastructural in addition to inducing phyllody, thereby contributing to abnormal plant growth. This work highlights the functional pleiotropy of pathogenic effectors and underscores the need to consider their impacts on multiple physiological processes in the context of phytoplasma pathogenicity.

Under nitrogen limiting conditions, legume plants interact with nitrogen fixing bacteria known as rhizobia, resulting in the formation of a new organ, the nodule. This process is accompanied by dramatic changes in gene expression, which operate at different levels. A previous study revealed that histone methylation is differentially modulated during nodulation. However, the histone methyl transferases and demethylases involved in this modulation have not been characterized. In this study we report the identification of the Medicago truncatula putative histone lysine demethylase MtPKDM9B , which is subject to alternative splicing (AS), and the differential modulation of AS variants at translational level during nodule symbiosis. Knockdown of MtPKDM9B impaired infection by rhizobia, nodule development, bacterial viability and the expression of the leghemoglobin coding gene MtLHB1 . MtPKDM9B is the putative ortholog of Arabidopsis EARLY FLOWERING 6 ( ELF6/AtPKDM9B) gene involved in the removal of the repressive mark H2K27me3. A combination of ChIP-seq and RNA-seq experiments revealed that MtPKDM9B is required for demethylation of H3K27me3 in regions nearby or contained within gene bodies of symbiotic genes and the upregulation of the cognate mRNAs in response to rhizobia, including those encoding the putative ubiquitin ligase MtPUB2, the MYB transcription factor MtMYB040 and the auxin conjugating enzyme MtGH3 (Gretchen Hagen 3). Our findings illustrate how AS and translational regulation of this plant specific histone lysine demethylase contributes to the removal of the repressive mark H3K27me3, promoting transcriptional activation of symbiotic genes required for the formation of functional nitrogen fixing nodules.

Summary Canonical ribonucleases such as Argonaute (AGO) participate in RNA-based gene regulatory processes. They are important for small RNA (sRNA) association and the formation of RNA-induced RNA silencing complexes. Potato ( Solanum tuberosum ) has 14 AGO -encoding genes, including the Solanum -specific StAGO15 , which is activated upon infection by filamentous pathogens. The resistant phenotype conferred by ectopic expression of StAGO1 5 was abolished in plants in which a 21-nt sequence in the PAZ-encoding region of StAGO15 was targeted using an artificial miRNA. We then analyzed the sRNAs associated with StAGO15 in Phytophthora infestans leaf infection. Small RNAs were coimmunoprecipitated, subjected to deep sequencing, and analyzed against the P. infestans and potato genomes, in which cleavage sites were predicted by applying the R package, smartPARE. The most notable difference between pathogen-and water-inoculated StAGO15 samples was observed in the transposon (TE) category. Here, two categories of nonautonomous TE dominated: the miniature inverted-repeat transposable elements ( MITEs ) and short interspersed nuclear elements. The StAGO15 -associated sRNAs bind and target complementary endogenous potato RNAs. One example is siRNA122, which acts on a cleavage site in the resistance gene PGSC0003DMG400015681 ( StRES1 ). siRNA122 is derived from a MITE ; upon deletion of this source sequence, resistance is restored. Our findings provide new insights into the plant immune surveillance network and demonstrate that deleting a 21 nt RNA sequence from a MITE transposon restored a defense response against two important filamentous pathogens. Significance Statement Potato is a major food crop in the world with increasing importance in the developing countries due to its high adaptive capability to variable climate conditions. The potato plant is known to be attacked by many pathogens and insects during its growing season. Here we demonstrate the importance of a small RNA affecting the defense level to the late and early blight diseases which provides new possibilities for crop improvement.

Mesorhizobium ciceri, a nitrogen-fixing symbiont of chickpea (Cicer arietinum), remains genetically challenging to manipulate using conventional homologous recombination approaches, which are labor-intensive and often leave undesirable selection markers. In this protocol, we describe a streamlined genome-editing strategy using a broad-host-range Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) system adapted for M. ciceri. We demonstrate the efficacy of this method by targeting nodC, which encodes the N-acetylglucosaminyltransferase required for chitin backbone synthesis in Nod factors, the primary signaling molecules involved in symbiotic molecular communication. This protocol details the systematic design of single-guide RNAs (sgRNAs) and the construction of a homology-directed repair (HDR) template. The HDR template was designed to facilitate site-specific integration of a green fluorescent protein (GFP) reporter flanking the nodC cleavage site. Following delivery of the Cas9/sgRNA/HDR construct through biparental mating, putative mutants were identified using a fluorescence-based screening approach. Successful disruption of the 1.3 kb nodC locus within the nodulation (nod) cassette was initially screened by visualization of GFP expression in mutant colonies using fluorescence microscopy. The disruption was further validated by restriction digestion, amplification of the integrated cassette from genomic DNA, and Sanger sequencing. GFP expression was additionally quantified by reverse transcription quantitative polymerase chain reaction. To validate the functional impact of the mutation, chickpea infection assays were performed, demonstrating impaired nodulation in plants inoculated with ΔnodC M. ciceri compared with the wild-type strain. Overall, this protocol provides an efficient and reproducible framework for precise gene disruption and functional genomics studies in Mesorhizobium.

Coffee (Coffea arabica L. and Coffea canephora Pierre ex A. Froehner) is a globally important perennial plantation crop that sustains the livelihoods of millions of smallholder farmers while making substantial contributions to agricultural economies worldwide global agricultural economies. Nevertheless, sustainable coffee production is increasingly constrained by climate change, declining soil fertility, emerging pests and diseases and the environmental costs associated with intensive use of synthetic agrochemicals. Recent advances in plant microbiome research have transformed the understanding of coffee from an individual organism to a holobiont, where the host plant and its associated microorganisms operate as an integrated biological system. The coffee phytobiome encompasses diverse microbial communities inhabiting the rhizosphere, rhizoplane, endosphere, phyllosphere, anthosphere, carposphere and spermosphere, together with complex multitrophic interactions involving shade trees, soil fauna, insects and the surrounding environment. These interactions collectively regulate nutrient acquisition, carbon assimilation, water-use efficiency, hormonal balance, stress tolerance, immune responses and overall plant productivity. This review critically examines current advances in multitrophic plant-microbe interactions that influence physiological adaptation in coffee, with particular emphasis on sustainable production under Indian agroecological conditions. It highlights the functional functions of plant growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF), endophytic microorganisms and other beneficial microbes in enhancing root development, nutrient cycling, and resilience to biotic and abiotic stresses. Furthermore, the review evaluates recent progress in metagenomics, metatranscriptomics, metabolomics and microbiome engineering for harnessing indigenous microbial resources. Finally, it outlines future research priorities integrating plant physiology, microbial ecology, systems biology, and precision agriculture to develop climate-resilient, resource-efficient, and environmentally sustainable coffee production systems.

Endophytic beneficial microorganisms are widely used in agriculture for promoting plant growth and enhancing plant defense mechanisms. This study aimed to characterize endophytic fungi isolated from the roots of coffee plants cultivated in organic agroforestry systems and evaluate their potential as biocontrol agents against fungal pathogens, as well as their ability to promote plant growth. Biocontrol activity was assessed using in vitro dual-culture assays on potato dextrose agar, measuring the inhibition of pathogen growth. Plant growth promotion was evaluated by co-cultivating Arabidopsis thaliana seedlings with fungal isolates on Murashige and Skoog medium. Isolates were further subjected to both qualitative and quantitative biochemical characterization. A total of 18 endophytic fungal strains were identified and classified in five genera: Colletotrichum, Fusarium, Simplicillium, Lasiodiplodia and Trichoderma. Among these, ten Trichoderma isolates demonstrated strong antagonistic activity against selected fungal pathogens and significantly enhanced the growth of Arabidopsis seedlings in vitro. These beneficial effects were associated with the production of siderophores and indole-3-acetic acid, as well as the apparent nitrogen availability -- likely mediated through interactions with nitrogen-fixing bacteria.

Biotic and abiotic stresses alter the physiology of perennial plants, with consequences for fungal endophytes and disease expression. In grapevine, drought inhibits esca disease expression, but the underlying molecular interactions between the plant and fungi are unknown. We combined wood metatranscriptomics, metabolomics, and metabarcoding to investigate these interactions in 30‐year‐old grapevines and eight wood‐pathogenic fungi under conditions of drought or esca leaf symptom expression. Both esca and drought decreased grapevine transpiration, but with different underlying mechanisms that induced specific transcriptomic and metabolic signatures. Similar pathways were also activated, including the phenylpropanoid and stilbenoid synthesis pathways. These stress responses could potentially confer cross‐tolerance and elicit different fungal molecular responses. Across all fungi, the total level of putative virulence factors increased significantly under both stresses. Under drought, only the relative abundance of Phaeomoniella chlamydospora and gene expression involved in anti‐oxidative mechanisms, growth, and reproduction increased. Under esca expression conditions, only the relative abundance of Fomitiporia mediterranea and gene expression involved in wood degradation, competition, detoxification, and growth increased. Under drought, induced grapevine defenses and reduced transpiration, together with the low abundance and putatively weak virulence of F. mediterranea may account for the inhibition of esca leaf symptom.

Alternaria tenuissima leaf spot disease severely hinders sustainable walnut production in Xinjiang. To dissect the molecular basis of walnut disease resistance and mine resistance-associated genes, we investigated the physiological and transcriptomic responses of a resistant wild walnut and a susceptible cultivar, 'Xinxin No.2'. We found that the wild walnut displayed slower lesion spread and markedly higher defense enzyme activity; its chitinase activity stayed 30%-45% above that of 'Xinxin No.2' throughout pathogen infection. Transcriptomic data revealed significant enrichment of the amino sugar and nucleotide sugar metabolism pathway, proving chitin hydrolysis is vital for host defense. Genome-wide screening obtained 24 JrCHI genes classified into GH18 and GH19 families, whose promoters carry numerous jasmonic acid (JA) and salicylic acid (SA) response elements. JrCHI6 and JrCHI24 were screened as key resistance genes with elevated expression post-infection. Exogenous hormone treatments verified methyl jasmonate alone induced the expression of the two genes, while SA barely altered their transcript abundance. As JA signaling governs plant defense against necrotrophic pathogens, JrCHI6 and JrCHI24 are inferred to drive JA-mediated immunity to A. tenuissima. Transient overexpression of JrCHI6 and JrCHI24 improved leaf spot resistance in 'Xinxin No.2' and increased expression of JA/ET defense markers JrPR3 and JrPR4; in contrast, silencing these genes greatly compromised wild walnut resistance. This work systematically identified the walnut JrCHI gene family and verified JrCHI6 and JrCHI24 as core functional genes mediating chitinase-dependent resistance against A. tenuissima.

Main conclusionPlant-microbial partnerships and their underlying molecular mechanisms are indispensable, natural drivers of improved nutrient acquisition and stress tolerance in the face of climate-driven environmental challenges. Modern multi-omics tools, when coupled with artificial intelligence and synthetic biology, enable the precise design of targeted bioinoculants and synthetic microbial consortia. Translating these advanced microbiome-based strategies into scalable, field-level agricultural applications provides a sustainable path toward securing global food production while maintaining soil health. Global climate change imposes multifaceted abiotic and biotic stresses on crops, disrupting physiological and molecular processes and threatening agricultural productivity. Plant-associated microbes represent an underexplored yet powerful ally in enhancing crop resilience. This review presents current knowledge of plant-microbe interactions and the molecular mechanisms governing plant stress physiology, with an emphasis on climate-resilient and sustainable farming. Hence, ever-changing environmental cues pose a significant burden on agricultural productivity, and plant-associated microbial communities modulate a cascade of physiological and molecular responses, including production of phytohormones, signaling, regulation of reactive oxygen species homeostasis, and activation of plant immune responses to help plants withstand stress and enhance productivity. Moreover, root exudates, phytohormones, and quorum sensing mediate the central communication networks, facilitating plant-microbe cross talk. Additionally, the advances in OMICs approaches aid in disentangling the molecular underpinnings of these interactions by providing mechanistic insights and potential candidate gene targets for crop improvement and stress resilience. In the post-genomic era, integrating artificial intelligence and big data analysis to optimize microbiome-based strategies for sustainable agriculture is a new frontier for disentangling plant-microbe symbiosis to improve soil health, enhance crop yields, and improve stress tolerance. Thus, by integrating the ecological, physiological, and molecular perspectives, this review highlights the transformative potential of harnessing plant-microbe symbiosis for climate-resilient and sustainable agriculture.

Symbiotic microbes play critical roles in facilitating the rapid adaptation of invasive insects to the defense systems in host plants. The host pine, Pinus tabuliformis, contains high levels of D-pinitol, which is inherently unsuitable for Dendroctonus valens, an invasive beetle in its introduced range in China. Our previous study has reported that gallery microbiota could degrade D-pinitol to enhance the larval adaptation. However, the adaptive strategies of adults and larvae under differential D-pinitol stress are not yet fully understood. In this study, we explored the D-pinitol-degrading capacity and potential sources of gallery microbiota and compared the structures and functions of D-pinitol-degrading microbes in the guts between adults and larvae. We confirmed that gallery microbiota, rather than native pine endophytes, are responsible for D-pinitol degradation and the formation of a low-pinitol microhabitat, and we postulate that adult beetles are the major contributors to gallery microbiota. Additionally, adult guts harbored a higher abundance of Erwinia and Serratia compared with larval guts and exhibited a stronger D-pinitol-degrading capacity. Our results indicate that adults and larvae employ distinct strategies to overcome differential D‑pinitol stress from host pines. Specifically, adult beetles enrich more D‑pinitol‑degrading microbes in their guts, whereas larvae mainly rely on the external degradation of gallery microbiota. These findings provide additional evidence supporting that the adults and larvae utilize detoxifying microbes to metabolize deterrent compounds and adapt to host pines at different developmental stages.

Main conclusionPlant immunity depends on coordinated control of signaling gain, molecular persistence, RNA fate, interorganismal exchange and timely attenuation, defining testable routes to durable disease control. Plant immunity is often reviewed as a sequence of receptor activation, transcriptional reprogramming, and antimicrobial output. That organization is useful, but it obscures a central mechanistic problem: immune performance depends on coupled control of protein abundance, RNA fate, interorganismal exchange and response termination. Here, we develop an evidence-weighted protein-RNA control-loop framework that is explicitly differentiated from receptor-, proteostasis-, RNA-silencing- and extracellular-vesicle-centered reviews. The framework organizes plant-pathogen interactions around five experimentally tractable control variables: signaling gain, molecular persistence, RNA routing, interorganismal exchange and memory versus cost. We evaluate how pattern-recognition receptors and nucleotide-binding leucine-rich-repeat receptors set signaling gain; ubiquitination, SUMOylation, proteasomal turnover and autophagy determine persistence; RNA-binding proteins, alternative splicing, N6-methyladenosine (m6A), translation and decay route immune messages; and vesicular or non-vesicular ribonucleoprotein carriers mediate cross-kingdom RNA exchange. For each layer, we distinguish association from physical mechanism, causal perturbation, and crop-level validation. Receptor synergy, resistosome signaling, selected proteolytic circuits and several cross-kingdom RNA interference mechanisms are strongly supported, whereas generalized roles for immune m6A, stress-granule routing, vesicle-exclusive RNA transport and durable epigenetic memory remain incompletely demonstrated. The framework yields testable predictions and a stage-gated roadmap for engineered receptors, host- and spray-induced gene silencing, proteostasis or RNA-binding-protein engineering, and multi-omics breeding. Durable resistance should therefore be judged not by maximal defense activation, but by sufficient gain, correct routing, appropriate persistence and timely attenuation with acceptable yield and environmental costs.

Microbial pathogens pose a serious challenge to food security under climate change. Microbiomes can filter out incoming pathogens. However, the specific microbial taxa and mechanisms that indicate the response of the microbiome and plant health to the invasion of pathogens remain largely unknown. Here, we combine multiple field surveys and experiments with DNA and RNA sequencing using tobacco (Nicotiana tabacum) and the bacterial wilt pathogen Ralstonia solanacearum as the model system, and f ind that Pseudomonadaceae constitute an important root-associated component against pathogen invasions. Based on this finding, we develop synthetic microbial consortia, which support plant health by establishing protective microbial barriers and systemically reprogramming root immunity and metabolism. Targeted metabolite profiling further identifies vanillin as a candidate bioactive component of SynCom-induced root exudates, with its concentration increasing by approximately 240% under SynCom inoculation. Exogenous vanillin inhibits the growth and motility of R. solanacearum and also suppresses several other soilborne pathogens. Together, our work establishes the root microbiome as an important component of plant immune defense and highlights microbiome inoculation as a potential tool to enhance crop resistance against increasing pathogen pressures under climate change.

Arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) are common root-associated fungal groups in natural ecosystems. However, how their colonization and community composition vary among seasons under natural drought conditions remains unclear. We investigated Populus euphratica and Haloxylon ammodendron in the Ebinur Lake Wetland National Nature Reserve, China. Microscopic observation and high-throughput sequencing were combined to characterize AMF and DSE colonization and community composition across spring, summer, and autumn under low, moderate, and high drought conditions. The results showed that drought strongly affected fungal colonization, with responses varying between host species. AMF colonization in P. euphratica increased to 90% under high drought conditions, and its response to drought differed among seasons, whereas DSE colonization responses were less season-dependent. AMF communities changed markedly across drought conditions, whereas DSE communities remained comparatively stable. Soil environmental variation was driven mainly by seasonality, and the colonization rates of both fungal groups were positively associated with soil ammonium nitrogen. Co-occurrence structure also varied seasonally, with the autumn network showing the greatest connectivity and complexity and a predominance of positive correlations. Overall, drought, seasonality, and host identity jointly shaped the colonization, community composition, and association patterns of root endophytic fungi in desert plants. These findings reveal contrasting responses of AMF and DSE to environmental stress and provide ecological insights relevant to dryland restoration.

Treating plants with plant-derived DNA triggers a strong and usually highly self/nonself-specific immune response. Several studies reported an induction of early signaling responses, resistance gene expression, and increased resistance to certain biotic stressors for self-DNA-treated Arabidopsis and also for crops like bean, lettuce, tomato, maize, or peach. These results indicate that self-DNA might bear potential as a tool for future strategies for a preventive biological pest control. However, it remains to be studied how DNA treatment affects different major defense hormones, to which degree responses at the early signaling levels predict the resistance at the phenotypic level, and whether the results of the earlier, lab-based studies would translate to the agronomic field. Here, we treated common bean (Phaseolus vulgaris) plantlets with self-DNA and with nonself-DNA from a congeneric species (lima bean, Phaseolus lunatus) and from a more distantly related species in the same family (Acacia farnesiana). We observed a self/nonself-specific induction of jasmonic acid only by self-DNA that was associated with decreased feeding by a chewing herbivore. By contrast, salicylic acid was induced by self- and nonself-DNA to similar degrees, which was also reflected in reduced population densities of several pathogenic fungi and bacteria. Most importantly, under open field conditions, a single self-DNA-treatment increased yield approximately 1.5-fold in the rainy season and 3.2-fold in the dry season. We suggest that self-DNA merits attention as a tool for future, preventive strategies in the biological control of pests and plant diseases.

Summary Statement TGERa, a PERU allelic variant identified in the diploid potato inbred lines, functions as the receptor for the Phytophthora PAMP Pep‐13, whereas its paralog TGERb is nonfunctional. TGERa/PERU confers enhanced resistance to Phytophthora pathogens in heterologous plants.

African rice (Oryza glaberrima) was independently domesticated in West Africa around 3000 years ago, and has long been intertwined in the history of the region. Asian rice (Oryza sativa), which was introduced in Africa when European settlers arrived, gradually replaced African rice and has since dominated rice cultivation in the continent. Domesticated rice species are affected by bacterial leaf blight (BLB), which is caused by the pathogen Xanthomonas oryzae pv. oryzae (Xoo). Here we show that the bacterial leaf blight pathogen in Africa (AfXoo) belongs to a distinct phylogroup from the one circulating in Asia (AsXoo), and has a different evolutionary history. Analysis of 87 AfXoo genomes identified five main populations, including highly clonal ones, and a more diverse and recombinant population. Tip-dating analysis revealed that the AfXoo population went through a period of expansion, then decline and more recent recovery. We hypothesize this followed the rise and fall of African rice, and that the introduction of O. sativa served as a bottleneck leading to the emergence of current AfXoo populations. We show that AfXoo has a highly conserved repertoire of type III effectors (T3E), but that nonetheless there is variation especially between populations. In the case of transcription activator-like effectors (TALEs), variation can arise quickly through rearrangements, and we hypothesize that the TALE repertoire of AfXoo has been selected to allow the bacteria to colonize both species of cultivated rice found in the continent. Our research provides an attempt to decipher the genetic history of bacterial blight in West Africa, and its past and present impact on rice cultivation in the region.

SUMMARY Light is essential for plant growth and development. Sustainably feeding a constantly-growing human population will likely involve adapting crop plants to intercropping and high planting density by rational manipulation of light signaling. Here, we edited the tomato ( Solanum lycopersicum ) genome to generate lines with a light-stable version of ELONGATED HYPOCOTYL 5 (HY5), a master transcription factor involved in the integration of light and hormone signaling. Removing the tomato HY5 N-terminal domain required for interaction with CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) prevented light-dependent protein degradation and resulted in a gain-of-function phenotype of short seedlings. Elongation growth was also compromised under proximity shade conditions either simulated by enriching white light (W) with far-red light (W+FR) or achieved by growing plants at a higher density. Transcriptomic analysis of gene expression changes after exposure to W+FR for 24h revealed a reduced number of shade-responsive genes in edited lines compared to unedited, wild-type controls, many of which are related to growth and hormone (notably auxin) biosynthesis and signaling. The reduced elongation observed in edited lines correlated with enhanced resistance to infection by viral, bacterial and fungal pathogens, both under low and high density conditions. These results indicate that our editing approach allows the generation of gain-of-function tomato plants in which HY5 is camouflaged to avoid COP1 recognition and eventual degradation. Our findings therefore provide a biotechnological tool to create more compact and pathogen-resistant plants amenable to high planting densities.

Metabolic bioengineering has emerged as a transformative approach for reshaping plant defense by targeting intrinsic biosynthetic pathways to enhance immunity in modern agriculture. Moving beyond proof-of-concept metabolomics to broad-spectrum programmable pathway engineering addresses gaps in plant rational design and optimizes resilience in response to diverse environmental cues. This review aims to comprehensively highlight the transition of innovative approaches to phenolics, alkaloids, flavonoids, terpenoids, and benzoxazinoids, inferring adaptive reprogramming that mediates the growth-defense balance and functions as molecular sentinels in plants. Furthermore, decoding the volatile metabolome reveals a dynamic signaling interface that influences defense responses and stress-induced plant-microbe interactions, with the shikimate, jasmonate, and salicylate pathways functioning as central hubs for microbial deterrence and priming immune memory. Recent developments in multi-scalar genome-editing strategies, including CRISPR-driven combinatorial edits, enzyme orthogonalization, fluxomics, and spatially resolved multi-omics, reconfigure central and specialized metabolic fluxes toward improved defense function and regulation. Additionally, emerging tools, such as WUSCHEL2 and BABY BOOM transcriptional modules, and artificial engineering strategies integrating deep learning model-driven predictions facilitate rapid development of synthetic genetic circuits and support a predictive engineering of plants. Moreover, Mass spectrometry imaging (MSI) in spatial metabolomics enables to obtain structures and locations of unidentified endogenous metabolites within cells and tissues. Overall, this review emphasizes a diverse array of primary and secondary metabolites, spanning molecular concepts to recent advances in plant immune mechanisms. It also illustrates new frontiers in programmable metabolic engineering that accelerate the understanding of plant-microbe-metabolite cross-talks, offering strategies to improve plant resistance and advance sustainable agricultural solutions.

Abstract European apple canker ( Neonectria ditissima ) is major constraint to apple production, with limited chemical control and no microbiome-based solutions currently available. Recent studies linked Sphingomonas taxa in leaf scar tissues with reduced canker severity, suggesting a potential for targeted microbiome manipulation. However, whether Sphingomonas can establish in planta and influence resident host microbiomes remains unresolved. Apple ‘M9’ rootstocks were inoculated at planting with consortia of apple-derived Sphingomonas strains. Inoculation did not affect plant height, girth or biomass, nor increase the relative abundance of Sphingomonas in roots, rhizosphere or leaf-scar compartments. Nevertheless, Sphingomonas inoculation affected alpha diversity in roots and rhizosphere without significant effects on beta diversity and relative abundance of taxa belowground. In contrast, root inoculation significantly altered fungal community composition in leaf scar tissues, reducing the relative abundance of known plant pathogenic genera including Gnomoniopsis , Phomopsis , Ilyonectria , Neofabraea , and Perenniporia . This study demonstrates that root-applied microbiome amendments can exert cross-compartment effects on microbial communities in a perennial crop, even in the absence of sustained colonisation by the inoculated taxa. Sphingomonas induced reduction in potential fungal pathogens in leaf scars reveal a decoupling between inoculant persistence and microbiome restructuring, highlighting a new possibility for microbiome engineering in woody hosts.

Abstract Plant-virus protein-protein interactions (PPIs) play crucial roles in viral infection and host immune responses. However, large-scale experimental characterization of plant-virus PPIs is costly and labor-intensive, suggesting the need to develop computational methods. Recent advances in biological sequence language models are offering unprecedented opportunities to develop machine learning-based plant-virus PPI predictors. In this study, we collected publicly available PPI data between Arabidopsis thaliana (A. thaliana) and viruses, establishing a robust dataset for method development. Based on this dataset, we developed AraVirusPPI, a machine learning framework for predicting A. thaliana-virus PPIs. AraVirusPPI employs the protein language model ESM Cambrian (ESMC) to encode sequence features and combines these representations with Extreme Gradient Boosting (XGBoost) to build the prediction model. Furthermore, we systematically evaluated multiple protein and genomic language models combined with various classification algorithms, thereby justifying the optimal model architecture adopted in AraVirusPPI. We applied AraVirusPPI to predict PPIs in the A. thaliana-Cabbage leaf curl virus (CabLCV) system. Predicted viral target proteins were compared with experimental datasets from A. thaliana-Turnip mosaic virus (TuMV) and A. thaliana-Pseudomonas syringae (Psy) systems. Network topology and functional enrichment analyses showed that the predicted CabLCV targets in A. thaliana were more similar to TuMV than Psy targets. These results suggest potential shared and distinct host-targeting patterns among pathogens and provide computational evidence supporting the biological significance of the proposed AraVirusPPI predictor.

Plant-pathogenic fungi secrete small proteins, termed effectors, to reprogramme host metabolism and suppress immune responses during infection. Although transcriptional waves of effector expression have been described in several pathosystems, the cis-regulatory elements underlying their temporal coordination remain largely unknown. Here, we investigate the temporal regulation of effector genes in the biotrophic smut fungus Ustilago maydis, a model organism for fungal plant pathogenesis. By integrating transcriptome reanalysis with comparative promoter motif enrichment across biotrophic fungi, we identify distinct promoter motifs associated with defined infection phases. In U. maydis, three candidate cis-regulatory elements correlate with early, proliferative and late infection stages, respectively. Positional enrichment relative to transcription start sites supports their regulatory relevance. Functional dissection of the native pep1, stp1, and cmu1 promoters revealed strongly context-dependent contributions of these elements and synthetic promoter assays showed that the early-phase motif acts as an activating and the proliferative motif as a repressive element. Together, our findings support a combinatorial cis-regulatory code in which activating and repressive promoter elements are integrated in a promoter-specific manner to shape temporal effector expression during U. maydis infection. These elements provide a framework for engineering synthetic fungal promoters with tunable transcriptional outputs.

Wheat leaf rust, caused by Puccinia triticina, poses a continuous threat to global food security. To dissect durable resistance mechanisms, we compared early infection responses between the near-immune cultivar 'Yunmai 34' (YM) and the susceptible 'Chinese Spring' (CS). While CS exhibited massive transcriptional reprogramming upon infection, YM displayed a relatively narrow scale-changes yet could arrested the pathogen prior to haustorium formation. This resistance might be associated with a constitutive immune state, evidenced by elevated basal expression of pathogenesis-related (PR) genes and defence regulators in the absence of pathogen challenge. Comparatively, CS mobilised a massive differentially expressed genes including those involved in hormone pathways and immune receptors yet insufficient to suppress the pathogen infection. Integrated analysis of differential expression and co-expression networks identified two defence-associated genes encoding an LRR-domain-containing protein (TaLRRop) and an aspartyl-tRNA synthetase (TaAspRS). Functional validation confirmed both as positive regulators contributing to PR- and ROS-associated basal defence. Collectively, these findings demonstrate that a constitutive immune state at the transcriptional level, rather than the overall magnitude of induced responses, underlies effective early resistance to leaf rust, offering valuable target candidates for resistance breeding.

Single-cell proteins (SCPs) broadly comprise microbial biomass or biomass-derived proteinaceous products produced from bacteria, yeasts, filamentous fungi, or microalgae. In the broader field of cell-derived alternative proteins, animal- and plant-cell-derived platforms are also being explored because they can reproduce the structural, compositional, and sensory equality of conventional food materials. In contrast, microbial SCPs are distinguished by diverse cellular structures, metabolites, cell-wall components, and bioactive fractions that may confer functionality beyond nutritional supplementation. This review summarizes recent progress in SCP-related platforms by first contrasting animal- and plant-cell-derived proteins with microbial SCPs, and then focusing on bacterial, yeast, filamentous fungal, and algal SCPs. Recent animal, human, and cell-based studies indicate that microbial SCPs and related microbial biomasses may modulate the gut microbiota, intestinal barrier function, immune and oxidative-stress responses, lipid metabolism, muscle protein synthesis, disease resistance, and product-quality traits. Nevertheless, microbial SCP development remains constrained by microbial-group-specific safety and processing issues, including nucleic acid reduction, toxin and contaminant control, and cell-wall-associated digestibility. Finally, this review summarizes regulatory frameworks for SCPs in the United States and the European Union, where commercialization remains product-specific rather than governed by a harmonized SCP category. Overall, SCPs represent promising but heterogeneous food and feed platforms that require integrated evaluation of nutritional value, functionality, safety, processing, and regulatory compliance.

The antibiotic resistance crisis has become a major threat to global public health. Discovering natural antibacterial compounds with unique mechanisms from traditional medicinal plants is an effective strategy to overcome this challenge. Through activity-guided fractionation, three compounds were isolated from Turpinia arguta leaves, identified as piperyamine A, cynaroside, and gallic acid. Cynaroside exhibited the strongest antibacterial activity, with MIC values of 31.25 μg/mL against Staphylococcus aureus (Gram-positive) and 62.5 μg/mL against Vibrio parahaemolyticus (Gram-negative). Phenotypic experiments, including scanning electron microscopy, electrical conductivity measurements, and alkaline phosphatase (ALP) activity assays, were performed to evaluate antibacterial effects of cynaroside. The results indicated that cynaroside exerted antibacterial effects by disrupting the integrity of bacterial cell walls and cell membranes, with markedly different responses between Gram-positive and Gram-negative bacteria. Specifically, the increase in electrical conductivity was more pronounced in V. parahaemolyticus (Gram-negative), while the peak ALP activity was higher in S. aureus (Gram-positive). Integrated metabolomic and transcriptomic analyses were conducted to elucidate the differential antibacterial mechanisms. In S. aureus, cynaroside treatment was associated with suppression of pyrimidine metabolism and histidine metabolism, negatively regulating 11 metabolites with pyrC as the hub gene; in V. parahaemolyticus, it mainly inhibited glyoxylate and dicarboxylate metabolism and branched-chain amino acid degradation, negatively regulating tricarboxylic acid cycle intermediates with fdh3B as the hub gene. This study reveals the differential antibacterial mechanisms of cynaroside isolated from Turpinia arguta against Gram-positive and Gram-negative bacteria, laying a theoretical foundation for the development of species-selective natural antibacterial agents.

Fermented agricultural by-products may improve poultry health through microbiota-host interactions, but the underlying microbial and transcriptional responses remain incompletely understood. We assigned 160 Wuliangshan black-bone chickens to diets containing 0%, 5%, 10%, or 15% fermented feed from 8 to 120 d of age. The fermented feed was produced from soybean hulls, rapeseed meal, and vegetable waste by sequential fermentation with Bacillus subtilis, Lactiplantibacillus plantarum, and Limosilactobacillus reuteri. Ileal microbial communities were profiled by 16S rRNA gene sequencing, followed by shotgun metagenomics and ileal mucosal transcriptomics in the control and 10% groups. Fermented feed increased average daily gain (ADG) and ileal villus height and reduced feed conversion ratio (FCR). The 10% diet increased the relative abundance of Lactiplantibacillus from 1.05% to 74.47%, while reducing Enterococcus, Streptococcus, Staphylococcus, and Escherichia-Shigella. Metagenomic analysis revealed enrichment of microbial pathways and carbohydrate-active enzymes associated with starch and plant cell-wall polysaccharide degradation. Ileal butyrate concentration increased, whereas predicted virulence determinants, including cereulide, β-hemolysin/cytolysin, and LPS/LOS-related factors, decreased. The resistome shifted toward efflux-related determinants without broad enrichment of antimicrobial resistance genes. Ileal mucosal transcriptomics revealed lower expression of proinflammatory and innate immune genes and higher expression of genes involved in growth signaling and nutrient transport. Spearman correlation analysis showed that Lactiplantibacillus abundance and butyrate concentration were associated with higher ADG, villus height, and expression of growth-related genes and with lower FCR. Lactiplantibacillus abundance was also positively correlated with selected nutrient transport genes and negatively correlated with selected immune-related genes, whereas the depleted potentially opportunistic genera generally showed opposite correlation patterns. Collectively, fermented feed improved growth performance and intestinal health in association with Lactiplantibacillus enrichment, reduced predicted microbial virulence potential, attenuated mucosal immune activation, and enhanced host responses related to growth and nutrient transport.

Extracellular vesicles (EVs) are lipid-bound structures that transport bioactive molecules and are increasingly recognized as key mediators of plant-pathogen interactions. Although EV secretion has been reported in filamentous pathogens, their roles in plant immunity, microbial interactions and cargo-sorting mechanisms remain poorly understood. This study aimed to characterize the functional roles and composition of EVs produced by P. nicotianae, and to investigate shared EV-associated protein categories and putative cargo-sorting signals, including KFERQ-like (Lys-Phe-Glu-Arg-Gln) motifs, across filamentous pathogens. EVs were isolated and characterized using transmission electron microscopy (TEM) and Nanoparticle tracking analysis (NTA). TEM confirmed the presence of membrane-bound vesicles with both single- and double-membrane morphologies, while NTA demonstrated a reproducible and heterogeneous particle population. Functional assays demonstrated that P. nicotianae EVs elicit immune-associated responses in Nicotiana benthamiana, including cell death, ROS accumulation, and callose deposition. In addition, the EVs exhibited antimicrobial activity against five soil-associated bacterial isolates. Proteomic analysis identified 2,324 proteins, revealing a diverse EV cargo enriched in metabolic enzymes, protein-modifying enzymes, and transporters, along with virulence-associated proteins. Comparative analysis across selected filamentous pathogens identified shared EV-associated protein categories, including ATP synthase subunits, elongation factors, and heat shock protein 70 (HSP70), present across all examined species. Notably, approximately 78% of EV-associated proteins from nine filamentous pathogens contained putative KFERQ-like motifs. Collectively, these findings show that P. nicotianae secretes structurally heterogeneous EV-like particles with biological activity in plant and bacterial assays, and suggest potential roles in host interaction, microbial competition, and putative conserved features that may be involved in cargo loading.

The Lateral Organ Boundaries Domain protein family comprises a group of plant-specific transcription factors involved in diverse developmental and stress-response processes. We identified 106 NtLBD genes in tobacco (Nicotiana tabacum L.) and 50 SmLBD genes in eggplant (Solanum melongena L.), and classified them into Classes I and II. Duplication analysis identified 26 segmental and 3 tandem duplicated gene pairs, while interspecies synteny analysis revealed 17 tobacco-Arabidopsis thaliana and 31 eggplant-Arabidopsis thaliana syntenic pairs. Expression profiling showed broad tissue expression, with relatively high transcript abundance in roots and stems, and selected LBD proteins were localized to the nucleus. VIGS constructs targeting NtLBD4/NtLBD31 in tobacco and SmLBD19/SmLBD48 in eggplant were used for preliminary functional assessment. In tobacco, these treatments were associated with reduced expression of ABA-responsive genes, less visible injury under low-temperature stress, and increased susceptibility to bacterial wilt. In eggplant, they were associated with increased expression of ABA biosynthesis and signaling related genes, reduced visible cold injury, and enhanced bacterial wilt resistance. Expression changes in related LBD members further suggested possible functional redundancy or compensatory regulation. These findings indicate that selected LBD genes and their associated regulatory networks participate in ABA responses, low-temperature adaptation, and pathogen defense in tobacco and eggplant, and they further underscore the potential utility of these genes as targets for improving stress tolerance in Solanaceous species, although additional experimental confirmation is warranted.

Drought severely limits global wheat production, and plants can mitigate this stress by recruiting beneficial rhizosphere microbiomes. However, the role of host genotype in shaping this recruitment and influencing microbial inoculant efficacy is poorly understood. Here, we aimed to elucidate the interplay among host genotype, drought stress, and inoculation with Trichoderma citrinoviride. A factorial pot experiment investigated the responses of two wheat genotypes (JN 14-95, constitutively drought-tolerant; JN 72, drought-responsive) to water regimes and inoculation with T. citrinoviride. Metagenomic analysis was performed to characterize rhizosphere bacterial community structure and functional potential. Host genotype was the primary driver of bacterial community structure (42.3% of variation), exceeding water stress (23.8%) and inoculation (11.5%). JN 14-95 adopted a "physiological autonomy" strategy with constitutive high root-to-shoot ratio and inferred enrichment of auxin and SOD biosynthesis genes. JN 72 employed a "microbial outsourcing" strategy, enriching beneficial bacteria (Pseudomonas, Bacillus, Streptomyces) and showing inferred enrichment of central carbon metabolism genes. T. citrinoviride amplified these genotype-specific responses, increasing the total dry matter by 21.3% in JN 14-95 and 30.3% in JN 72 under drought. Our findings suggest a need to move from uniform inoculation practices toward genotype-informed microbiome management. The two strategies provide a framework for leveraging host genetics in sustainable agriculture, highlighting that breeding and microbiome management should be integrated.

Plants rely on plant-microbiota interactions to balance growth, defence, and metabolism under stress. Still, the mechanisms governing growth-resistance-quality trade-offs in the shade-tolerant medicinal herb Pseudostellaria heterophylla in understory cultivation systems remain unclear. Here, we integrated phenotypic/photosynthetic/quality assays, rhizosphere microbiome sequencing, leaf transcriptomics, and plant-microbe co-culture experiments to study responses to graded shading. Shading enhanced yield and disease resistance, reshaped the microbiome (enriching beneficial taxa, suppressing pathogenic Alternaria), and induced transcriptional reprogramming-upregulating starch-sucrose/glycerophospholipid metabolism and downregulating flavonoid biosynthesis genes (CHI/CHS) to reduce root-exuded flavonoids that inhibit beneficial bacteria. Functional assays confirmed that these bacteria promote growth and suppress Alternaria-mediated disease under low light. Our findings demonstrate trade-offs in shading coordinates along a bidirectional microbiota-root-shoot axis, establishing a framework for sustainable understory medicinal plant cultivation.

Mangrove ecosystems are dynamic coastal environments that offer essential ecological services, socioeconomic benefits, and resilience against climate change. Central to their functionality there are complex microbial communities-primarily bacteria and fungi-that drive key biogeochemical processes such as organic matter decomposition, nitrogen fixation, carbon sequestration, and sulfur cycling. This review aims to synthesize current knowledge on mangrove microbiomes, focusing on microbial taxonomic diversity, ecological roles, and environmental responsiveness. The effect of abiotic factors such as salinity, tidal regimes, vegetation type, and anthropogenic pressures on microbial community structure and function is also assessed. Key findings highlight the presence of both conserved microbial taxa across biogeographic regions and functional adaptations to local conditions, underscoring the global ecological significance of these microbial assemblages. Particular attention is given to microbe-mediated nutrient cycling, symbiotic plant-microbe interactions, and microbial contributions to pollutant degradation, including microplastics and heavy metals. Additionally, recent advances in omics-based approaches have expanded our understanding of microbial functionality and unveiled promising avenues for biotechnological applications, such as enzyme production and bioactive compound discovery. The review also identifies critical knowledge gaps, including the need for long-term monitoring, methodological standardization, and integrated multi-omics frameworks. Overall, recognizing microbial communities as foundational components of mangrove health is essential for effective conservation, ecosystem restoration, and sustainable resource management in the face of accelerating global environmental change.

Helicobacter pylori (H. pylori) is a human gastric pathogen that colonizes the stomach by adapting to acidic pH, penetrating the gastric mucus layer, adhering to epithelial and mucosal receptors, and delivering virulence factors that promote inflammation and immune evasion. Antibiotic-based regimens have markedly reduced the burden of H. pylori-associated disease; however, increasing antimicrobial resistance, treatment failure, recurrence, and disruption of the gut microbiota remain important clinical challenges. Future therapeutic strategies should therefore combine bacterial control with modulation of the gastric environment that sustains chronic infection and impaired host responses. Chitosan-based micro- and nanoparticles are promising delivery systems for drugs and biologically active compounds because of their biodegradability, cationic surface charge, and mucoadhesive properties. This Mini Review summarizes chitosan-based formulations proposed for the delivery of antibiotics, plant-derived antimicrobial compounds, and antimicrobial peptides, as well as systems targeting H. pylori adhesins, bacterial biofilm, or urease activity. We also discuss an experimental concept in which the immunomodulatory vaccine strain Mycobacterium bovis Bacillus Calmette-Guerin (BCG) is encapsulated in chitosan micro- or nanoparticles for gastric and/or intestinal delivery. Available in vitro and in vivo evidence suggests that chitosan-based systems may increase local exposure to active components, support mucosal immune responses, and potentially facilitate epithelial repair. Nevertheless, most data remain preclinical, and clinical efficacy in humans has not been demonstrated. The review highlights the need for broader independent validation, careful biosafety assessment, and standardized formulation parameters before these approaches can be considered for translation.

Phosphorus is an essential macronutrient for plant growth and development, especially in P-sensitive crops such as rapeseed (Brassica napus). However, the role of phosphorus (P) availability in plant disease resistance mediated by the root-associated microbiome remains poorly understood. Here, we investigated how P homeostasis regulates rapeseed resistance to Sclerotinia sclerotiorum through modulation of the root-associated microbiome. P deficiency significantly inhibited plant growth and increased susceptibility to S. sclerotiorum in multiple rapeseed ecotypes, including spring, semi‑winter, and winter types. Microbiome profiling revealed that Massilia was a key P-responsive biomarker genus significantly enriched under P-sufficient conditions. Both foliar application and root inoculation with Massilia effectively suppressed S. sclerotiorum infection in rapeseed. Mechanistically, Massilia colonization strongly activated the expression of pathogenesis‑related (PR) genes, antioxidant genes, and jasmonic acid (JA) signaling genes. Overall, this study establishes a P-mediated tripartite interaction linking root microbiota assembly and plant immunity. These results highlight that optimizing P supply to enrich beneficial microbes such as Massilia can enhance rapeseed resistance to S. sclerotiorum, providing a sustainable strategy for disease management.

IntroductionFungal polyketides constitute a structurally diverse class of secondary metabolites involved in ecological adaptation, stress responses, antagonistic interactions, and host-associated processes. However, their roles in mycorrhizal fungi remain poorly understood. In this study, we investigated the repertoire and potential functions of polyketide synthases (PKSs) in the ericoid mycorrhizal fungus Oidiodendron maius.Methods and resultsGenome-wide analyses identified 45 PKS-coding genes and 59 predicted biosynthetic gene clusters associated with secondary metabolite production. Transcriptomic analyses revealed that several PKS genes were differentially regulated during symbiosis with Vaccinium myrtillus and under cadmium exposure. Among these, the gene OmPKS197601 was strongly upregulated under both conditions and was selected for functional characterization through Agrobacterium tumefaciens-mediated gene disruption. Three independent OmPKS197601 knockout mutants were generated and evaluated for their ability to establish mycorrhizal symbiosis and tolerate cadmium and zinc stress. Knock-out mutants were also tested for their ability to antagonize the growth of selected fungal species. No significant differences were observed between the wild-type strain and the three OmPKS197601 knock-out mutants in either mycorrhization efficiency or metal tolerance. In contrast, dual-culture assays with the M2 knock-out mutant demonstrated reduced antagonistic activity against both plant pathogenic and saprotrophic fungi, including Heterobasidion annosum, Pythium sp., Kuehneromyces mutabilis, and Stereum hirsutum.ConclusionsOverall, these findings reveal the extensive genetic potential of O. maius for polyketide biosynthesis and suggest that regulated expression of PKS genes may contribute to the ecological fitness and biocontrol potential of ericoid mycorrhizal fungi.

BackgroundThe southern green stink bug (SGSB), Nezara viridula, is a globally distributed hemipteran pest that damages many economically important crops. Its midgut supports digestion, defense, symbiosis, and interactions with orally delivered control agents, yet the cellular composition of this tissue remains poorly characterized. We therefore developed a single-cell transcriptomic atlas of the N. viridula midgut.ResultsSingle-cell RNA sequencing of two biological replicates yielded a quality-filtered data set of 13,763 cells. Unsupervised clustering identified 12 transcriptionally distinct populations with putative annotations, including a stem cell/enteroblast (SC/EB)-like population, seven enterocyte-related populations, goblet-like cells, enteroendocrine cells, visceral muscle cells, and an extracellular-matrix-associated epithelial population. Enterocyte-related populations accounted for more than 77% of recovered cells. Putative annotations were assigned primarily from marker gene enrichment and homology to markers reported in other insects. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses identified population-associated functional enrichment patterns, and pseudotime analysis suggested transcriptional relationships between the SC/EB-like population and several enterocyte- and secretory-associated populations without establishing developmental lineages. Immune- and defense-associated transcripts were preferentially enriched in the pEC2 population, and genes associated with symbiont recognition, insecticide action, xenobiotic transport, and orally delivered double-stranded RNA showed population-biased expression. Descriptive comparisons with published insect midgut data sets identified shared and data-set-specific patterns among annotated populations.ConclusionThis atlas provides the first single-cell transcriptomic resource for a stink bug midgut and establishes a descriptive cellular framework for SGSB midgut biology. The dataset prioritizes candidate genes and cell populations for future spatial validation, functional testing, and studies of hemipteran midgut physiology, symbiosis, immunity, and pest-management-relevant traits. © 2026 Society of Chemical Industry.

Drought stress is one of the major factors limiting the growth and productivity of agricultural crops, including shallot (Allium ascalonicum L.). This study aimed to investigate the role of symbiotic associations between dark septate endophytes (DSEs) and shallot plants in improving growth performance, physiological responses, and yield under varying field capacity. A factorial completely randomized design was employed with two factors: DSE inoculation (without DSE, Cladophialophora nyingchiensis S51, and Diaporthe pandanicola TM1) and field capacity levels (100%, 80%, 50%, and 30% of available water). Observed parameters included vegetative growth, physiological responses, root colonization, and bulb yield. The results demonstrated that DSE inoculation significantly enhanced shallot plants' tolerance to drought stress. Cladophialophora nyingchiensis S51 exhibited superior performance in promoting vegetative growth, increasing plant height by 38%, root length by 122%, leaf biomass by 27%, and root biomass by 130% under severe drought conditions (30% field capacity). In contrast, Diaporthe pandanicola TM1 was more effective in enhancing root biomass (110%) and bulb formation (31%) under severe drought conditions (30% field capacity). Physiologically, both DSE inoculations increased chlorophyll content, reduced malondialdehyde (MDA) accumulation, and enhanced proline production, indicating improved adaptive responses to water deficit. Furthermore, both DSE inoculation increased bulb number and bulb weight across all field capacity levels. These findings demonstrate that C. nyingchiensis S51 and D. pandanicola TM1 have considerable potential as biological agents to improve drought tolerance and enhance shallot productivity in sustainable agricultural systems.

Abstract Plants coordinate phenylpropanoid metabolism across different tissues to defend against pathogens, but the underlying transcriptional mechanisms remain unclear. Here, we identify the kiwifruit transcription factor AcMYB15 as a regulator that lignin deposition and salicylic acid (SA) accumulation, conferring bacterial canker resistance in kiwifruit through a tissue-specific regulatory module during Pseudomonas syringae pv. actinidiae (Psa) infection. In leaves, AcMYB15 directly binds to the CAACCC motif in the AcPAL1 promoter to activate its transcription, promoting localized lignin accumulation. In stems, which are the primary conduits for systemic Psa colonization, AcMYB15 forms a protein complex with the stem-enriched transcription factor AcWRKY24. Formation of the AcMYB15-AcWRKY24 protein complex enhanced transcriptional activation of the stem-prevalent AcPAL2 by strengthening its DNA-binding preferences for the CAACCA motif. Functional characterization indicates that AcPAL1 and AcPAL2 exert distinct defensive roles: AcPAL1 overexpression primarily leads to increased lignin deposition, whereas AcPAL2 overexpression predominantly enhances SA accumulation. Therefore, the AcMYB15-AcWRKY24 synergistic module provides a coordinated defense mechanism in the stem by simultaneously promoting lignin deposition and SA accumulation, thereby conferring enhanced resistance to bacterial canker. Our study uncovers a two-tiered transcriptional mechanism that coordinately regulates the tissue-specific accumulation of lignin and SA during Psa infection. These findings provide a solid theoretical basis and potential genetic resources for the genetic improvement and molecular breeding of disease-resistant fruit crops.

The genus Xanthomonas comprises a phylogenetically and ecologically diverse group of bacteria, historically characterized by economically important plant-pathogenic members affecting a wide range of agricultural crops. However, an increasing number of non-pathogenic and endophytic lineages have been recognized within the genus, expanding understanding of its taxonomic and ecological breadth. While it has been known for the last 30 years that non-pathogenic Xanthomonas (NPX) are present in diverse plants, studies from our lab over the last decade on the rice microbiome have revealed the presence of multiple, diverse NPX species as a community associated with a particular plant host. In this study, we identify two novel citrus plant-associated NPX species and provide evidence for the existence of an NPX community composed of multiple, diverse species, as in rice. Biochemical analysis, along with phylogenomic and genome-based taxonomic investigations, revealed that these strains represent two novel species within the genus Xanthomonas , providing evidence for a citrus-associated NPX community composed of multiple, diverse species, analogous to that described in rice. The lack of a canonical type III secretion system, along with multiple antimicrobial biosynthetic loci, as in the rice NPX community, points to their importance in plant microbiomes. Hence, such systematic studies of the NPX community need to be extended to all other plants. Accordingly, we propose that strain LMG 8992 T (DSM =122509 T ) be classified as a novel species, Xanthomonas pateli sp. nov., and strain LMG 8993 T (DSM = 122444 T ) be classified as a novel species, Xanthomonas hingoranii sp. nov.

Impact of long‐term (17‐day) exposure to growth‐stimulating (250 mM) and growth‐inhibitory (750 mM) NaCl concentrations on biochemical characteristics related to C₄ pathway, anatomy and ultrastructure of chloroplasts of leaf tissues in the euhalophyte S. altissima was investigated. Immunological and biochemical studies identified C₄ enzymes, phosphoenol pyruvate carboxylase (PEPC), NAD‐malic enzyme (NAD‐ME), and NAD‐malate dehydrogenase (NAD‐MDH). Anatomical studies revealed Kranz anatomy with mesophyll (MCs) and Kranz cells (KCs) at the leaf periphery. Water‐storage cells (WSCs) and vascular bundle chlorenchyma cells (VBCCs), both located in the leaf middle, contained chloroplasts, as did MCs and KCs. Electron microscopy revealed granal chloroplasts containing starch granules, while immunogold labeling revealed Rubisco in chloroplasts of all chlorophyllous tissues. The highest Rubisco content was found in KC chloroplasts. These results are consistent with the notion that S. altissima is a C₄ plant; however, they do not exclude its belonging to an intermediate C₃‐C₄ form. The growth‐stimulating NaCl concentration increased Rubisco content in all chlorophyllous tissues and activity of NAD‐ME in leaves activating, probably, the C₄ pathway. In contrast, the growth‐inhibitory NaCl concentration reduced Rubisco content, decreasing it below the level observed in the absence of NaCl. An increase in cell size was observed in the chlorophyllous tissues in response to both the growth‐stimulating and growth‐inhibitory salinity, leading to an increase in leaf succulence. The greatest increase in size was observed in WSCs. Chloroplasts from different tissues differed in their ability to maintain native structure and robustness of Rubisco‐chloroplast binding under salinity, suggesting chloroplast heterogeneity and different Rubisco sensitivities to NaCl.

IntroductionSoil salinity severely compromises rice (Oryza sativa L.) growth and grain yield by disrupting ionic and osmotic homeostasis. Although transcriptome profiling has advanced understanding of salt-stress responses in rice, the conservation of co-expression networks across genetic backgrounds and the relationship between network connectivity and transcriptional induction remain poorly characterized.MethodsWe performed integrated RNA sequencing and weighted gene co-expression network analysis (WGCNA) on three japonica rice varieties under control (0 mM), moderate (85.6 mM), and severe (136.9 mM) NaCl stress.ResultsAll three varieties exhibited basal salt tolerance. Differential expression analysis identified 2,067 and 4,900 non-redundant differentially expressed genes (DEGs) under moderate and severe stress, with 205 and 1,093 DEGs shared across the three varieties, respectively. WGCNA identified 32 co-expression modules. The brown module showed the strongest dose-dependent response to salt stress and was enriched in phenylpropanoid biosynthesis, plant hormone signal transduction, and MAPK signaling; its eigengene was highly concordant across varieties (mean r = 0.987), consistent with a shared leaf-level transcriptional program. Across brown-module genes, intramodular connectivity and salt-induced fold change were positively but only moderately correlated (Pearson r = 0.377): OsDREB1A was the most connected gene but not the most strongly induced, whereas LOC_Os10g35080 was the most strongly induced yet ranked 14th in connectivity. In the 15-node hyperosmotic salinity response subnetwork, no single hub dominated, and OsDREB1A occupied a peripheral position.DiscussionConnectivity and induction thus reflect complementary aspects of the salt-stress response, and candidate hub genes for molecular breeding should be prioritized by both criteria jointly, followed by validation across diverse genetic backgrounds.

Salinity disrupts ionic balance, osmotic regulation, redox homeostasis, and hormone signaling, thereby constraining plant growth and metabolism. Plants employ adaptive responses to salinity stress, including ion transport regulation, compatible solute accumulation, antioxidant defenses, and signaling reprogramming; however, these mechanisms are often insufficient under high salinity. Beneficial microbes, including plant growth‐promoting rhizobacteria, endophytes, fungi, and halotolerant taxa, are increasingly associated with improved plant performance under saline conditions. These associations are consistently linked with changes in ion homeostasis, osmotic adjustments, redox balance, hormone signaling, and root architecture. Importantly, most available evidence derives from transcriptomic, biochemical, and physiological observations, which do not establish direct mechanistic regulation. This review critically evaluates microbial contributions to plant salinity responses by explicitly distinguishing between experimentally validated mechanisms, correlative associations, and hypothesis‐driven models. We integrate insights from molecular genetics, biochemistry, and multi‐omics approaches while highlighting their limitations in establishing causality. Particular emphasis is placed on experimental strategies required to establish causal mechanisms, including isotope tracing, genetic perturbation, and synthetic community approaches.

Cajal bodies (CBs) are nuclear condensates known for their role in sn- and snoRNA maturation in animal, but their function in plant stress responses remains poorly understood. We compared wild-type (WT) Arabidopsis thaliana with the CB-lacking ncb-1 mutant during hypoxia and reoxygenation, focusing on nuclear retention of transcripts. In both genotypes, hypoxia caused nuclear accumulation of poly(A+) RNA and extensive reprogramming of gene expression. However, the transcriptional response, including induction of hypoxia-responsive genes, was considerably weaker in ncb-1, both during stress and recovery. In nuclear RNA, introns with low (intron retention index, IRX 50%) retention showed opposite, genotype-dependent dynamics. During hypoxia, efficiently spliced introns (IRX < 10%) accounted for over 70% of introns detected in nuclear RNA. Relative to normoxia, this proportion increased two- to threefold in WT but only moderately in the mutant. Together with poly(A+) RNA accumulation, this suggests that largely spliced transcripts, including those of genes involved in protein transport and ribosome biogenesis, are held in the nucleus during stress. Conversely, strongly retained introns, often terminal and located in genes related to hypoxia and other abiotic stresses, were most abundant in WT nuclear RNA under normoxia. Their proportion dropped sharply during hypoxia, and much less so in ncb-1. This indicate that WT plants maintain a nuclear reserve of incompletely spliced stress-related transcripts that are processed upon stress. The mutant also showed reduced interaction between U2 snRNA and U2B″, which leads to splicing defects. Our results indicate that CBs regulate which transcripts are held in the nucleus and how they are processed, contributing to plant tolerance of oxygen deprivation and recovery.

The application of nanotechnology presents a promising strategy for mitigating heavy metal toxicity in crops, yet the integrative mechanisms underlying nanoparticle-mediated stress alleviation across the plant-soil system remain poorly understood. Herein, we investigated the transcriptomic and rhizosphere microbial responses of maize to cadmium (Cd) stress and the responses associated with fungal-mediated iron-based nanoparticles (NPs). Characterization confirmed the synthesis of spherical, Fe-O dominant NPs with uniform distribution. Under Cd stress, transmission electron microscopy revealed severe ultrastructural damage in leaf cells, which was associated with pronounced cellular alterations, whereas NPs application was associated with preservation of leaf ultrastructure. RNA-sequencing analysis demonstrated that Cd stress triggered extensive transcriptional reprogramming, suppressing defense-related and phenylpropanoid biosynthetic genes while activating stress-signaling pathways. NPs treatment alone was associated with a distinct transcriptional response, including changes in defense and metabolism-related genes without pronounced stress-associated transcriptional signatures. In NPCd co-treated plants, NPs were associated with reversal of Cd-induced transcriptional changes, particularly in starch/sucrose and phenylpropanoid metabolism and ethylene and jasmonate related signaling pathways. Rhizosphere microbiome analysis further revealed that NPs application significantly altered bacterial diversity and community composition, with treatment-associated shifts in several bacterial genera, including Sphingomonas and Methylobacterium. Collectively, the results showed that fungal-mediated NPs application was associated with improved Cd tolerance in maize, accompanied by preservation of leaf ultrastructure, reduced Cd-associated superoxide accumulation, transcriptional reprogramming of defense and metabolism related pathways, and changes in rhizosphere bacterial communities. These findings highlight the potential of fungal-mediated NPs as a promising strategy for improving crop performance under Cd stress.

We previously identified seven members of the domain of unknown function 966 (DUF966) family in rice and showed that OsDSR2 and OsDSR3 regulate tolerance to drought, salinity and alkali stresses. However, bioinformatic characterization of this family remains limited, and the biological functions of the remaining members are largely unexplored. In this study, we performed a systematic bioinformatic analysis of the structure and potential function of the rice DUF966 family and focused on the unidentified member OsDSR6 in salinity tolerance. Phylogenetic and collinearity analyses showed that DUF966 genes are more evolutionarily conserved in monocot species. Promoter analysis indicated that family members commonly contain light-responsive elements, abscisic acid-responsive elements (ABREs) and methyl jasmonate (MeJA)-responsive elements. Transcriptional regulatory prediction identified 25 putative transcription factors and 91 microRNA-target interactions associated with this family. Among them, NIGT1 (LOC_Os02g22020) and LOC_Os05g37730 were predicted to bind the promoter regions of OsDSR3 and OsDSR7, respectively, OsDSR6 was predicted to be regulated by osa-miR399a/b/c. OsDSR6 exhibited high expression levels in rice leaves and leaf sheaths. Its expression was rapidly downregulated by salt and drought stress but upregulated by abscisic acid (ABA) treatment. The OsDSR6-encoded protein is localized to the plasma membrane. Functional analysis showed that overexpression of OsDSR6 increases rice sensitivity to salt stress, and the transcript levels of stress-responsive genes, including Lea3, Rab16c, P5CS and OsNCED4, were decreased in OsDSR6-overexpressing plants. Collectively, these findings extend current understanding of the rice DUF966 gene family and identify OsDSR6 as a potential target for molecular breeding of salt-tolerant rice.

The kinase proteins are a superfamily in plants and are involved in diverse biological and molecular functions for better adaptation of land plants. Protein kinase diversification is hypothesized to enhance plant adaptation to abiotic stress, particularly salinity. This study tests whether evolutionary expansions of stress-responsive kinase subfamilies correlate with functional variation in salt tolerance. The study identified a total of 49,611 genes in 32 land plants across mosses, bryophytes, lycophytes, gymnosperms, and angiosperms. All identified genes were classified into 26 major classes (I-XXVI) based on domain architectures, e.g., protein kinase tyrosine (PkT), leucine-rich repeat (LRR), LRR-protein kinases (LRR-Pk), LRR-Pk tyrosine kinases (LRR-PkT), and protein kinase (Pk) and the X domain representing other than the listed domains. RNA-seq-based expression profiling under biotic and abiotic stresses and in various plant tissue of Arabidopsis thaliana (Ath), Glycine max (Gma), Gossypium hirsutum (Ghi), Zea mays (Zma), and Oryza sativa (Osa) highlighted potential OGs including OG12, OG35, and OG44 showing response in all plants. Furthermore, upregulation of OG12_LR_GhPk08 in Ghi under both biotic, (e.g., Xanthomonas citri) and abiotic (e.g., salt, heat, and cold) stress, as well as in specific tissues like roots and seedlings, suggest a potential role in stress responses. Molecular docking analysis demonstrated the preferential binding of ATP to Ghi kinase proteins, indicating their conserved phosphorylation mechanisms. Furthermore, SNP and InDel analyses in salt-resistant (Mac7) and salt-susceptible (Coker 312) cotton genotypes revealed genetic variations potentially linked to differential stress responses. Moreover, the qRT-PCR-based expression analysis of two genes (Gohir.D08G10000; OG12_LR_GhPk08, Gohir.D05G155340; OG35_Pk_GhPk02) out of three, showed salt-responsive transcription under the tested condition in Mac7 and Coker 312 cotton genotypes, supporting their prioritization as candidate genes for further functional validation and characterization. Thus, the study provides a deep insight into diversity and functional responses of kinase genes in land plants, particularly, in cotton leading to future research on kinase studies.

Abstract Soil salinization is a global threat to the agricultural productivity of the world. Halotolerant plant growth-promoting rhizobacteria (HT-PGPR) offer a prospective sustainable approach for mitigating salt stress, but comprehensive understanding of their molecular mechanisms remains lacking. In this study, a HT-PGPR strain T6-2 was isolated and an integrated phylogenomic, genomic and functional approach was undertaken to elucidate its salt tolerance and plant growth promotion strategies. Phylogenetic and genome-based analyses strongly established the strain as Kosakonia cowanii . Whole-genome sequencing revealed genes involved in ion transport, compatible-solute biosynthesis (glycine betaine, trehalose, proline and polyols) and plant growth-promoting traits, including IAA production, organic phosphorus solubilization and nitrogen assimilation. RT-qPCR demonstrated threshold-dependent induction of key osmoprotectant and ion-transporter genes, while targeted quantification showed a staged shift in compatible solutes: sorbitol and betaine predominated under low-to-moderate salinity, whereas proline accumulated preferentially under higher salt stress. In vitro assays confirmed IAA production and organic phosphorus solubilization. Strain T6-2 innoculation also enhanced wheat seeed germination. Under 100 mM NaCl stress, strain T6-2 inoculation triggered strong, crop-dependent growth stimulation: strong biomass accumulation in soybean and selective root-associated effects in maize and no consistent growth benefit in wheat. Our integrated data conform to a multidimensional model in which strain T6-2 ameliorates both ionic and osmotic stress, affects phytohormone composition, and modulates nutrient supply.This mechanistic characterization positions Kosakonia cowanii strain T6-2 as a promising microbial inoculant to enhance crop tolerance to saline agroecosystems.

Cadmium (Cd) is a highly toxic heavy metal that disrupts plant growth, photosynthesis and cellular homeostasis. Although Cd uptake, transport, and detoxification have been extensively studied in plants, how lignification-associated cell-wall remodeling contributes to Cd tolerance remains poorly understood, particularly in highly lignified woody grasses. Here, we used moso bamboo (Phyllostachys edulis) as a model to investigate whether lignification-associated responses participate functionally in the Cd stress response. Dose-response analysis showed that Cd caused progressive reduction of seedling growth and photosynthetic performance. At an early stage of exposure, Cd also induced redox imbalance and transcriptomic reorganization, including strong enrichment of phenylpropanoid- and lignin-associated pathways. These changes were accompanied by increased 4-coumarate:CoA ligase activity, accelerated lignin accumulation, stronger tissue lignification and thicker cell walls. Pharmacological perturbation provided additional support for the functional relevance of these responses. Treatment with 3,4-(methylenedioxy)cinnamic acid, which was used to suppress lignification-associated phenylpropanoid metabolism, intensified Cd-induced injury. Conversely, exogenous p-coumaric acid partially alleviated stress symptoms and promoted lignification-associated cell-wall responses. These contrasting treatments were also associated with altered Cd allocation between roots and leaves, with increased lignification stimulating root Cd sequestration and reduced leaf accumulation. Together, these findings indicate that lignification-associated cell-wall changes are closely associated with Cd tolerance in moso bamboo and may contribute to the Cd response, at least in part, by raising root Cd retention and restricting Cd accumulation in aerial tissues. This work therefore extends current understanding of heavy-metal stress biology in highly lignified woody species and provides a physiological basis for considering bamboo responses in the context of ecological restoration.

Cold stress severely restricts the growth and development of cotton. Plants must adjust their developmental and physiological processes to cope with such stress. Protein phosphorylation, a vital reversible post-translational modification, dominates intracellular signal transduction upon environmental stimuli. In this study, we analyzed the phosphoproteome of cotton under cold stress to delineate how phosphorylation modifications mediate cold responses. Phosphoproteomic analysis showed that differentially phosphorylated proteins were significantly enriched in pathways associated with post-transcriptional regulation, including RNA splicing, pre-mRNA processing, mRNA stability, and translation. We further performed functional analyses of the RS2Z subfamily gene GhRS2Z32, which encodes a serine/arginine-rich protein, and the GRP family gene GhGRP7, which encodes a glycine-rich protein. Their transcript abundance and phosphorylation levels increased under cold treatment. Gene silencing in cotton enhanced cold tolerance, whereas heterologous overexpression in Arabidopsis compromised freezing tolerance, as evidenced by decreased survival rates, increased membrane damage, and elevated reactive oxygen species accumulation. These findings suggest that GhRS2Z32 and GhGRP7 act as negative regulators of cold tolerance in cotton. Overall, this study advances the understanding of genes encoding SR proteins and GRPs and offers a new theoretical basis for improving cotton cold tolerance.

BackgroundSoil salinization poses a significant threat to agricultural production, necessitating innovative agronomic strategies to mitigate its impact. Salinity stress is a critical abiotic factor that hampers tomato growth by causing ionic imbalances, oxidative damage, and disruptions in physiological systems. This study investigated the effect of sodium silicate (Na₂SiO₃) in reducing salt stress in tomato plants exposed to 75 and 150 mM NaCl.ResultsThe results indicated that salinity substantially decreased plant growth, the content of photosynthetic pigments, and the uptake of essential mineral nutrients. Concurrently, it heightened oxidative stress markers such as malondialdehyde (MDA), electrolyte leakage, hydrogen peroxide (H₂O₂), and hydroxyl radicals (•OH). Treatment with Na₂SiO₃, particularly at a concentration of 2 mM, significantly improved growth performance and photosynthetic pigment content. Silicon treatments enhanced the antioxidant defense mechanisms by increasing the activity of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and peroxidase (POX). Additionally, levels of ascorbate (AsA) and glutathione (GSH) were raised, leading to reduced oxidative damage and stabilized cellular membranes. The application of silicon also improved ionic homeostasis by decreasing sodium ion (Na⁺) concentration while enhancing the absorption of nitrogen (N), phosphorus (P), potassium (K⁺), and silicon.ConclusionsIn conclusion, the study demonstrated that silicon effectively alleviates salt stress in tomato plants by improving redox balance, enhancing antioxidant capacity, and regulating ion transport. The most effective concentration of Na₂SiO₃ was found to be 2 mM. The exogenous application of sodium silicate, especially at this concentration, helps to mitigate salinity-induced impairments in tomato plants by enhancing physiological performance, antioxidant defense, and ionic balance. This suggests its potential use as a sustainable agricultural strategy under salt-affected conditions.

Soil salinization severely limits forage crop productivity, yet the regulatory networks that govern salt stress adaptation in alfalfa, a moderately salt-tolerant leguminous forage, remain largely unexplored. Here, we examined the physiological and transcriptomic dynamics of alfalfa leaves under 200 mM NaCl stress across three time points. Salt stress induced a progressive elevation of the Na+/K+ ratio, biphasic activation of antioxidant enzymes and concurrent accumulation of malondialdehyde. Time-course RNA-seq analysis identified 3631 differentially expressed genes (DEGs) and 132 core salt-responsive transcription factors (TFs). Pathway and functional annotation analyses indicated that these DEGs were prominently involved in cell wall biogenesis, redox homeostasis, and the carotenoid biosynthesis pathway, with carotenoid accumulation strongly activated under salt stress. Using weighted gene co-expression network analysis (WGCNA), nine distinct co-expression clusters were constructed. Notably, the brown module, which showed a positive correlation with Na+ accumulation and the Na+/K+ ratio, was significantly enriched in the plant hormone signal transduction pathway, within which 72.7% of the enriched genes belonged to the TIFY family. Among them, a core hub gene, MsTIFY11B, was isolated for functional characterization. Subcellular localization demonstrated that MsTIFY11B is exclusively localized to the nucleus. Heterologous expression in yeast showed that MsTIFY11B overexpression enhanced tolerance to salinity and alkalinity, whereas it conferred negligible protection against mannitol-induced drought stress. Taken together, our findings provide a comprehensive temporal framework of the alfalfa transcriptomic response to salinity and suggest that MsTIFY11B may contribute to salt-alkali tolerance, making it a promising candidate for further functional characterization and potential application in the development of stress-adapted alfalfa varieties.

Abstract Cadmium stress is one of the most critical factors affecting rice growth and development due to environmental pollution. Alternative polyadenylation (APA) plays essential roles in regulating plant gene expression and protein translation. Comprehensive understanding of APA response mechanisms under cadmium stress holds significant importance. We performed APA dynamics analysis in rice under cadmium stress using RNA-seq data from the SRA public database. The results revealed: 1) Cadmium stress induced widespread poly(A) site switching with differences between roots and shoots and temporal variations in APA patterns during prolonged stress exposure. Notably, the numbers of APA events raised significantly with the increasement of cadmium concentrations. What’s more, low and ultra-low cadmium concentrations preferentially promoted proximal poly(A) site selection across multiple genes, accompanied with significant transcriptional level changes. 2) APA regulated genes coupled with down-regulated expression were enriched in the photosynthetic components in rice shoots. Concurrently, significant transcriptional regulated genes with APA events were observed in reactive oxygen species scavenging, signal transduction pathways and protein synthesis machinery. 3) Expression profiles of polyadenylation factors were regulated under cadmium stress, suggesting their potential involvement in mediating APA alterations in downstream genes. 4) Among genes selecting distal poly(A) sites, we identified enriched miRNA target sites within extended 3'UTR regions, suggesting that miRNA binding to these extended regions may destabilize transcript isoforms, thereby contributing to their reduced expression levels. These findings provide valuable candidate genes and theoretical frameworks for elucidating APA-mediated regulatory networks in response to cadmium stress.

Drought and salt stress severely constrain tomato growth and yield. The B-box (BBX) zinc finger transcription factor family is widely known for its roles in core developmental processes such as photomorphogenesis and flowering regulation, and its regulatory functions in abiotic stress responses have attracted increasing attention in recent years. However, the precise molecular mechanisms by which tomato BBX family members mediate drought and salt stress tolerance remain to be systematically elucidated. In this study, we functionally characterized SlBBX6, a tomato subfamily I BBX gene encoding a protein containing B-box and CCT domains. Functional validation using overexpression (OE) and CRISPR/Cas9-mediated knockout (KO) lines demonstrated that SlBBX6 negatively regulates drought and salt stress tolerance in tomato: under drought, OE lines exhibited reduced survival rates and exacerbated oxidative damage; under salt stress, they showed accelerated chlorophyll degradation and enhanced lipid peroxidation, whereas KO lines displayed enhanced tolerance to both stresses. Quantitative real-time PCR (qRT-PCR) analysis revealed that under drought stress, SlBBX6 transcriptionally represses SlAREB1, SlDREB2A, SlCAT2, and SlNCED1, attenuating abscisic acid (ABA) biosynthesis and signaling, stress responses, and antioxidant defense; under salt stress, it transcriptionally represses SlAREB1, SlDREB2A, SlP5CS1, and SlSOS1, attenuating ABA signaling, stress responses, osmotic adjustment, and the Na⁺ extrusion pathway. This negative regulatory effect exhibits developmental stage specificity. Collectively, these findings identify SlBBX6 as a negative regulator of drought and salt stress responses in tomato.

Plants, as sessile organisms, continuously encounter diverse environmental challenges, often experiencing multiple abiotic stresses during their development and growth. Among these stress events, heat has emerged as an increasingly severe factor because of global warming and currently poses a major threat to plant development, crop productivity, and agricultural sustainability. As a heat stress response, plants have evolved multilayered sensing and signaling systems that perceive heat cues and activate downstream protective and adaptive programs. This review summarizes recent knowledge regarding heat stress responses of Arabidopsis thaliana and major crops, including maize, rice, and wheat. We first outline the morphological, physiological, and molecular consequences of heat stress and then examine current models of heat perception and downstream regulatory networks. Particular emphasis is placed on second messenger signaling, transcriptional control, epigenetic regulation, post-transcriptional regulation, and heat memory. We also highlight stem cell homeostasis as an emerging component of heat adaptation that links stress signaling with developmental stability. Finally, we evaluate current strategies for improving crop heat tolerance, particularly during the reproductive stage, and discuss priorities for future research and breeding in a warming climate.

Salt stress is frequently encountered by plants and is a major constraint on crop production. Wheat (Triticum aestivum L.) is a staple cereal crop that is important for human health and food security. However, the response mechanisms of roots from different wheat varieties to salt stress remain unclear. In this study, we analyzed the complex response mechanisms of the salt-tolerant variety Longjian 114 (LJ114) and the salt-sensitive variety Chinese Spring (CS) to salt stress using integrated transcriptomic and metabolomic analyses. The results showed that LJ114 maintained better growth and exhibited higher antioxidant enzyme activities and greater osmotic adjustment capacity under salt stress. Differentially expressed genes in LJ114 were predominantly enriched in flavonoid biosynthesis, alpha-linolenic acid metabolism, and zeatin biosynthesis under salt stress. Metabolomic profiling identified 1366 differential metabolites, with LJ114 specifically enriched in tyrosine metabolism, tryptophan metabolism, glycerophospholipid metabolism, and fatty acid metabolism under salt stress. Integrated analyses further highlighted ABC transporters and alanine, aspartate, and glutamate metabolism as hub pathways, with notably higher GAD expression and GABA accumulation under salt stress. These findings suggest new insights into the molecular mechanisms underlying wheat salt tolerance and propose potential candidate targets for enhancing crop salinity tolerance.

Increasing evidence indicates that long non-coding RNAs (lncRNAs) play a regulatory role in plant responses to environmental stress. However, the epigenetic mechanisms behind the regulation of salt stress by lncRNAs remain largely elusive. We previously discovered that the lncRNA MtCIR2 of Medicago trnucatula negatively regulated seed germination under salt stress by altering endogenous ABA and GA concentrations via histone ubiquitination. Here we evaluated the roles of MtCIR2 in regulating response of M. truncatula seedlings to salt stress. We found that over-expression and mutation of MtCIR2 reduced and enhanced tolerance to salt stress due to enhanced and suppressed foliar Na accumulation in over-expression and Mtcir2 mutant plants relative to their wild-type counterparts under salt stress. The MtCIR2-depenent Na accumulation was accounted for by the suppression of MtSOS2 that encodes a kinase responsible for Na exclusion from plants. We discovered that MtCIR2 physically interacted with BMI1, a core component of Polycomb Repressive Complex 1 (PRC1), leading to down-regulation of MtSOS2. ChIP assays revealed that MtCIR2 facilitated H2A ubiquitination at the chromatin of MtSOS2, thus repressing its expression, which in turn suppressed the SOS-dependent Na exclusion, and rendered the plants sensitive to salt stress. These results unravel a novel mechanism by which lncRNA epigenetically regulates SOS-mediated Na accumulation via histone ubiquitination in plant response to salt stress.

Crop productivity is a major concern in modern agriculture due to fluctuating environmental conditions, such as salinity. This study aims to explore the green‐synthesized potassium nanoparticles (K‐NPs) mediated metabolic regulations, including improved ion homeostasis, photosynthetic efficiency, and antioxidative defense mechanisms in soybean (Glycine max var. Soygold) under salinity stress. The K‐NPs were green synthesized using the leaf extract of Cordia myxa L. and then characterized using UV–Vis spectroscopy, high‐resolution transmission electron microscopy (HRTEM), and zeta potential. Soybean seedlings were treated with 0, 30, 60, and 90 mg L⁻¹ of K‐NPs along with salinity stress of 30, 60, and 90 mg L⁻¹ NaCl at 3, 7, and 14 days of plant growth. After the treatments, morphological, biochemical and antioxidant activities were measured. The plant root/shoot lengths were enhanced by 9% and 59%, respectively, while the number of leaves was increased by 32%, fresh biomass by 84%, and dry weight by 105% under K‐NPs treatments compared to control. K‐NPs at 30 and 60 mg L⁻¹showed non‐significant results under 60 and 90 mg L⁻¹ NaCl concentrations, but highly significant results were observed at 90 mg L⁻¹ K‐NPs under 30 and 90 mg L⁻¹ NaCl concentrations. The 90 mg L⁻¹ K‐NP concentration improved the root/shoot lengths of soybean seedlings by 33% and 132%, respectively. The number of leaves were increased by 38%, the plant fresh biomass by 22%, and dry weight by 133% as compared to lower concentrations under salinity stress. Furthermore, phenolic contents, secondary metabolites, and ionic homeostasis were also increased in the presence of 90 mg L⁻¹ K‐NPs under 90 mg L⁻¹ NaCl salinity stress. When exposed to salt stress, K‐NPs treatment increased antioxidant enzymes (SOD, CAT, and POX) more than the salt stressed control. Taken together, these results suggest that K‐NPs are effective nanomaterials for plant growth enhancement by regulating the defense mechanisms of soybean to cope with salt stress conditions. The findings could help in the designing and optimization of nanomaterial‐based fertilizers in order to achieve sustainable agriculture.

Soil salinity is a major abiotic stress limiting rice growth and grain productivity worldwide. Basic leucine zipper (bZIP) transcription factors serve as central regulators of plant environmental stress responses, yet the biological function and molecular regulatory mechanism of rice OsbZIP60 (LOC_Os07g44950) underlying salinity tolerance remain largely uncharacterized. In this study, we systematically characterized the salt-stress regulatory function of OsbZIP60 in rice. Tissue expression profiling revealed that OsbZIP60 was ubiquitously transcribed across all examined rice tissues, and its encoded protein predominantly localizes to the cell nucleus. Transcript abundance of OsbZIP60 was significantly induced by salt, the osmotic phase, abscisic acid (ABA), and oxidative stress signals. Phenotypic assays demonstrated that overexpression of OsbZIP60 substantially enhanced rice salt tolerance, whereas the bzip60 knockout mutant exhibited aggravated salt hypersensitivity. Physiological quantification revealed that OsbZIP60 promoted the accumulation of osmoprotectants, alleviated salt-triggered oxidative damage, and elevated the activities of core antioxidant enzymes under saline conditions. Moreover, OsbZIP60 maintained intracellular Na+/K+ homeostasis and positively modulated the transcript levels of a series of salt-responsive downstream genes. Collectively, our results demonstrate that OsbZIP60 acts as a positive regulatory hub that coordinates osmotic adjustment, antioxidant defense, and ion balance to confer salt tolerance in rice, providing a promising genetic target for molecular breeding of salt-tolerant rice varieties.

Salinity stress severely limits crop production, particularly in arid and semi‐arid regions. This study explored the potential of zinc oxide nanoparticles (ZnO‐NPs) as a priming agent to enhance castor (Ricinus communis) seedling growth under salt stress (100 mM NaCl), focusing on metabolic reprogramming and storage reserve mobilization. ZnO‐NPs priming significantly improved seedling vigor by enhancing lipid mobilization, reducing triacylglycerol (TAG) accumulation, and promoting fatty acid breakdown, especially in the cotyledons. This was accompanied by upregulation of key enzymes in the glyoxylate and tricarboxylic acid (TCA) cycles, facilitating the conversion of stored lipids into soluble sugars for energy. Furthermore, priming restored ATP levels, particularly in the radicle, thereby improving energy metabolism. ZnO‐NPs also mitigated oxidative stress, reducing lipid peroxidation and preserving membrane integrity. Transcriptomic analysis revealed upregulation of genes involved in lipid metabolism, stress responses, and energy conversion, supporting the biochemical changes observed. These findings highlight ZnO‐NPs as a promising strategy to enhance seedling establishment and crop resilience under salt stress, contributing to sustainable agricultural practices and improved salt tolerance in oilseed crops.

In nature, plants are subjected to an interplay of biotic and abiotic stresses that impede their growth and overall productivity. The study used the Medicago truncatula plant model to explore the relationship between salinity and Phoma medicaginis infection, focusing on its effect on plant defence mechanisms. Low sodium chloride (NaCl) concentrations (50–100 mM) enhanced growth and plant resistance to P. medicaginis infection, while high concentrations reduced growth and resistance, demonstrating a synergistic effect. The increase in total polyphenol was linked to an increase in NaCl concentration, regardless of the presence of the fungal pathogen in the plants. The study found that under salt stress, potassium levels in both infected and noninfected M. truncatula plants decreased, while sodium ions increased in infected leaves and roots. Salinity stress and P. medicaginis infection affected M. truncatula membrane fatty acid composition, increasing saturated fatty acids (C18:0 and C18:1) and decreasing unsaturated ones (C18:3), possibly as an adaptive response to modulate membrane fluidity. Furthermore, the study analysed gene expression involving allene oxide synthase 1, catalase, chalcone reductase, lipoxygenase 2 and phenylalanine ammonia‐lyase in M. truncatula, revealing their role in the early response to salt–P. medicaginis stress and suggesting a crosstalk between jasmonic acid and salicylic acid pathways in shaping M. truncatula's resilience to P. medicaginis infection under salt stress conditions.

Drought and salinity are two major factors limiting soybean productivity worldwide, and both stresses can negatively affect plant growth in similar manners by disrupting cellular redox homeostasis and metabolism. However, the regulatory networks coordinating transcriptional control and metabolic adaptation under both types of stress remain poorly understood. Here, we demonstrate that the R2R3-MYB transcription factors GmMYB60a and GmMYB60b serve as core negative regulators of drought and salt tolerance in soybean. Loss-of-function mutations in GmMYB60a/b clearly increased stress tolerance, which was associated with reduced reactive oxygen species accumulation and increased antioxidant capacity. The GmMYB60 proteins directly bind to and transcriptionally repress GmCYS20, a key gene involved in cysteine biosynthesis, thereby reducing cellular cysteine and glutathione pools. Notably, the GmCYS20 protein physically interacts with GmMYB60 in the nucleus and weakens its ability to bind DNA, resulting in the formation of a transcription-metabolic feedback loop that stabilizes redox homeostasis under stress. Furthermore, the stress-responsive NAC transcription factor GmNAC3 directly suppresses GmMYB60 expression, thereby linking environmental signals to metabolic regulation. Together, these findings reveal a hierarchical GmNAC3-GmMYB60-GmCYS20 regulatory module that coordinates cysteine homeostasis and oxidative stress responses by integrating transcriptional repression with metabolic feedback. This work provides mechanistic insights into soybean stress adaptation and identifies promising genetic targets for improving soybean resistance to drought and salinity.

Abstract Concurrent drought and heat waves impose a non-additive physiological constraint on soybean (Glycine max (L.) Merr.), creating a physiological trade-off between hydraulic preservation, which favors stomatal closure to prevent cavitation, and evaporative cooling, which requires sustained stomatal opening to mitigate thermal damage. This review synthesizes physiological and molecular evidence to develop a conceptual framework for understanding how arbuscular mycorrhizal fungi (AMF) may influence plant responses under combined stress. Available evidence indicates that improved plant water status is the most direct explanation for higher stomatal conductance in mycorrhizal plants under combined stress, which may in turn support transpirational cooling. Signaling responses may also contribute, but their roles have not been tested independently of differences in plant water status, particularly in soybean. Ultimately, integrating AMF-mediated physiological resilience into agricultural management may support strategies for maintaining crop performance under increasingly frequent climate extremes.

Salinity is one of the most harmful abiotic factors limiting crop productivity. Chitosan is a natural polysaccharide biostimulant able to alleviate salt stress, but its efficiency depends on its physicochemical properties, which remain insufficiently understood. The present study aimed to determine whether two chemically distinct forms of chitosan, i.e., high-molecular-weight chitosan (ChT) and chitosan oligosaccharide lactate (ChL), differentially mitigate the adverse effects of NaCl-induced salinity in cucumber (Cucumis sativus L.). Plants were grown hydroponically under control conditions or in the presence of 50 mmol L-1 NaCl, with or without medium-applied ChT or ChL (10 mg L-1). Salinity reduced growth and the accumulation of photosynthetic pigments, whereas chlorophyll fluorescence parameters remained largely unaffected, indicating limited impairment of PSII photochemistry. Chitosan, particularly the ChT form, partially alleviated NaCl-induced growth inhibition; ChT increased shoot and root fresh weight and leaf area under salt stress, whereas the effects of ChL were not statistically significant. Chitosan also enhanced free proline accumulation and was associated with changes in selected tricarboxylic acid (TCA) cycle intermediates, particularly malate and α-ketoglutarate, indicating an association with altered central carbon metabolism under salt stress. In contrast, neither chitosan form substantially improved the ionic status of cucumber, as excessive Na accumulation and reductions in K concentration and the K/Na ratio persisted despite treatment. These findings suggest that the beneficial effects of chitosan, particularly ChT, in salt-stressed cucumber were associated more closely with metabolic and osmotic responses than with restoration of ion homeostasis or direct protection of the photosynthetic apparatus, with the two chitosan forms showing different response patterns under the experimental conditions used.

Cytorrhysis describes the shrinkage of plant cells caused by water loss, during which the protoplast and cell wall contract while remaining connected. Cytorrhysis can occur naturally during drought and freezing and can be experimentally induced by exposure to osmotic substances. Although traditionally studied separately, the cytorrhysis process itself shares the same underlying mechanisms: water efflux along chemical potential gradients and the mechanical response of the cell wall-plasma membrane continuum. Cytorrhysis has been documented across diverse taxa - including algae, bryophytes, ferns and seed plants - indicating that it represents a fundamental response of walled cells to dehydration. Besides osmotic effects, the extent of cytorrhysis is also strongly affected by biomechanical cell structure. Cell size, wall thickness, elasticity and tissue architecture influence the likelihood of cellular collapse. Small, thick-walled cells withstand larger pressure differences and develop negative turgor pressure whereas large, thin-walled cells collapse more readily. During freezing, extracellular ice imposes temperature-dependent dehydration forces, and biomechanical constraints may generate negative turgor pressure. Recent advances, including cryo-microscopy, differential scanning calorimetry and psychrometric water-potential measurements, allow more precise quantification of freeze dehydration and structural responses. Functionally, cytorrhysis can be protective or damaging. Moderate, reversible cytorrhysis helps maintain membrane integrity e.g. in desiccation-tolerant species. In freezing environments, cells undergoing cytorrhysis survive temperatures far below those tolerated by supercooling cells. However, excessive dehydration may cause cell death. Integrating classical observations with modern biophysical approaches provides a renewed perspective on cytorrhysis and highlights structural traits that could be exploited to enhance plant resilience to drought and freezing stress in a changing climate.

The Ankyrin-repeat proteins (ANKs) play a key role in plant development and in response to abiotic stress. This research identified family members of the ANK genes in Sorghum bicolor at the whole-genome level, analyzed their sequence characteristics, evolutionary relationships, and expression patterns, and provided a scientific basis for elucidating the functionality of SbANK genes and for salt-tolerant breeding. Using bioinformatics methods, this study conducted a comprehensive identification of the SbANK gene family, analyzing its physicochemical properties, domain composition, chromosomal distribution, colinearity relationships, promoter cis-acting elements, and conserved protein motifs. Transcriptomic data and qRT-PCR were used to detect changes in their expression under salt stress. A total of 186 ANK family members were identified in the Sorghum bicolor genome, classified into 13 subfamilies and unevenly distributed across 10 chromosomes. Intra-species colinearity analysis revealed 7 pairs of duplicated genes, while inter-species colinearity analysis showed that S. bicolor and Oryza sativa share 88 pairs of orthologs, far exceeding the number found in Arabidopsis thaliana (11 pairs). Promoter analysis indicated that SbANK genes are enriched with cis-acting elements associated with hormone responses (particularly MeJA elements, accounting for 51.7%) and stress responses (particularly anaerobic-inducible elements, accounting for 60.9%). Transcriptomic expression analysis revealed that SbANK genes exhibit distinct tissue specificity, with the ANK-IQ subfamily highly expressed in leaves and the ANK-M subfamily showing the most widespread response under salt stress. Expression levels of the 10 candidate genes showing the most significant responses to salt stress were analyzed using qRT-PCR. The results indicated that SbANK91, SbANK135, and SbANK136 were significantly upregulated under 200 mmol/L NaCl treatment. The SbANK family is distinguished by a large number of member genes and structural diversity, with the ANK-M subfamily being the primary group responding to salt stress. SbANK91, SbANK135, and SbANK136 are identified as putative candidate genes for salt stress responses.

High soil salinity constrains global agricultural productivity, necessitating effective mitigation strategies. Plant growth-promoting bacteria (PGPB) offer a promising approach to enhance crop salt tolerance. This study evaluated halotolerant bacteria from O. dillenii to improve tomato (Solanum lycopersicum L.) growth under salinity. Bacteria from plant organs and rhizosphere were screened at 0-3 M NaCl; 52.38% grew above 1.5 M, and six strains-P. flexa S4, S6, R7, C5; B. amyloliquefaciens R4; and M. yunnanensis C11-tolerated up to 2.5 M. Under 120 mM NaCl, biopriming with P. flexa S6 and C5 increased germination to 75% and vigor index by 149.2%. Under 200 mM NaCl, P. flexa S6 boosted root biomass by 167.5%, C5 improved root length and diameter by 25.6% and 30.98%, and R7 enhanced shoot growth while reducing leaf proline by 78%. These selected strains effectively alleviate salt stress and promote tomato growth, promising an application in sustainable agricultural systems.

Mango (Mangifera indica L.), a tropical fruit crop with significant commercial value, is particularly vulnerable to salt stress. Salinity exposure causes severe physiological disruptions, including leaf burning, chlorosis, and stunted growth, especially in the early phases of plant development, and can be mitigated by salt tolerance. The current study evaluated the effectiveness of exogenous melatonin in mitigating salinity stress at 60 mM NaCl. The findings showed that applying melatonin at an optimal level of 150 μM improved photosynthetic efficiency, relative water content, and membrane stability index, thereby enhancing plant performance. Furthermore, melatonin treatment significantly reduced the build-up of malondialdehyde, a marker of membrane lipid peroxidation, while maintaining increased levels of phenol, proline, and total sugars. Increased activity of superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase enzymes, which together reduced oxidative stress, indicated a stronger antioxidant defense system, strongly linked to enhanced tolerance. Correlation analysis further revealed strong positive relationships among physiological and biochemical parameters, whereas malondialdehyde exhibited negative correlations with all other traits. Also, principal component analysis of six rootstock genotypes under salinity showed that melatonin enhanced stress tolerance in Kurukkan, K 106, and Olour by boosting antioxidant enzymes and preserving photosynthetic pigments. Overall, our findings demonstrate that melatonin increases mango rootstock resilience in saline environments by preserving cellular stability and regulating redox equilibrium.

Colonising Mars requires sustainable food production systems that use local resources. However, the Martian regolith presents significant challenges for plant cultivation. We investigated whether soybean ( Glycine max (L.) Merr.) could grow and form symbiotic nodules with Bradyrhizobium japonicum in MGS-1, a high-fidelity Martian regolith simulant. Soybean plants grown in an MGS-1:vermiculite mix (1:2) and supplemented with a nutrient solution were shorter, produced fewer leaves, and accumulated less biomass than sand-grown plants. More importantly, no nodulation occurred in MGS-1, despite successful nodulation in sand-grown controls. To identify factors inhibiting nodulation, we tested increasing concentrations of MgSO₄ (50–150 mM), a major salt in MGS-1, and osmotic stress (300 mM Mannitol). While both treatments reduced nodule numbers, nodulation persisted across all MgSO₄ concentrations and under osmotic stress, indicating that neither factor fully accounts for the absence of nodules in MGS-1. The shoot-to-root dry weight ratio increased under MgSO₄ stress but decreased under mannitol, suggesting distinct stress responses. Our findings demonstrate that soybean can survive in MGS-1 with nutrient supplementation and an additional substrate to improve MGS-1 compactness and porosity. Still, soybean grown in MGS-1 exhibits severe developmental constraints and complete inhibition of biological nitrogen fixation. Overcoming these limitations will require strategies to mitigate salinity, alkalinity, and physical compaction, as well as engineering symbiotic conditions compatible with sustainable agriculture on Mars.

Maintaining the cytosolic K+/Na+ homeostasis is crucial for plants' salt tolerance. Therefore, elucidating the regulatory mechanism governing K+/Na+ transport is pivotal to engineering salt-tolerant crops. Although many regulators have been shown specifically to modulate either Na+ or K+ transporters, little is known about integrative regulators that orchestrate the transport of both cations. In this study, we reveal that a rice basic helix-loop-helix (bHLH) transcription factor, BES1-INTERACTING MYC-LIKE1 (OsBIM1), negatively regulates K+/Na+ homeostasis under salt stress. Loss of OsBIM1 function conferred salt tolerance and K+/Na+ homeostasis in rice, whereas its overexpression produced the opposite effect. The K+ transporter OsHAK5 and transcription factor OsWRKY53 were identified as the targets of OsBIM1. By binding to E-box elements in their promoter regions, OsBIM1 not only repressed OsHAK5 expression and the root K+ uptake, but also trans-activated OsWRKY53 to suppress transcription of the Na+ transporter gene OsHKT1;5 and the Na+ unloading from xylem. OsBIM1 expression was attenuated by salt stress, and the loss of OsBIM1 function leads to synergistic upregulation of OsHAK5 and OsHKT1;5, thereby enhancing salt tolerance. As a positive regulator of brassinosteroid (BR) signaling, OsBIM1 was also found to be indispensable for BR-induced attenuation of salt tolerance and involved in BR-mediated regulation of OsHAK5 and OsWRKY53 expression. This study reveals an integrative regulator of K+/Na+ transport and a negative regulatory mechanism by which BR affects salt tolerance, providing a promising strategy for breeding salt-tolerant rice.

Bell peppers (Capsicum annuum L.) are of great importance and have high productive potential in Brazil, especially in the Northeast region; however, their production is limited due to salinity problems in water and soil. The objective of this study was to evaluate sources and methods of calcium application for mitigating salt stress in bell pepper plants grown in a substrate-based hydroponic system. The experiment was conducted using a split-plot design in a 2 × 2 × 3 layout with four replicates, consisting of two application methods (substrate and spray), two ECiw levels (0.5 and 5.0 dS m-1), and three calcium sources (no calcium-NCa; calcium nitrate-Ca(NO3)2; calcium complexed with amino acids-Ca-AA). Forty-two days after transplanting, gas exchange analysis, chlorophyll a, b, and total chlorophyll indices, chlorophyll fluorescence, fruit number and weight, and yield per plant were measured. Ca-AA showed higher photosystem II efficiency when applied via the substrate and higher stomatal conductance (28.10%), resulted in higher fruit yield compared to the other treatments, and contributed to a reduction in apical rot. Meanwhile, the application of Ca(NO3)2 improved electron transport in photosystem II by 5.46%, gas exchange, and increased chlorophyll b and total chlorophyll indices by 29.62% and 10.93%, respectively. It is concluded that the application of Ca sources, especially Ca-AA, has the potential to mitigate the effects of salt stress on bell pepper plants in a substrate-based hydroponic system, improving their photosynthetic and productive performance.

Understanding the physiological and biochemical responses of spinach to salt stress through amino acid supplementation is crucial for improving crop resilience under increasing soil salinity conditions. Salt stress represents one of the most severe abiotic constraints limiting vegetable crop productivity worldwide, yet comprehensive studies examining organ‐specific metabolic reprogramming across entire plant systems remain limited. However, knowledge about how different amino acids mediate these responses through distinct metabolic pathways is limited. We investigated mineral nutrition, antioxidant defense systems, and secondary metabolite profiles of spinach supplemented with three amino acids (asparagine, phenylalanine, and tryptophan) under salt stress conditions. Amino acid type, salt treatment, and organ significantly influenced all measured parameters (p ≤ 0.0001). Asparagine consistently demonstrated comprehensive protective effects, excluding toxic ions while promoting calcium uptake, maintaining photosynthetic capacity, and dramatically enhancing flavonol biosynthesis (quercetin and rutin accumulation increased several‐fold) under combined stress compared to other treatments. Phenylalanine excelled in ionic homeostasis restoration, achieving superior Na/K ratio reduction and enhanced phenylpropanoid pathway activation through elevated cinnamic acid biosynthesis. Tryptophan uniquely triggered exceptional divalent cation accumulation (6–10‐fold increases in magnesium, calcium, and phosphorus) and maximally enhanced antioxidant enzyme activities, though with notable protein synthesis trade‐offs. Organ‐specific accumulation patterns revealed leaves as primary sites for photosynthetic pigments and phenolic compounds, roots as storage organs for specialized flavonoids and catechins, and petioles showing exceptional rutin accumulation. These findings demonstrate that amino acid selection fundamentally reshapes metabolic priorities in salt‐stressed spinach through divergent yet complementary biochemical strategies. We conclude that amino acid selection significantly influences spinach resilience to salt stress through divergent metabolic reprogramming strategies, with each amino acid offering distinct advantages depending on cultivation priorities. Differential metabolic responses between amino acids provide insights for precision agriculture applications, while the quantitative biochemical patterns identified offer valuable parameters for optimizing amino acid supplementation strategies under saline conditions.

Soil salinization has become a major constraint on afforestation and forest regeneration in Northeast China, limiting the growth and development of Larix gmelinii. Enhancing the salt tolerance of L. gmelinii is therefore of considerable importance, and ectomycorrhizal fungi (ECMF) inoculation has been shown to improve plant stress resistance. However, the mechanisms by which ECMF enhance salt tolerance in L. gmelinii remain poorly understood. In this study, L. gmelinii seedlings inoculated with an ECMF consortium consisting of Boletus edulis and Chroogomphus rutilus were subjected to 120 mM NaCl stress. Photosynthetic characteristics, antioxidant enzyme activities, osmotic adjustment substances, ion contents, and metabolite profiles were analyzed to elucidate the physiological mechanisms underlying ECMF-mediated salt tolerance. The results showed that ECMF inoculation promoted the growth of L. gmelinii under salt stress. ECMF alleviated the decline in net photosynthetic rate caused by stomatal limitation and maintained photosynthetic performance. Under salt stress, L. gmelinii primarily scavenged reactive oxygen species through increased peroxidase activity, activation of ascorbate and aldarate metabolism, and enhanced flavonoid biosynthesis. Furthermore, ECMF inoculation enhanced galactose metabolism and amino acid biosynthesis, resulting in increased soluble sugar and soluble protein contents and improved osmotic adjustment capacity. In addition, ECMF improved ion homeostasis by promoting K⁺ translocation to shoots while restricting Na⁺ transport and enhancing Na⁺ retention in roots. These findings demonstrate that ECMF enhance salt tolerance in L. gmelinii through coordinated regulation of photosynthesis, antioxidant defense, osmotic adjustment, and ion homeostasis, providing a theoretical basis for the application of ECMF in saline-alkali land afforestation.

Extremophilic fungi are a relatively unexplored source of stress-resilient microbes for sustainable agriculture. Here, we describe Talaromyces pseudofuniculosus FAR, isolated from the highly acidic site of Piccolo Inferno in southern Italy and identified by ITS and β-tubulin (BenA) sequencing. Despite its origin from an extreme environment, T. pseudofuniculosus FRA remained fully functional under non-stress conditions and displayed tolerance to salinity and heavy metals while maintaining key plant growth-promoting traits, including phosphate solubilization, auxin-related compound production and hydrolytic activities. In soybean (Glycine max L.), the live fungus promoted plant growth under both non-stress conditions and agriculturally relevant salinity and copper stress, and successfully colonized the roots. A cell-free extracellular extract (exCFS) promoted early seedling development in vitro, but its effects were not sustained in pot experiments. GC-MS analysis provided a qualitative characterization of the extracellular metabolite profile and revealed condition-dependent differences between control, NaCl and CuSO4; growth conditions, indicating environment-dependent modulation of fungal metabolism. Overall, these findings highlight the physiological and metabolic plasticity of T. pseudofuniculosus FRA and support its potential as a fungal biostimulant for saline- and metal-impacted agricultural soils.

The homeodomain-leucine zipper IV (HD-Zip IV) transcription factor subfamily plays essential roles in epidermal development, cuticle formation, lipid metabolism, and environmental adaptation in plants. Despite its biological importance, the HD-Zip IV family has not been systematically characterized in apple (Malus domestica). Here, we identified 17 apple HD-Zip IV genes and named them MdHDZIV1-MdHDZIV17 based on their locations on the chromosomes. The 17 genes showed a nonuniform distribution on eight chromosomes, while the occurrence of both tandem and segmental duplications indicated that family expansion involved more than one duplication mechanism. All MdHDZIV proteins contained the conserved HD, LZ, START, and SAD domains but lacked the MEKHLA domain, consistent with typical HD-Zip IV structural features. Phylogenetic analysis classified MdHDZIV proteins into five groups together with HD-Zip IV members from Arabidopsis thaliana and rice, indicating evolutionary conservation of this subfamily. Collinearity and Ka/Ks analyses revealed that duplicated MdHDZIV gene pairs were mainly subjected to purifying selection. Promoter scanning revealed diverse cis-regulatory motifs associated with hormonal signaling, environmental stress, light response, and epidermal regulation, including ABRE, ARE, W-box, MYC, G-box, and L1-box motifs. Integration of transcriptomic profiling with qRT-PCR validation revealed pronounced tissue-dependent differences in the expression of MdHDZIV genes in leaf, fruit skin, and branch bark. Under PEG6000-induced osmotic stress and NaCl-induced salt stress, 10 candidate MdHDZIV genes displayed gene-specific and stress type-specific expression patterns, with MdHDZIV3 showing strong induction under PEG6000 treatment. Functional validation in apple calli showed that MdHDZIV3 overexpression enhanced PEG tolerance, increased fresh weight, elevated SOD and POD activities, and reduced MDA accumulation under osmotic stress. These findings provide a genome-wide framework for understanding the apple HD-Zip IV gene family.

Summary Statement This study reveals GmCCD4 coordinates branch development and stress responses through ABA metabolism and signalling in soybean, and natural variants of GmCCD4 provide promising targets for improving soybean architecture and resilience.

Salinity is a major constraint to pasture establishment and productivity, particularly in marginal soils. This study evaluated the effect of halophilic and halotolerant plant growth‐promoting bacteria (PGPB) on germination and early vegetative growth of Rhodes grass (Chloris gayana cv. Reclaimer) under saline stress. Seven bacterial strains, previously isolated from hypersaline environments, were tested during germination of caryopses and spikelets at 0, 100, and 300 mM NaCl. The three most effective strains (Halomonas sp. 3R12, Pseudomonas sp. AN23, and Pseudarthrobacter sp. ER25) were further evaluated in a hydroponic system at 0 and 300 mM NaCl for morphological, biochemical, ionic, and photosynthetic parameters. Halomonas sp. 3R12 enhanced germination across all salinity levels, while Pseudomonas sp. AN23 and Pseudarthrobacter sp. ER25 showed seed‐type‐dependent effects. Under salt stress, bacterial inoculation increased plant height and root dry weight, reduced leaf Na⁺ accumulation, and improved K⁺/Na⁺ and Ca²⁺/Na⁺ ratios, indicating restricted Na⁺ translocation. Halomonas sp. 3R12 also promoted higher antioxidant activity and proline content, while Pseudarthrobacter sp. ER25 and Pseudomonas sp. AN23 improved the photosynthetic performance. These bacteria maintained the photosynthetic efficiency (Fv/Fm, Phi2) in plants under stress, in contrast to non‐inoculated plants, which showed activation of regulated energy dissipation mechanisms and reduced electron flow. Overall, Halomonas sp. 3R12 and Pseudarthrobacter sp. ER25 were the most effective strains supporting their potential as bioinoculants for improving forage grass performance in salt‐affected soils.

Endo-β-1,4-mannanases (MANs) are glycoside hydrolase family 5 (GH5) enzymes that degrade cell wall mannan polysaccharides and participate in plant growth and stress adaptation. This gene family has not been systematically characterized in common wheat (Triticum aestivum L.). Here, we identified 24 TaMAN genes (TaMAN1-TaMAN24) genome-wide and analyzed their phylogeny, gene structures, chromosomal distribution, synteny, and promoter cis-acting elements. Expression profiles under biotic and abiotic stresses were investigated using public databases, salt-stress RNA-seq, and RT-qPCR. TaMAN proteins (386-475 aa) were mainly predicted to localize in the extracellular space. Phylogenetic analysis divided them into three groups, with Groups II and III representing monocot-specific expansions. Family expansion was driven primarily by whole-genome duplication, supplemented by tandem duplication on homoeologous group 6. Promoters were enriched in hormone- and stress-responsive cis-acting elements (ABRE, as-1/CGTCA-motif, W box). TaMAN1, TaMAN5, TaMAN8, TaMAN9, TaMAN16 and TaMAN19 were significantly induced by powdery mildew, while TaMAN3, TaMAN4 and TaMAN19-TaMAN22 rapidly responded to salt stress. This study provides candidate genes for disease-resistant and salt-tolerant wheat breeding.

Abstract Salt stress severely restricts citrus productivity in arid and semi-arid regions by disturbing ionic balance, water relations, and physiological functioning. The present study investigated salinity-induced changes in growth, ionic composition, physiological traits, and stress-responsive metabolites in four commercially important citrus cultivars, namely sweet orange (Blood Red), grapefruit (Star Ruby), kinnow mandarin, and lemon (Baramasi). Plants were exposed to five salinity levels ranging from 0.12 to 7.0 dS m−1 under controlled pot conditions using a completely randomized design with five replications. Increasing salinity progressively reduced plant growth, chlorophyll stability, biomass accumulation, tissue hydration, and leaf health, while membrane injury increased markedly under severe stress. Salinity also disrupted ionic homeostasis through enhanced accumulation of Na+ and SO42− ions coupled with depletion of Ca2+, Mg2+, and K+ in leaves and roots. Accumulation of anthocyanins and flavonoids increased under saline conditions, suggesting activation of stress-responsive biochemical protection mechanisms. Marked cultivar-specific differences were observed in salinity tolerance. Sweet orange and kinnow maintained lower Na+ accumulation, better K+/Na+ balance, higher chlorophyll stability, improved relative water content, and greater biomass retention under stress conditions. In contrast, grapefruit and lemon exhibited greater ionic imbalance, membrane injury, and physiological disruption. The findings indicate that maintenance of ionic homeostasis, chlorophyll stability, and adaptive biochemical modulation plays an important role in salinity tolerance in citrus cultivars. Among the cultivars evaluated, sweet orange and kinnow exhibited comparatively stronger adaptive resilience under progressive salinity stress.

Pyrophosphate serves as a vital reagent for the chemical remediation of soil heavy metal contamination. However, reports regarding how pyrophosphate regulates plant cadmium (Cd) tolerance at the physiological level remain scarce. In this study, we combined phenotypic, physiological, transcriptomic, and metabolomic approaches to comprehensively elucidate the response of Arabidopsis to Cd stress mediated by sodium pyrophosphate (Na-PPi). The results demonstrated that oxidative stress triggered by Cd significantly suppressed the growth of Arabidopsis seedlings. In contrast, exogenous Na-PPi effectively lowered the contents of MDA, H2O2, and O2•-, and concurrently diminished Cd accumulation in the plants under the Cd stress condition. Meanwhile, Na-PPi significantly altered the expression of genes associated with "phenylpropanoid biosynthesis" and "cell wall biosynthesis". Furthermore, Na-PPi application under Cd stress significantly enhanced the expression of the nitrate transporter genes NRT2.1 and NRT2.6 and promoted nitrogen accumulation in Arabidopsis plants. Metabolomic analysis revealed that Na-PPi significantly altered the metabolic profile of Arabidopsis under Cd stress, increasing the accumulation of polydatin while decreasing the levels of nicotinic acid, baicalin, camalexin, and glutathione. These findings provide a crucial scientific basis for a deeper understanding of pyrophosphate-mediated heavy metal soil remediation from a plant-level perspective.

Ubiquitination is a reversible post-translational modification that orchestrates a wide spectrum of fundamental processes throughout the plant life cycle. Executed by a hierarchical E1-E2-E3 cascades, this modification tags targets with ubiquitin to modulate their turnover, activity, or subcellular compartmentalization. Among the diverse E3 ligase families, plant U-box (PUB) proteins stand out as a prominent class that determines substrate selection and has emerged as a focal point of stress biology. In this review, we first delineate the structural features of PUB proteins, highlighting their conserved domains and associated regulatory motifs. We then systematically dissect their multifaceted functions in abiotic stress adaptation, encompassing drought, salinity, extreme temperatures, oxidative stress, heavy metal toxicity, with particular emphasis on their integration with ABA signaling networks. We further outline critical knowledge gaps and propose future strategies to decode the regulatory architecture of PUBs. Collectively, this review provides a theoretical foundation and new insights for facilitating the genetic improvement of crop resilience in the face of continuously intensifying environmental stresses through the manipulation of PUB-mediated ubiquitination networks.

Small heat shock proteins (sHSPs) act as molecular chaperones that protect other proteins from damage caused by stress‐induced denaturation. Bermudagrass (Cynodon dactylon L.) is a broadly adopted forage and turfgrass known for its capability to withstand various abiotic stresses. However, the biological pathways by which sHSPs promote drought tolerance in bermudagrass remain unclear. In this study, 99 sHSPs were characterized in the bermudagrass genome. Drought stress led to the induction of the majority of these genes with CdsHSP16.970 showing the most significant induction. Overexpression (OE) of CdsHSP16.970 promoted root elongation and improved seedling growth performance in transgenic Arabidopsis lines under osmotic stress, with reduced electrolyte leakage (EL) and lower malondialdehyde (MDA) deposition compared with the control. Meanwhile, several stress‐related genes were significantly induced in CdsHSP16.970‐OE plants when subjected to osmotic stress compared to the control group. Two basic leucine zipper transcription factors, CdbZIP04 and CdbZIP65, were also induced by drought stress in bermudagrass. Further investigation using electrophoretic mobility shift assay, yeast one‐hybrid and dual‐LUC assays revealed that they directly and specifically bind to the upstream regulatory region of CdsHSP16.970, consequently promoting its expression. In summary, our results suggest that the CdbZIPs–CdsHSP16.970 cascade positively regulates the osmotic stress signaling pathway in bermudagrass.

Background/Objectives: Phytocystatins are plant-specific inhibitors of papain-like cysteine proteinases that participate in plant development and responses to biotic and abiotic stresses. The roles of rice (Oryza sativa L.) phytocystatins under cold stress remain poorly understood. Xiaomagu (XMG), a cold-tolerant japonica rice landrace, provides valuable material for exploring phytocystatin transcript responses to low-temperature stress. This study characterizes 12 rice phytocystatin genes isolated from XMG and investigates their transcript profiles under cold and abscisic acid (ABA) treatments. Methods: Twelve phytocystatin genes were isolated from the cold-tolerant japonica rice landrace Xiaomagu. Sequence characterization and conserved domain analysis were performed for these phytocystatin family members. Real-time quantitative PCR was performed to examine transcript abundance of phytocystatin genes: low-temperature stress was applied to leaf and root tissues, while ABA treatment was carried out for leaf tissues only. Results: Conserved domain analysis revealed structural differences among the identified phytocystatin members. Real-time quantitative PCR detected distinct transcriptional responses to cold stress among these phytocystatin genes. In leaves, OsCST1, OsCST8, and OsCST12 were up-regulated, whereas OsCST11 was down-regulated. In roots, OsCST2, OsCST3, OsCST6, and OsCST12 showed increased transcript levels, while OsCST1 and OsCST11 were down-regulated. OsCST2, OsCST6, and OsCST11 exhibited significantly altered transcript abundance under ABA treatment. Conclusions: This study characterizes transcript-level responses of 12 phytocystatin genes from the cold-tolerant rice landrace Xiaomagu (XMG) under cold and ABA treatments. The detected expression changes reflect stress-responsive transcriptional regulation of rice phytocystatin family genes. Further in planta functional assays and comparisons with cold-sensitive genotypes are needed to elucidate their precise roles in rice cold adaptation.

Salinity stress is a major global constraint to agriculture, limiting plant growth and productivity. Exogenous application of ZnSO₄ offers a potential strategy to mitigate its detrimental effects owing to zinc's vital role in plant metabolism. A preliminary screening using varying concentrations of ZnSO₄·7H₂O (0.025%, 0.05%, 0.1%, 0.2%, and 0.5%) under 50 and 100 mM NaCl in Capsicum annuum L. identified 0.1% as the most effective dose, while 0.5% caused phytotoxicity. To validate these findings, a pot experiment was conducted under natural conditions to evaluate the physio‐chemical, ultrastructural, yield and metabolomic responses of Capsicum annuum L. to 0.1% and 0.5% ZnSO₄ under salinity stress. The results revealed that 0.1% ZnSO₄ significantly enhanced stomatal aperture and density, chlorophyll content, photosynthetic efficiency, and relative water content, leading to improved biomass and yield. Untargeted quantitative UHPLC–HRMS metabolomics showed upregulation of phenylpropanoids, amino acids, and fatty acid amides, indicating enhanced antioxidant defense and metabolic signaling for stress tolerance. Conversely, 0.5% ZnSO₄ disrupted cellular homeostasis, increased ROS accumulation and impaired energy metabolism, reflecting oxidative damage. Overall, these findings underscore how Capsicum annuum L. dynamically perceives and responds to salinity and exogenous ZnSO₄ via coordinating physio‐chemical performance and molecular signaling to achieve ecological resilience.

Salinity is a growing problem for cereal cultivation because it imposes multiple stresses, including osmotic, ionic, nutritional, and oxidative constraints, on the crop. Sorghum (Sorghum bicolor L. Moench) is considered a climate-smart C4 cereal for food, feed, fodder, forage, and bioenergy, but recent studies indicate that salinity continues to hinder establishment, biomass formation, reproductive growth, and yield. This review compiles the literature on the impacts of salinity on sorghum from 2021 to 2026, with a focus on germination, vegetative growth, physiological and biochemical responses, ion homeostasis, genetic control, productivity, mitigation, and future breeding priorities. In total, 160 records were identified, 118 records were screened after duplicate removal, and 44 recent sources were included in the synthesis. Across comparable sorghum studies, saline/NaCl treatments of approximately 60-200 mM commonly reduced germination by about 20-40%, root and shoot elongation by 25-50%, and biomass by 20-55%, while tolerant genotypes generally maintained higher K+/Na+ balance, 40-60% greater biomass retention, or two- to five-fold stronger ion homeostasis indicators than sensitive lines under similar conditions. Salt stress also lowers leaf expansion, chlorophyll stability, gas exchange, dry matter accumulation, panicle fertility, and grain filling. Tolerant genotypes show greater antioxidant potential, osmotic adjustment, photosynthetic stability, and root system resilience. Recent omics and genome-wide association studies suggest that salinity tolerance in sorghum is polygenic and involves genes related to ion transport, stress signalling, antioxidant regulation, osmolyte metabolism, and growth maintenance. This review recommends a shift from descriptive trait lists to full-cycle field validation, multi-trait selection indices, and integrated packages combining breeding with seed priming, soil water management, amendments, and beneficial microorganisms.

Soil salinization poses a significant threat to potato crops, particularly in arid and semi-arid regions. This study investigated the effects of foliar application of cerium oxide nanoparticles (CeO₂-NPs) at concentrations of 0, 10, 20, and 40 mg L⁻¹ on the growth, biochemical, and physiological responses of potato plants under both non-saline and saline conditions. Saline treatments included control, moderate salt stress (MSS) = 75 mM and severe salt stress (SSS) = 150 mM NaCl salinity. The results demonstrated that both MSS and SSS substantially affected the plant performance. However, application of CeO₂-NPs at the 40 mg L⁻¹ significantly enhanced growth, biomass, physiology, and photosynthetic traits of potato plants. The severity of saline stress was associated with increased generation of reactive oxygen species (ROS). However, treatment with 40 mg L⁻¹of CeO₂-NPs resulted in marked increase in soluble sugars (47.35%), and soluble protein (23.34%), alongside significant reduction in proline accumulation (22.13%), H₂O₂ contents (14.67%), O₂⁻ contents (32.12%), MDA contents (33.38%), and Na⁺ ions concentration (35.95%) relative to non-stressed control. Furthermore, this optimal NPs treatment enhanced the activity of key enzymes including sucrose enzyme activity (27.79%), glutamine synthetase (82.10%), sucrose phosphate synthase (33.26%), sucrose synthase activity (51.33%), nitrate reductase activity (48.20%), and starch branching enzyme (49.35%) as compared to control (0 mg L⁻¹). Principal component analysis (PCA) and heatmap analysis revealed significant interrelationships among measured traits, underscoring their collective role in determining the crop capacity to sustain growth under salt stress. In conclusion, foliar application of CeO₂-NPs at an optimal concentration holds an effective approach for improving the salinity tolerance in potato plants.

Spices contain diverse plant secondary metabolites with antimicrobial and antioxidant activities that can influence plant–microbe interactions. Among these metabolites, curcumin and piperine are major bioactive constituents of turmeric and black pepper, respectively. In this study, we demonstrate that curcumin and piperine enhance alcoholic fermentation in the budding yeast Saccharomyces cerevisiae . Curcumin significantly promoted alcoholic fermentation under high-glucose conditions (20–40% glucose), which impose substantial osmotic stress on yeast cells. Under these conditions, curcumin enhanced yeast growth, improved osmotic stress tolerance, and increased resistance to cell wall digestion, suggesting enhanced stress adaptation during fermentation. Furthermore, deletion of HOG1 , which encodes the central MAP kinase of the High-Osmolarity Glycerol (HOG) pathway responsible for the osmotic stress response, abolished the fermentation-enhancing effect of curcumin, indicating that Hog1 function is required for this effect. Consistent with this result, transcriptomic and RT- qPCR analyses revealed increased expression of stress adaptation-related genes, including known HOG pathway-responsive genes. In contrast, the expression of genes involved in amino acid catabolism was decreased at 12 h after the initiation of fermentation, while metabolomic analysis revealed increased intracellular levels of amino acids, including glutamate and asparagine, at 24 h. These coordinated transcriptional and metabolic changes are consistent with reduced amino acid catabolism and a shift toward a metabolic state favorable for sustained fermentation. Together, these findings suggest that curcumin enhances alcoholic fermentation under osmotic stress by promoting stress adaptation that supports yeast growth and sustained fermentation. Importance Plant secondary metabolites are widely recognized for their roles in plant defense, whereas their beneficial effects on microbial physiology and metabolism remain poorly understood. This study demonstrates that the spice-derived compounds curcumin and piperine enhance alcoholic fermentation in the budding yeast Saccharomyces cerevisiae . Curcumin, in particular, improved yeast growth and stress tolerance under high-glucose conditions that impose substantial osmotic stress. Its fermentation-enhancing effect required Hog1 function and was accompanied by increased expression of HOG pathway- responsive genes and changes in amino acid metabolism. These findings demonstrate that plant-derived compounds can not only exert antimicrobial effects but also beneficially modulate yeast stress adaptation and fermentation performance. The use of natural bioactive compounds to improve fermentation robustness under high-sugar conditions may contribute to increased productivity and reduced costs in fermented food and bioethanol production.

Salt stress is a major abiotic constraint that limits plant growth by inducing oxidative stress, disrupting photosynthetic processes, and damaging cellular membranes. Although Rosa rugosa is considered relatively tolerant to adverse environments, differences in antioxidant defense and physiological responses among its cultivars under sustained salt stress remain insufficiently understood. Comparative evaluation of these responses is important for identifying salt-tolerant germplasm and understanding stress-adaptation mechanisms. In this study, two R. rugosa cultivars, Siji and Fenghua, were subjected to irrigation treatments with solutions containing 0, 50, 100, or 150 mM NaCl for 28 days under controlled conditions. Morphological traits, salt injury, photosynthetic pigments, osmolytes, ROS accumulation, antioxidant enzyme activities, and lipid peroxidation were measured to assess cultivar-specific differences in salt-stress responses. Salt stress caused concentration-dependent growth inhibition, increased salt injury, and reduced chlorophyll content, while oxidative-stress responses differed between the two cultivars, with stronger adverse effects observed in Fenghua. Siji maintained better growth performance, higher fresh and dry mass of sampled leaves, greater chlorophyll retention, better maintenance of several osmolytes, and stronger antioxidant enzyme activities under the higher NaCl treatments. In contrast, Fenghua showed greater ROS accumulation and higher MDA under the higher NaCl treatments, which are consistent with greater oxidative stress and membrane lipid peroxidation. Exploratory correlation and principal component analyses further showed that antioxidant defense, osmotic adjustment, chlorophyll retention, and reduced oxidative damage were closely associated with salt tolerance in Siji. These results indicate that greater antioxidant capacity, osmotic adjustment, and membrane protection were associated with the superior salt-stress performance of 'Siji' compared with 'Fenghua'. The study identifies Siji as a promising R. rugosa candidate for further salt-tolerance evaluation and provides useful physiological and biochemical indicators for salt-tolerance screening in ornamental rose breeding.

Seed germination of species in arid zones is shaped by climatic extremes, which include high temperatures, prolonged drought and episodic rainfall. Halophytic plants, such as samphires, face additional pressure from fluctuating salinity. It is anticipated that current physiological thresholds will be surpassed for many species because of climate change, resulting in disrupted plant recruitment patterns as seed germination windows narrow or shift. Our study aimed to determine the timing of germination opportunities for the threatened samphire Tecticornia lylei and understand the effects that climate change may have on future recruitment. We assessed the germination responses of seeds to light, temperature, water and salinity stress. The comparative effects of salinity and water stress under increasing temperatures were also observed, as were responses to freshwater exposure characteristic of episodic rainfall. We found seeds removed from fruits commenced germination more rapidly, but other treatments assessed had minimal effect on final germination percentages. Tecticornia lylei germinated optimally in cooler conditions under constant darkness, but also had capacity for strong germination in fluctuating light/dark photoperiods. Low numbers were observed to germinate at higher temperatures, and under water stress which caused greater delays than salinity stress, with increasing temperatures exacerbating these effects. However, ungerminated seeds responded rapidly to freshwater treatment, even after exposure to stress levels that surpassed thresholds for germination, this being suggestive of the action of a priming effect. While Tecticornia lylei has a broad thermal window for germination in very low stress environments, it narrows substantially as osmotic stress increases. High rainfall events during cooler seasons are therefore critical to reduce salinity and increase soil moisture for sufficient time for seeds to germinate in high numbers.

Salt stress is a key abiotic limiting factor restricting agricultural productivity, making the screening and breeding of salt-tolerant plants essential for alleviating soil salinization. Ulmus pumila, a native Chinese tree with fast growth, good wood properties and high tolerance to salt stress and drought, was investigated to reveal its salt adaptation and underlying mechanisms. This study applied phenotypic identification, physiological determination and high-throughput sequencing to analyze growth traits and rhizosphere microbial communities under low, moderate and high salt stress. Our findings revealed that salt stress significantly inhibited seedling height, ground diameter, biomass and other growth indexes in a salinity-dependent manner. Distinct mineral element allocation existed between stems and leaves, with leaves as the main accumulation organ, and U. pumila adapted to salt stress by regulating element distribution and maintaining ion homeostasis. Bacterial communities were more sensitive to salt stress than fungi: Proteobacteria increased while Actinobacteria decreased with elevated salinity, and Ascomycota-dominated fungal communities remained structurally stable. Salt stress reshaped microbial co-occurrence networks, with bacteria adapting via species turnover and fungi maintaining function through core taxa. Ulmus pumila resists salt stress via integrated strategies of growth regulation, physiological adjustment and rhizosphere microbial synergy, providing a scientific basis for saline-soil vegetation restoration, afforestation and soil sustainable improvement.

Reducing cadmium (Cd) accumulation in plants by disrupting transporter genes involved in Cd uptake or transport often compromises normal plant growth due to the co‑transport of essential elements. Therefore, identifying genes that specifically reduce Cd accumulation without impairing micronutrient uptake remains challenging. Here, we identified and functionally characterized two duplicated Moso bamboo genes, PeNRAMP5-1 and PeNRAMP5-2. Both genes are predominantly expressed in roots, downregulated by Cd, and encode plasma membrane‑localized transporters. Immunostaining revealed non polar distribution of PeNRAMP5 proteins in all root cells. Yeast assays demonstrated their ability to transport both Cd and Mn. Expression them in rice mutant osnramp5 increased root Cd uptake but reduced Cd accumulation in shoots and grains. In contrast, root Mn uptake and accumulation in shoots and grains were largely restored. When expressed in the mutant atnramp1 or wild‑type Arabidopsis, these genes markedly enhanced root Mn uptake and its accumulation in shoots. Conversely, although Cd accumulation in roots increased, shoots Cd accumulation was strongly inhibited. These results suggest that PeNRAMP5 can limit Cd accumulation in shoots while restoring Mn uptake in plants. This work identifies PeNRAMP5 as a promising genetic resource for inter‑species application to minimize Cd accumulation and maintain Mn nutrition in plants.

Background: Domain of Unknown Function 1645 (DUF1645) is a conserved but poorly characterized plant gene family whose evolutionary history and roles in stress adaptation remain unclear. We performed an integrated genomic, evolutionary, transcriptomic, and Quantitative Trait Locus (QTL) characterization of the DUF1645 family in rice (Oryza sativa). Methods and Results: We identified 14 intronless, non-redundant OsDUF1645 genes distributed across eight chromosomes. Phylogenetic and collinearity analyses suggested that family expansion within Poaceae involved ancestral segmental and localized tandem duplication events. Promoter analysis identified stress- and phytohormone-responsive cis-acting elements, including ABRE, MBS, and MeJA-associated motifs. Public transcriptome datasets revealed diverse OsDUF1645 expression patterns under abiotic and hormonal treatments. Integration with the Quantitative Trait Loci Annotation Rice Online (Q-TARO) QTLome identified physical co-localization of multiple OsDUF1645 loci with stress- and agronomic-trait QTLs, including salinity-, drought-, root architecture-, and water-deficit-associated regions. On Chromosome 1, OsDUF1645.1, OsDUF1645.2, OsDUF1645.3, and OsDUF1645.4 overlapped QTL intervals associated with salinity-related physiological traits, including Na+ uptake and Na+ balance, and drought-related root traits. On Chromosome 5, the tandemly arranged OsDUF1645.8, OsDUF1645.9, and OsDUF1645.10 co-localized with QTLs related to root architecture and water-deficit responses. qRT-PCR validation under salinity, osmotic stress, and cadmium exposure confirmed distinct stress-responsive expression profiles; OsDUF1645.6 exhibited broad multi-stress responsiveness, whereas OsDUF1645.3 was downregulated under several conditions. Conclusions: The OsDUF1645 family exhibits substantial functional diversification, supported by distinct regulatory architectures, expression profiles, and QTL associations. These findings provide a framework for prioritizing OsDUF1645 candidates for functional validation and their potential application in molecular breeding and development of climate-resilient rice cultivars.

The expansin (EX) gene family plays a crucial role in the growth and development of various plants, as well as responses to biotic and abiotic stresses. However, genome-wide analysis of the EX gene family and their functions in drought and salt stress tolerance has not been examined in Rosa rugosa. In this study, a total of 30 RrEX genes were identified and located on seven different chromosomes. Phylogenetic analysis classified these genes into four subfamilies: EXPA (24 members), EXPB (3 members), EXLA (1 member), and EXLB (2 members). The average amino acid length was 269.17 aa, with isoelectric points ranging from 4.79 to 9.97. Most members exhibited high aliphatic indices and protein stability, suggesting their adaptability to diverse environments. The synteny analysis provided insights into the evolution of the EX gene family in rose. Toxicity and autoactivation assays confirmed that BD-RrEXPA1 was non-toxic to yeast cells and lacked autoactivation activity, indicating its suitability for yeast two-hybrid screening. The transgenic Arabidopsis lines overexpressing RrEXPA1 improved seed germination and root length under abiotic stress. In addition, the overexpression lines showed reduced malondialdehyde (MDA) levels and increased chlorophyll content and superoxide dismutase (SOD) activity. These results suggest that RrEXPA1 may enhance stress tolerance by promoting root elongation and modulating physiological responses. This study provides important insights into the role of RrEXs in salt and drought stress and lays the foundation for further studies on the regulatory mechanisms of abiotic stress.

The Na ⁺ /H⁺ antiporter (NHX) gene family plays a central role in maintaining cellular ion homeostasis and responding to abiotic stress in plants. However, the systematic identification and functional characterization of this gene family in woody plants of the genus Forsythia remain unexplored. To address this, genome-wide identification and systematic comparative analysis of the NHX gene family were performed in three Forsythia species, Forsythia suspensa, F. viridissima, and F. ovata, with a focus on elucidating F. suspensa NHX gene expression under various types of stress: salt, drought (PEG-simulated), and exogenous abscisic acid (ABA). In total, 18 NHX members were identified and were classified into three subclasses based on phylogenetic analysis, resulting in 12 vacuolar membrane-localized 'Vac'-class members, four plasma-membrane-localized 'PM'-class members, and two endosomal system-localized 'Endo'-class members. Those proteins within the same evolutionary clade exhibit highly conserved structural features in terms of motif arrangement and gene structure. Synteny analysis identified whole-genome or segmental duplication as the primary driving force for the expansion of this family, with all of the duplicated gene pairs having undergone strong purifying selection. Based on interspecies synteny analysis, F. suspensa and F. viridissima exhibited the highest degree of NHX gene family homology. Expression analysis revealed that the F. suspensa NHX gene family exhibits distinct and tissue-specific responses to drought, salt, and exogenous ABA stress. Among the NHX members, FsNHX3 and FsNHX6 exhibited the most pronounced and sustained transcriptional upregulation across the three stress treatments, suggesting their potential involvement in stress response. These findings provide valuable insights for the further exploration of the functions and molecular mechanisms of the NHX gene family in Forsythia under salt and drought stress, offering valuable candidate genetic resources for breeding Forsythia varieties with enhanced tolerance to salinity and dehydration.

The sea cucumber Apostichopus japonicus holds significant economic importance in China's marine aquaculture. However, climate change-particularly extreme summer temperatures and hypoxia-has caused substantial economic losses in major production regions. These stressors impair the physiological, immunological, and survival ability of A. japonicus, with compounded high temperature and low oxygen stress exerting particularly severe effects. Using enzyme activity assays, RNA-seq, and ATAC-seq, we investigated how elevated temperature and hypoxia affect immune, antioxidant, and metabolic responses in A. japonicus (mean body weight 106 ± 15.3 g) under three conditions: optimal growth, low oxygen, and combined high temperature with low oxygen. Integrative analysis of transcriptomic and epigenomic data identified key co-expression modules and pathways associated with stress responses. Notably, the NF-κB signaling pathway emerged as a central regulator of immune and inflammatory responses under combined stress. Furthermore, genes involved in immune function (Orct2, bmp2), epigenetic regulation (DNMT), and protein homeostasis (ST8SIA1) exhibited coordinated changes in both chromatin accessibility and expression, revealing that chromatin remodeling underpins stress-induced transcriptional reprogramming. These findings demonstrate that A. japonicus responds to escalating environmental stress through hierarchical layers of signal transduction, epigenetic remodeling, and physiological output, with important implications for climate-adaptive aquaculture management.

Salt stress is a major environmental factor limiting crop growth and productivity. We found a long-chain acyl-CoA synthetase (LACS) gene ZmLACS9 that was associated with salt tolerance traits in maize (Zea mays). Mutation of ZmLACS9 impaired plant growth, while overexpression of ZmLACS9 improved plant growth vigour under salt stress conditions. Metabolome and transcriptome analyses indicated that ZmLACS9 may regulate the expression of lipid trafficking-related genes, thereby influencing the concentrations of galactolipids and phospholipid in the photosynthetic membrane under salt stress. Under salt stress conditions, ultrastructural observation and chlorophyll fluorescence parameters showed that the mutation of ZmLACS9 led to significant impairment of the number of thylakoid layer structures and PSII activity, while overexpression of ZmLACS9 markedly improved the number of chloroplast thylakoid grana lamella and PSII activity. Furthermore, yeast one-hybrid and LUC transient expression assays found that ZmWRKY17 could bind to the promoter of ZmLACS9. Collectively, our study provides candidate genes for breeding maize varieties with higher stress resistance.

ABSTRACT Global warming is increasing the frequency and intensity of high-temperature episodes, limiting plant productivity. However, the molecular mechanisms integrating primary metabolism with the heat stress response remains poorly understood. Here, we show that lysine biosynthesis contributes to the coordination of physiological, metabolic and transcriptional responses to heat stress in Arabidopsis thaliana . We compared wild-type, the lysine-biosynthesis mutant dapat , and the salicylic acid (SA)-biosynthesis and signaling mutants sid2-1 and npr1-3 under prolonged warming (6□°C above control for 7 days) and heat shock (38 °C for 6 h), followed by recovery. We assessed growth, gas exchange, photosynthetic performance, free SA, salicylic acid glucoside (SAG), salicylic acid glucose ester (SGE), and total SA content, primary metabolite profiles, heat-stress-responsive gene expression and transcriptome-wide changes by RNA sequencing. Before heat stress, dapat mutant presented a distinct metabolic state, marked by amino-acid accumulation, altered organic-acid profiles, reduced soluble sugars and elevated endogenous SA. This metabolic configuration persisted during prolonged warming, whereas WT and SA-pathway mutants underwent more dynamic reprogramming. Heat shock, by contrast, elicited a more convergent response across genotypes. Despite reduced basal PSII efficiency, dapat maintained photosynthetic performance during prolonged warming and recovered. Its transcriptional response, however, differed from that of WT and SA-pathway mutants: selected heat-responsive genes were constitutively or more strongly expressed, whereas some canonical heat- stress regulators showed weaker induction after heat shock. RNA-seq further revealed a largely conserved core heat-shock response but genotype-dependent regulation of defense, hormone and amino-acid-metabolism programs, particularly during recovery. Together, these findings indicate that impaired DAPAT activity establishes a metabolically primed but energetically constrained state that reshapes gas exchange, photosynthetic acclimation and heat-responsive transcription. Lysine homeostasis therefore emerges as a regulatory node linking primary metabolism and SA accumulation with SA-dependent and SA-independent components of heat-stress acclimation.

Increasing soil salinization in the Songnen Plain region, Northeast China, has hindered common bean (Phaseolus vulgaris L.) cultivation. The specific regulatory mechanisms of sugar metabolism in common bean leaves under alkaline salt stress remain unclear. To address this research gap, in China's Songnen Plain, we subjected the high‐yielding and multi‐resistant granular bean variety “Qingyun 1” seedlings to stress treatments at 0, 24, and 48 h time points. Alkaline salt stress was simulated by applying 100 mM NaHCO₃ and Na₂CO₃ mixed at a 9:1 M ratio. With prolonged treatment time, common bean leaves gradually wilted and yellowed. Stomatal conductance, total chlorophyll, and carotenoids significantly decreased by 16.45%–75.77%. Superoxide dismutase, peroxidase, and catalase activities and malondialdehyde and H₂O₂ contents significantly increased by 42.04%–91.36%, all reaching extremely significant levels (p < 0.01). Under progressive photosynthesis inhibition, the dynamic regulatory network of sugar metabolism helped sustain common bean leaf responses to alkaline salt stress. Integration of the dynamic changes in the activities of hexokinase (HK), α‐galactosidase (galA), and 6‐phosphofructokinase with the levels of sugars resulted in persistently suppressed HK phosphorylation, enhancing galA activity accelerated stachyose and raffinose metabolism and Suc and Fru release promotion in the leaves under alkaline salt stress. This ensured sustained high SS concentrations, ultimately providing plants with additional energy and carbon sources. Thus, the key genes encoding galA could be important candidates for exploring alkaline salt stress response mechanisms in common bean. These findings provide insights and theoretical support for breeding new alkaline salt‐tolerant common bean varieties.

Flowering time is a critical developmental process that directly influences reproductive success, environmental adaptation, and agricultural productivity in Brassica crops. Recent climate instability, including drought, temperature fluctuations, salinity, and irregular photoperiods, has intensified the need to understand the molecular mechanisms regulating flowering adaptation. WRKY transcription factors are key integrators of flowering time. They connect environmental cues, hormone networks, and circadian rhythms to floral regulator genes. This review summarizes the structural, evolutionary, and functional characteristics of WRKY transcription factors involved in flowering regulation in Brassica species. Current evidence indicates that WRKY transcription factors contribute to flowering-time regulation through direct or indirect modulation of floral regulators, including FT, SOC1, and LFY. However, much of the mechanistic evidence originates from Arabidopsis, and the direct regulatory relationships between WRKY proteins and major flowering genes, particularly FLC, remain insufficiently characterized in Brassica species. Transcriptomics, epigenomics, proteomics, single-cell sequencing, and CRISPR/Cas genome editing further reveal that WRKY proteins participate in regulatory networks associated with photoperiodic signalling, vernalization, gibberellin pathways, stress-responsive flowering, and hormonal crosstalk. Comparative genomics demonstrates substantial expansion of WRKY gene families through genome triplication and polyploidization. However, limited functional validation and incomplete regulatory network mapping remain major challenges. Integrating multi-omics, artificial intelligence-assisted systems biology, genomic selection, and precision genome editing will accelerate development of climate-resilient, early-maturing Brassica cultivars.

Mulberry (Morus alba L.) is an important economic tree species, and its leaves are increasingly being used to prepare healthy dishes in addition to serving as food for silkworms. Drought is one of the main stress factors affecting the quality of mulberry leaves; however, the molecular mechanisms through which mulberry plants respond to drought stress are currently unclear. The AP2/ERF gene family is involved in plant growth, development and stress response processes. In response to drought stress, the expression of mulberry MnERF23 is induced. The overexpression of MnERF23 in Arabidopsis thaliana enhances drought tolerance, increases root length, and activates the expression of stress-related genes in transgenic plants. The transient overexpression of MnERF23 in mulberry leaves can reduce stress-related damage in transgenic seedlings and promote better growth. In this study, RNA-seq analysis of mulberry leaves transiently overexpressing MnERF23 in both normal and drought environments revealed 470 and 687 DEGs, respectively. In the DS-1305 vs. DS-ERF23 comparison, genes related to ROS clearance, cell wall precursors, and lignin synthesis were up-regulated. Further analysis revealed that Mn4CL3 is an up-regulated differentially expressed gene and that the promoter retains a DRE/CRT cis-acting element. The Y1H and dual-luciferase assay results reveal that MnERF23 can positively regulate Mn4CL3 in combination with DRE/CRT, synergistically enhancing the drought tolerance of mulberry. We elucidated the regulatory mechanism through which MnERF23-dependent DRE/CRT cis-acting elements promote Mn4CL3 expression and enhance drought tolerance in mulberry. This study contributes to the theoretical research on the regulatory network of plant abiotic stress, providing a theoretical basis and technical support for forest genetic breeding.

Soil salinization imposes severe ionic and osmotic stress on plants, threatening ecosystem sustainability. Biochar (BC) and arbuscular mycorrhizal fungi (AMF) have shown potential to alleviate salinity stress, but their interactive effects on the soil–plant‐microbe continuum have not been fully elucidated. This study utilized partial least squares path modeling (PLS‐PM) to assess the individual and combined contributions of BC and AMF to sodium (Na⁺) homeostasis in Suaeda salsa. Results indicate that BC primarily acted as a soil conditioner, reducing Na⁺ bioavailability by 49.19% (compared to the unamended saline control) (as indicated by the soil Na⁺/K⁺ ratio) through enhanced adsorption and cation exchange, thereby contributing to improved soil health (including an 87.23% increase in SOC). In contrast, AMF elicited a plant physiological response, characterized by the upregulation of antioxidant enzymes (such as a 114.85% increase in catalase) and osmotic regulators (e.g., a 90.22% increase in soluble sugars), which collectively mitigated oxidative stress and promoted vacuolar sequestration of Na⁺. A strong synergistic effect (E > 0) was observed. The PLS‐PM model quantitatively delineated the causal pathways, revealing that BC and AMF function through complementary soil–soil solution‐root‐leaf‐vacuole transport routes. This study reveals the mechanism underlying the interaction between biochar and AMF, while quantifying the pathways through which they synchronously regulate the rhizosphere chemical environment and internal detoxification in plants, providing new insights into how halophytes cope with environmental stress challenges.

The level of salt tolerance of rice varies with the developmental stage. The information on the tolerance of rice at each stage is valuable for breeding and planning in cultural management to obtain the optimal growth and yield in salt-affected areas. The objectives of this study were to compare the salt tolerance and physiological- and yield-related responses at three growth stages of the salt-sensitive Thai jasmine rice, KDML105, with its genetically improved variety RD73 (a registered commercial variety) and TSKC1-144 (a breeding line), both containing Pokkali-derived salt-tolerant QTL. The young hydroponically grown seedlings of TSKC1-144 treated with 150 mM NaCl were highly tolerant, while KDML105 was highly sensitive and RD73 was moderately tolerant. At the vegetative stage, KDML105 exhibited more growth and leaf physiological damage, showing the highest percentage reductions in the net photosynthesis rate (Pn), leaf relative water content (RWC), and shoot and total plant dry weight, but the highest increase in leaf electrolyte leakage (EL) and the highest leaf Na+/K+ ratio. During the reproductive phase, salt stress did not significantly induce physiological damage to the flag leaves, except for Pn, which was significantly reduced, particularly for KDML105. Both RD73 and TSKC1-144 exhibited lower biomass reductions and higher yields and yield components than KDML105. Compared with TSKC1-144, RD73 produced a lower grain number panicle-1, grain weight panicle-1, and lower 100-grain weight but 34% more panicles; therefore, it yielded a higher grain weight plant-1 (27.57 cf. 20.93 g). The most prominent trait that conferred a greater salt tolerance to RD73 and TSKC1-144 compared with KDML105 was the more efficient Na+ exclusion. Taking their tolerance at all growth stages into consideration, RD73 and TSKC1-144 are deemed suitable for growing under rain-fed conditions where the intensity of the soil salinity fluctuates throughout the growing season.

DExD/H-box RNA helicases constitute one of the largest families of adenosine triphosphate (ATP)-dependent nucleic-acid enzymes, with roles spanning the full breadth of RNA metabolism, from transcription to turnover. Their defining architectural feature is a conserved helicase core comprising two tandem RecA-like domains that couples ATP hydrolysis to duplex unwinding and ribonucleoprotein remodeling. Structural investigations have historically focused on human and yeast model systems, but recent methodological advances, particularly cryo-electron microscopy and deep learning-based structure prediction, have broadened the structural coverage of the DDX family, including full-length constructs and their incorporation into multiprotein assemblies. In plants, DDX helicases remain far less characterized despite their essential functions in development and stress adaptation. This review integrates structural biology insights with recent multi-omics datasets from cold-stressed rubber tree (Hevea brasiliensis), a tropical crop highly susceptible to chilling injury. Quantitative profiling has identified nine differentially abundant DDX proteins under cold stress, among which DDX39B shows the most pronounced response. Rubber tree DDX39B shares >95% sequence identity with orthologs from other tropical species and ~73% identity with human DDX39B; AlphaFold2 predictions reveal near-identical core helicase folds, consistent with strong functional constraint across vast evolutionary distances. These predictions also reveal plant-specific domain arrangements, including a putative DDX39B isoform bearing additional LysM and protein kinase domains. This work illustrates how computational structure prediction can bridge the sequence-to-function gap for stress-responsive proteins in non-model plant species and provides a conceptual framework for dissecting DDX helicase roles in plant abiotic stress tolerance.

Abstract Drought during germination and early seedling development can severely constrain wheat establishment, highlighting the need for seed-based strategies that enhance physiological resilience to water limitation. This study investigated whether zinc oxide nanoparticle priming (NZp) improves the physiological and growth responses of wheat ( Triticum aestivum L. cv. Jom) exposed to polyethylene glycol (PEG)-induced osmotic stress. Seeds were hydroprimed (Hp) or primed with ZnO nanoparticles (NZp) or zinc sulfate (IZp) at 50 mg L⁻¹ and subsequently germinated under 0, − 0.3, and − 0.6 MPa osmotic potentials. Drought stress impaired seedling performance, with radicle fresh weight and plumule length decreasing by 15.3% and 37.8% and 26.4% and 60.4%, at − 0.3 and − 0.6 MPa, respectively, relative to the non-stressed condition. Under non-stress conditions, NZp increased relative water content, germination, plumule length, radicle fresh weight, plumule fresh weight, and seedling vigor index by 13.6%, 5.26%, 15.0%, 18.0%, 15.0%, and 16.4%, respectively, compared with Hp. Under moderate drought (− 0.3 MPa), NZp increased soluble protein and radicle dry weight by 10.8% and 55.4% relative to Hp. Under severe drought (− 0.6 MPa), NZp reduced malondialdehyde levels by 20.9% and 36.8% compared with Hp and IZp, respectively. Overall, ZnO nanopriming strengthened early wheat drought resilience by coordinating water-status maintenance, membrane protection, and seedling growth, with its benefits being particularly evident under non-stress and moderate drought conditions.

Rice cultivation in multi-metal contaminated agricultural soils faces severe agronomic and food-safety challenges from co-occurring arsenic (As) and cadmium (Cd), which are increasingly exacerbated by climate change-driven abiotic extremes and episodic drought stress. Conventional single-strain bioinoculants typically struggle to resolve the opposing biogeochemical dynamics of As and Cd within the rice rhizosphere. Here, we present an evidence-integrated framework to prioritize candidate bacterial taxa for designing a climate-resilient Synthetic Microbial Community (SynCom). Leveraging public bioinformatics databases and targeted literature evidence synthesis, functional trait heuristics were constructed to evaluate candidate taxa across complementary capabilities: database-annotated As reduction (arsC) and efflux (acr3), Cd biosorption and efflux (cadA, czcA), extracellular polymeric substance (EPS) synthesis, ACC deaminase activity (acdS), and volatile organic compound (VOC) signaling. Bipartite network analysis revealed a structured architecture (connectance = 0.4048) with high distributed redundancy for EPS and VOC traits, while identifying single points of failure in enzymatic As(V) reduction (candidate P. putida), As(III) efflux (B. subtilis), and ACC deaminase (R. leguminosarum). Directional geochemical evaluation indicates that drought-induced root exudates exert opposing effects on As and Cd mobility—citrate enhances soluble Cd chelation while competitively mobilizing As from iron oxyhydroxides, whereas oxalate supports Cd immobilization via precipitation. The proposed SynCom provides a computationally prioritized foundation for rhizosphere microbiome engineering under dual-metal and drought stress. Rigorous in vitro, hydroponic, and greenhouse validations remain essential to confirm these modeled interactions.

Key messageAlternative splicing creates an SR45.1-specifi c phosphoregulatory region in which threonine 218 (T218) contributesto nuclear speckle organization, RNA regulatory responses, and salt tolerance in Arabidopsis. Alternative splicing expands protein and functional diversity in plants, yet the mechanistic basis by which closely related splice isoforms acquire distinct biological functions remains poorly understood. In our previous work, we showed that AS of the Arabidopsis splicing factor SR45 generates two isoforms with contrasting roles in salt stress response, where SR45.1, but not SR45.2, restores salt tolerance in the sr45 mutant background. The molecular basis underlying this isoform-specific functional divergence remained unclear. Here, we investigated the role of two conserved phosphosites, threonine 218 (T218) and serine 219 (S219), within the unique C-terminal region of SR45.1, a sequence absent from SR45.2. Site-directed mutagenesis was used to generate phospho-disruptive SR45.1 variants, which were then expressed in the sr45 mutant background. Functional analyses revealed that substitution of T218, but not S219, abolished the salt-tolerance function of SR45.1. Mechanistically, disruption of T218 altered SR45.1 nuclear speckle organization, resulting in fewer enlarged nuclear speckles compared with the numerous small speckles observed in functional lines. T218 disruption was further associated with altered AS of stress-related targets, including SOS4 and RD20, as well as reduced transcript accumulation of salt-responsive genes, including RD29A, RD29B, ADH1, and DREB2A. Together, our findings identify T218 phosphoregulation as a critical determinant of SR45.1 function, linking AS-generated isoform diversity to post-translational regulation, nuclear organization, and plant salt stress responses.