Osmotic stress reduces turgor pressure, relaxes the extracellular matrix, and may generate tensile forces at the cell wall-plasma membrane interface. While animal and yeast cells employ force sensor clusters for mechanosensing, turgor-sensing mechanisms in plants remain unclear. Here, we identify FERONIA (FER) as a cell wall-anchored turgor sensor in land plants. Turgor reduction triggers the assembly of FER into largely immobile nanodomains and activates its kinase activity, processes that depend on cell wall anchoring mediated by its malectin A domain. The extracellular domain is essential and sufficient for FER nanodomain formation, thereby initiating outside-in signaling. Together, our findings suggest that FER senses mechanical perturbations at the cell wall-plasma membrane interface during turgor reduction and provide mechanistic insight into osmosensing in walled organisms.

Both Ca2+-independent kinase OPEN STOMATA 1 (OST1) and Ca2+-DEPENDENT PROTEIN KINASEs (CPKs) play important roles in ABA-induced stomatal closure. We recently reported that OST1-mediated phosphorylation and activation of Ca2+ channels constituted mainly with CYCLIC NUCLEOTIDE-GATED CHANNEL 5 (CNGC5), 6, 9, and 12 (CNGC5/6/9/12) are required for ABA-induced stomatal closure in Arabidopsis. However, Ca2+-dependent protein kinases and underlying mechanisms that are involved in this Ca2+ signaling pathway are still largely unknown. In this study, we identified CPK3, 8, and 10 (CPK3/8/10) as Ca2+-dependent CNGC-activating kinases with CPK3 as the main one, and a conserved serine site at CNGCs' C termini is revealed to be the main CPK3-target sites, differing from OST-target sites at CNGCs' N termini. Double S-to-D (2D) and S-to-A (2A) point mutations at OST1- and CPK3-target sites respectively coactivate and cosuppress CNGCs, but individual S-to-D activation is impaired by S-to-A mutation at the other site. Abscisic acid (ABA)-induced stomatal closure and Ca2+ oscillations are impaired in Arabidopsis triple mutant cpk3/8/10, but are rescued fully by the CNGC6's 2D variant, largely by mixed A/D variant, and not by 2A variant. These results demonstrate that the cytosolic Ca2+ elevation derived from OST1-CNGC modules-mediated external Ca2+ influx activates CPK3 via Ca2+ binding, the Ca2+-bound CPK3 evokes more massive external Ca2+ influx through enhancing the activity of CNGCs by phosphorylation, and CPK3-evoked Ca2+ influx is required for the encoding of ABA-induced cytosolic Ca2+ signaling in Arabidopsis guard cells.

Plants are susceptible to various environmental stresses, but basic leucine zipper (bZIP) transcription factors play a key role in regulating stress responses. In this study, a drought response-related candidate gene (ZmbZIP92) was cloned from maize (Zea mays L.) following a comparative genomic analysis. This gene is highly homologous to the rice (Oryza sativa L.) gene OsbZIP62, exhibits tissue-specific expression patterns, and is significantly induced by drought, high salinity, and abscisic acid (ABA) treatments. Subcellular localization revealed that ZmbZIP92 is a nuclear protein. Additionally, yeast-based assays of ZmbZIP92 detected a lack of transcriptional self-activation. Dual-luciferase reporter assays demonstrated that ZmbZIP92 binds specifically to the G-box (CACGTG) cis-element. Overexpressing ZmbZIP92 in Arabidopsis thaliana significantly promoted root elongation, enhanced drought tolerance, and increased sensitivity to ABA, which was reflected by markedly inhibited seed germination. RNA sequencing and differential expression analyses indicated that multiple stress response-related pathways were enriched in ZmbZIP92-overexpressing plants, including ABA signaling, antioxidant response, transmembrane transport, and general plant signal transduction pathways. In summary, ZmbZIP92 positively regulates drought tolerance through its effects on the ABA signaling pathway and other stress response-related signaling networks.

CCT (CONSTANS, CO-like, and TOC1) proteins are plant-specific transcription factors characterized by a conserved C-terminal CCT domain, known to regulate both growth/development and stress resistance in plants. Although CCT genes have been studied in multiple plant species, systematic characterization in woody perennials, especially important forest species, is still limited. In this study, we performed a genome-wide analysis of the CCT gene family in Betula platyphylla (birch), a key species in temperate and boreal forests. A total of 22 CCT members were identified and subjected to comprehensive analyses, including phylogenetic classification, chromosomal distribution, gene/protein structural features, promoter cis-elements, computationally predicted miRNA–target relationships and phosphorylation sites, and expression profiles under diverse developmental and stress conditions. Notably, BpCCT9 was significantly upregulated under multiple abiotic stress conditions and acted as a nuclear-localized transcriptional repressor. Therefore, through heterologous overexpression and VP16-mediated transcriptional activation in Populus alba × P. glandulosa cv. ‘84K’, we functionally characterized BpCCT9 and elucidated its regulatory mechanism, demonstrating that BpCCT9 negatively influences cold tolerance in transgenic poplar and directly binds to and represses the promoters of the cold‑responsive transcription factors, PagCBF1 and PagPIF4. This study provided genetic resources for molecular breeding and mechanistic insights into the superior traits of birch, while also advancing the understanding of evolutionary and functional diversification of CCT genes in trees.

INTRODUCTION:Numerous studies have elucidated plants' drought response, yet how plant leaves perceive this stress remains unclear. OBJECTIVES:This study aims to deepen the understanding of how leaves perceive and respond to water loss. METHODS:Detached-leaf water loss rate was detected, which can effectively eliminate the effects of water absorption and water transport. Subsequently, a genome-wide association study (GWAS) was carried out on the detached-leaf water loss rate. RESULTS:There was a significant association between the TaWAK5 (cell wall-associated kinase) gene and the detached-leaf water loss rate. Phenotypic analyses of overexpression and CRISPR/Cas9-based knockout lines revealed the function of TaWAK5 in wheat response to drought stress. Subsequent study exhibited that drought induced the degradation of pectin into oligogalacturonides (OGs), and OGs have a higher affinity for TaWAK5 than pectin does. OGs can activate TaWAK5 kinase, leading to stomatal closure. Additionally, TaWAK5 phosphorylates TaSLAC1 (slow anion channel-associated 1), a key component regulating stomatal movement. A single nucleotide polymorphism site SNP-947 (G/A), at 947 bp of the cis-element in the TaWAK5 promoter region, is significantly associated with TaWAK5 expression, detached-leaf water loss rate and canopy temperature, and leads to the bZIP transcription factor TaPAN (PERIANTHIA) functioning as a transcriptional activator in haplotype Hap3/4, but not in Hap1/2 of TaWAK5. CONCLUSION:This study suggests that TaWAK5 perceives OGs to activate drought responses in wheat, highlighting a potential target for enhancing the drought tolerance of wheat.

Maize (Zea mays L.) is a globally important crop that is highly sensitive to cold stress during the early seedling stage, which severely constrains its development and growth. However, the molecular basis of cold adaptation in maize still remains largely unclear. In this study, we identified ZmKNOX13, a KNOX family transcription factor, as a key regulator for maize cold tolerance through genetic analysis of a cold-sensitive EMS mutant. Functional analyses using overexpression and CRISPR/Cas9 knockout lines demonstrated that ZmKNOX13 plays an important role in the maize cold stress response. We further showed that ZmKNOX13 physically interacts with ZmNAC70, and the zmnac70 mutant also exhibits a cold-sensitive phenotype. DNA affinity purification sequencing (DAP-seq) identified the cold-responsive bZIP transcription factor ZmLIP15 as a direct target of ZmKNOX13. Electrophoretic mobility shift assays (EMSAs) indicated that both ZmKNOX13 and ZmNAC70 directly bind to the ZmLIP15 promoter. Furthermore, dual luciferase reporter assay demonstrated that ZmNAC70 enhanced ZmKNOX13-mediated activation of the ZmLIP15 promoter under cold stress. Collectively, our findings uncover a novel transcriptional regulatory module that modulates maize cold stress response, and provide potential targets for the molecular improvement of cold-tolerant maize varieties

Root density, determined by root number, is a key trait for drought resistance and yield improvement. Here, we identified a drought-responsive root number regulator, TabHLH112-2A, in wheat (Triticum aestivum). Given its interaction with the essential factor of crown root initiation, TaMOR, TabHLH112-2A was subsequently designated TaMIP1. mip1 mutants had fewer crown and lateral roots than the wild type (WT). TaMIP1 specifically bound to the E-box cis-element and induced the expression of genes involved in auxin and ABA signaling pathways, root development, and drought stress response. Two TaMIP1 haplotypes were found in the natural population. Two nonsynonymous SNPs in the active domain led to enhanced transactivation activity of TaMIP1Hap-2A-2, resulting in higher root dry weight. The TaMIP1 and TaMOR haplotypes had additive effects on RDW, and the effect of TaMOR haplotypes was epistatic to that of TaMIP1 haplotypes. Furthermore, mip1 exhibited a lower survival rate under drought stress and a higher yield under well-watered conditions. Our findings elucidate the important roles of TaMIP1 in root density and the tradeoff between yield and drought resistance. The discovery of a regulatory module and combined haplotypes bring insights and genetic resources for drought resistance and high-yield breeding.

Maize (Zea mays L.), a vital food and economic crop, has widespread applications in industry, animal husbandry, and agriculture worldwide. Low-temperature stress during spring increases maize susceptibility to chilling injuries, ultimately reducing yield. The CIPK gene family plays a critical role in plant signal transduction under environmental stress; however, its function and molecular mechanisms in maize under low-temperature conditions remain largely unknown. In this study, we successfully cloned ZmCIPK14 from the maize inbred line B73. Quantitative real-time PCR analysis revealed that the relative expression level of ZmCIPK14 was highest in leaf tissue and was significantly upregulated under low-temperature stress. Heterologous expression in Escherichia coli BL21 and yeast demonstrated that ZmCIPK14 confers enhanced tolerance to low-temperature stress in both organisms. Subcellular localization revealed that ZmCIPK14 is localized to both the nucleus and plasma membrane, and protein interaction analysis identified ZmGST9 as a putative interacting partner. Transgenic Arabidopsis thaliana overexpressing ZmCIPK14 exhibited significantly enhanced tolerance to low-temperature stress compared with wild-type plants under the same conditions. Furthermore, these transgenic plants exhibited increased antioxidant enzyme activity and a stronger reactive oxygen species scavenging capacity, resulting in reduced cellular damage caused by low-temperature stress. This study provides a theoretical basis for understanding the molecular mechanisms underlying low-temperature responses in maize and contributes to the development of low-temperature-tolerant maize varieties.

Dicarboxylate transporters (DiTs) mediate the exchange of dicarboxylates across the chloroplast inner membrane, playing critical roles in C/N coupling, photorespiration, chloroplast redox homeostasis, and C4 photosynthesis. DiT1 and DiT2 are Na⁺-independent exchangers of the solute carrier 13 (SLC13) family, and exhibit overlapping yet distinct substrate specificities: DiT1 transports 2-oxoglutarate, malate, and oxaloacetate, while DiT2 additionally transports glutamate and aspartate. However, the structural determinants of their substrate specificity and transport mechanism remain unclear. Here, we determined cryo-electron microscopy structures of Arabidopsis thaliana DiT1 and DiT2.1 bound to diverse substrates in dual conformational states. Structural analyses revealed that AtDiT1 possesses a singular dicarboxylate-binding site that is electrostatically incompatible with amino acid substrates, whereas AtDiT2.1 has 2 distinct sites to accommodate C4- and C5-dicarboxylates, thus allowing amino acids to bind without electrostatic repulsion. Phylogenetic analysis identified an A226S substitution in the substrate-binding site of DiT1, emerging during evolution in the charophyte ancestor of land plants. This substitution enhances oxaloacetate binding affinity in DiT1, which may have improved adaptation to terrestrial environments. Additionally, 2 conserved positively charged residues in DiTs functionally mimic Na⁺ used by SLC13 co-transporters, thereby enabling a Na⁺-independent elevator-type transport mechanism. These findings provide critical structural and mechanistic insights into the functional divergence of plant DiTs.

Drought stress significantly constrains crop productivity and yield stability. Sorghum (Sorghum bicolor L. Moench), a C4 cereal widely cultivated in arid and semi-arid regions, exhibits high water-use efficiency and remarkable drought tolerance. Understanding both the impacts of drought and the plant’s response mechanisms is essential for enhancing drought resilience in this crop. In this study, physiological changes and differential protein accumulation were analyzed in leaves of the sorghum inbred line BT × 623 under 10% PEG-6000-induced drought stress. The physiological adaptation to drought was characterized by improved water retention and mitigation of oxidative damage through the synergistic action of antioxidant enzymes. Using two-dimensional electrophoresis (2-DE) and MALDI-TOF-TOF mass spectrometry, 43 protein spots were successfully identified, corresponding to 38 unique proteins differentially expressed under osmotic stress. These proteins function in diverse biological processes, including protein synthesis, processing, and degradation; photosynthesis; carbohydrate and energy metabolism; transcriptional regulation; stress and defense; lipid and membrane metabolism; and amino acid metabolism. Proteomic profiling revealed that the coordinated modulation of multiple functional groups, such as those involved in photosynthesis, energy metabolism, transcriptional adjustment, ROS scavenging, and protein turnover, underpins sorghum’s osmotic stress adaptation. These findings provide key insights into the drought resistance mechanisms of sorghum at both physiological and proteomic levels.

Plants cannot relocate when environmental conditions become unfavorable and therefore rely on sophisticated mechanisms to perceive, interpret, and respond to stresses. Stress sensing constitutes the first and often most decisive step in adaptation, because it determines how efficiently downstream signaling and protective responses are initiated. This review focuses on the biochemical and biophysical basis of abiotic-stress sensing, highlighting the fundamental molecular logic by which physical and chemical environmental changes are encoded, perceived, and converted into biological signals. We discuss representative sensing mechanisms associated with temperature stress, drought, salt and alkali stress, flooding, and soil compaction, and examine general messengers, including Ca2+ and reactive oxygen species, as central integrators across diverse abiotic stresses. Finally, we identify major outstanding questions, including the identity of the primary stress-associated cues, the spatial organization of sensing, and the perception of combined stresses, and highlight emerging approaches, including AI-driven biomolecular modeling, single-cell techniques, synthetic biology, and genome editing, as powerful tools to advance the mechanistic understanding of plant stress sensing.

Maize kernel size is regulated by multiple genetic pathways, including the evolutionarily conserved mitogen-activated protein kinase (MAPK) cascade, which plays crucial roles in plant growth and development. However, the function of MAPKK kinases in maize kernel development remains poorly understood. Here, we identified mitogen-activated protein kinase kinase 4 (ZmMKK4) as a positive regulator of kernel development in maize. ZmMKK4 is localized primarily in the nucleus and cytoplasm and shows high expression levels across multiple tissues, including kernels. Knockout mutants of zmmkk4 resulted in significantly reduced kernel size and weight, accompanied by severe impairments in kernel development. Furthermore, we observed a decrease in starch content accompanied by an increase in protein content in the mutant kernels. In summary, our study elucidates the role of ZmMKK4 in promoting kernel size and modulating starch and protein accumulation in maize, providing important insights into the molecular mechanisms underlying kernel development. These findings offer a theoretical foundation and a candidate gene for further research into maize kernel development and molecular breeding strategies.

Shoot branching is a key determinant of plant architecture and yield, and reduced shoot branching may enhance plant survival and promote effective seed maturation under drought stress. However, it remains unknown how plants alter shoot branching under drought. Here, we found that drought stress represses shoot branching in Arabidopsis. Abscisic acid (ABA)-responsive element-binding transcription factors (ABFs), central regulators of osmotic stress responses, transcriptionally activate the strigolactone (SL) biosynthesis genes MORE AXILLARY GROWTH 3 and 4 (MAX3/4), thereby inhibiting branching. The loss-of-function abf octuple mutant impairs MAX gene induction and branching repression under osmotic stress or ABA treatment, a defect that can be rescued by the SL analog GR-24. Our findings reveal a molecular pathway through which ABFs respond to drought stress signals to limit shoot branching by activating MAX3/4 expression, underscoring their central role in adapting plant architecture to adverse conditions.

As a globally essential food crop, the flowering time of Zea mays is fundamental to its reproductive development, yield formation, and adaptation to various agro-climatic conditions. Concurrently, drought represents a critical environmental factor that significantly limits maize yields worldwide. The AP2/ERF transcription factor superfamily is widely recognized for its crucial roles in regulating both developmental processes, such as seed development, plant height, flowering, and plant responses to abiotic stresses including drought, high temperature, low temperature. The Dehydration Responsive Element Binding protein (DREB) subfamily is a member of the AP2/ERF transcription factor family. In this study, we analyzed the function of the ZmDREB53 gene and elucidated its dual roles in the regulation of flowering and drought tolerance. Therefore, this study aims to systematically investigate the function of ZmDREB53 in two key aspects: the control of flowering time and the enhancement of drought tolerance, thereby addressing the significant knowledge gap surrounding this gene. In this study, a multi-layered analysis of phenotypic and physiological indices was performed on transgenic Arabidopsis thaliana plants, with systematic evaluation of their performance in growth, physiological, and yield-related traits. For growth and developmental traits, the number of flower buds, petal length, and silique length were determined in Wild-Type (WT) and overexpressing A. thaliana at the flowering stage. On 1/2 MS medium under mannitol-simulated drought stress, seed germination rates of the two A. thaliana lines were recorded after 6 days of treatment; primary root length and root hair density were measured following 18 days of treatment. Under normal growth conditions and after 20 days of natural drought stress, superoxide anion (O₂⁻) was detected via nitroblue tetrazolium (NBT) staining. Combined with 3,3'-diaminobenzidine (DAB) staining, reactive oxygen species (ROS) accumulation in transgenic and WT was quantitatively analyzed. Concomitantly, the content of hydrogen peroxide (H₂O₂), activities of antioxidant enzymes [peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD)], and level of malondialdehyde (MDA)—a core marker of lipid peroxidation—were determined. Furthermore. Furthermore, the expression profiles of key genes in the flowering pathway were analyzed via RT-qPCR, providing necessary molecular evidence. Our analysis revealed that ZmDREB53 exhibits predominant expression in maize seeds and is localized within the nucleus. Transgenic A. thaliana flowered 7–10 days earlier than WT plants, with no significant changes in flower or pod traits. Additionally, the transgenic plants exhibited stronger ROS scavenging ability and higher drought resistance compared to WT plants. Overall, ZmDREB53 functions as a positive regulator of flowering time in A. thaliana and contributes to enhanced drought tolerance. These findings offer valuable insights for elucidating the mechanisms of growth regulation in maize and provide a genetic resource for breeding improved varieties with stronger stress resistance.

Plant cyclic nucleotide-gated channels (CNGCs) belong to the cyclic nucleotide-binding domain (CNBD) channel family, but are phylogenetically classified in a distinct branch. In contrast to their animal counterparts of K+-selective or non-selective cation channels, plant CNGCs mainly mediate Ca2+ influx and are involved in various physiological processes, such as stomatal movements, pollen-tube growth and immune responses. Here, we present the cryo-EM structure and electrophysiological analysis of plant CNGC representatives, Arabidopsis CNGC1 and CNGC5. We found that CNGC1 and CNGC5 contain a unique extracellular domain featuring disulfide bonds that is essential for channel gating via coupling of the voltage-sensing domain with the pore domain. The pore domain selectivity filter possesses a Gln residue at the constriction site that determines the Ca2+ selectivity. Replacement of this Gln with Glu, typically observed in CNBD-type non-selective cation channels, could convert CNGC1 and CNGC5 from Ca2+-selective channels to non-selective cation channels permeable to Ca2+, Na+ or K+. In addition, we found that the CNGC1 and CNGC5 CNBD homology domain contains intrinsic-ligand-like interactions, which may devoid the binding of cyclic nucleotides and lead to gating independent of cAMP or cGMP. This research not only provides a mechanistic understanding of plant CNGCs' function, but also adds to the comprehensive knowledge of the CNBD channels.

The SNF1-regulated protein kinase 2s (SnRK2s) are activated by phytohormone abscisic acid (ABA) and osmotic stress to control plant growth and stress responses; however, assessing SnRK2 activity is challenging. Here, we present a protocol to detect SnRK2 activity in plants. We describe steps for performing immunoblotting with anti-phospho-S175-SnRK2 antibody, in-gel kinase assay, band-shift assay, and immunoprecipitated kinase assay. Immunoblotting and in-gel kinase assays are suitable for evaluating endogenous SnRK2 activity, whereas band-shift and immunoprecipitated kinase assays are applicable to assess tagged SnRK2 activity in transgenic lines. For complete details on the use and execution of this protocol, please refer to Li et al.,1 Li et al.,2 and Yuan et al.3.

Arabidopsis PHOSPHATE 1 (AtPHO1) and its closest homologue AtPHO1;H1 are phosphate transporters that load phosphate into the xylem vessel for root-to-shoot translocation. AtPHO1 and AtPHO1;H1 are prototypical members of the unique SPX–EXS family, whose structural and molecular mechanisms remain elusive. In this study, we determined the cryogenic electron microscopy structure of AtPHO1;H1 binding with inorganic phosphate (Pi) and inositol hexakisphosphate in a closed conformation. Further molecular dynamic simulation and AlphaFold prediction support an open conformation. AtPHO1;H1 forms a domain-swapped homodimer that involves both the transmembrane ERD1/XPR1/SYG1 (EXS) domain and the cytoplasmic SYG1/Pho81/XPR1 (SPX) domain. The EXS domain presented by the SPX–EXS family represents a novel protein fold, and an independent substrate transport pathway and substrate-binding site are present in each EXS domain. Two gating residues, Trp719 and Tyr610, are identified above the substrate-binding site to control opening and closing of the pathway. The SPX domain features positively charged patches and/or residues at the dimer interface to accommodate inositol hexakisphosphate molecules, whose binding mediates dimerization and enhances AtPHO1;H1 activity. In addition, a C-terminal tail is required for AtPHO1;H1 activity. On the basis of structural and functional analysis, a working model for Pi efflux mediated by AtPHO1;H1 and its homologues was postulated, suggesting a channel-like mechanism. This study not only reveals the molecular and regulatory mechanism underlying Pi transport mediated by the unique SPX–EXS family, but also provides potential for crop engineering to enhance phosphorus-use efficiency in sustainable agriculture. This study characterizes the plant inorganic phosphate transporter PHO1 through cryogenic electron microscopy and biochemical and physiological analysis, revealing the molecular and regulatory mechanism of transport mediated by the SPX–EXS family.

As one of the world's three major staple crops, maize is indispensable, particularly because of the critical role its essential amino acids play in both human and livestock nutrition. This study investigated the function of ZmERF118 in seed amino acid accumulation in Columbia wild-type (Col-0 WT) Arabidopsis plants. Subcellular localization experiments in tobacco leaves revealed that ZmERF118 acts as a transcription factor localized in the plant nucleus and encodes a 25.6kDa protein. Compared with Col-0 WT Arabidopsis, overexpression of this gene promoted seedling growth and increased seed size but inhibited embryo development in the seeds. Moreover, this gene positively regulated amino acid content in Arabidopsis seeds, with significantly elevated levels of all amino acids, except tryptophan, compared with the WT. In addition, the seed starch content was significantly lower than that of WT. In summary, overexpression of the ZmERF118 positively regulated amino acid content and negatively regulated starch content in Arabidopsis seeds.