Search

Heat stress (HS) poses a significant challenge to plant survival, necessitating sophisticated molecular mechanisms to maintain cellular homeostasis. Here, we identify SICKLE (SIC) as a key modulator of HS responses in Arabidopsis (Arabidopsis thaliana). SIC is required for the sequestration of RNA DEBRANCHING ENZYME 1 (DBR1), a rate-limiting enzyme of lariat intronic RNA (lariRNA) decay, into stress granules (SGs). The sequestration of DBR1 by SIC enhances the accumulation of lariRNAs, branched circular RNAs derived from excised introns during pre-mRNA splicing, which in turn promote the transcription of their parental genes. Our findings further demonstrate that SIC-mediated DBR1 sequestration in SGs is crucial for plant HS tolerance, as deletion of the N-terminus of SIC (SIC1–244) impairs DBR1 sequestration and compromises plant response to HS. Overall, our study unveils a mechanism of transcriptional regulation in the HS response, where lariRNAs are enriched through DBR1 sequestration, ultimately promoting the transcription of heat stress tolerance genes. SICKLE sequestrates DBR1 into stress granules to promote the accumulation of lariRNAs, which upregulate heat stress-related genes and enhance heat-stress tolerance. [ABSTRACT FROM AUTHOR]

SUMMARY: Plasma membrane (PM)‐associated abscisic acid (ABA) signal transduction is an important component of ABA signaling. The C2‐domain ABA‐related (CAR) proteins have been reported to play a crucial role in recruiting ABA receptor PYR1/PYL/RCAR (PYLs) to the PM. However, the molecular details of the involvement of CAR proteins in membrane‐delimited ABA signal transduction remain unclear. For instance, where this response process takes place and whether any additional members besides PYL are taking part in this signaling process. Here, the GUS‐tagged materials for all Arabidopsis CAR members were used to comprehensively visualize the extensive expression patterns of the CAR family genes. Based on the representativeness of CAR1 in response to ABA, we determined to use it as a target to study the function of CAR proteins in PM‐associated ABA signaling. Single‐particle tracking showed that ABA affected the spatiotemporal dynamics of CAR1. The presence of ABA prolonged the dwell time of CAR1 on the membrane and showed faster lateral mobility. Surprisingly, we verified that CAR1 could directly recruit hypersensitive to ABA1 (HAB1) and SNF1‐related protein kinase 2.2 (SnRK2.2) to the PM at both the bulk and single‐molecule levels. Furthermore, PM localization of CAR1 was demonstrated to be related to membrane microdomains. Collectively, our study revealed that CARs recruited the three main components of ABA signaling to the PM to respond positively to ABA. This study deepens our understanding of ABA signal transduction. [ABSTRACT FROM AUTHOR]

SUMMARY: The cytosol‐facing outer membrane (OM) of organelles communicates with other cellular compartments to exchange proteins, metabolites, and signaling molecules. Cellular surveillance systems also target OM‐resident proteins to control organellar homeostasis and ensure cell survival under stress. However, the OM proximity proteomes have never been mapped in plant cells since using traditional approaches to discover OM proteins and identify their dynamically interacting partners remains challenging. In this study, we developed an OM proximity labeling (OMPL) system using biotin ligase‐mediated proximity biotinylation to identify the proximity proteins of the OMs of mitochondria, chloroplasts, and peroxisomes in living Arabidopsis (Arabidopsis thaliana) cells. Using this approach, we mapped the OM proximity proteome of these three organelles under normal conditions and examined the effects of the ultraviolet‐B (UV‐B) or high light (HL) stress on the abundances of OM proximity proteins. We demonstrate the power of this system with the discovery of cytosolic factors and OM receptor candidates potentially involved in local protein translation and translocation. The candidate proteins that are involved in mitochondrion–peroxisome, mitochondrion–chloroplast, or peroxisome–chloroplast contacts, and in the organellar quality control system are also proposed based on OMPL analysis. OMPL‐generated OM proximity proteomes are valuable sources of candidates for functional validation and suggest directions for further investigation of important questions in cell biology. [ABSTRACT FROM AUTHOR]

A study published in the Plant Biotechnology Journal explores the role of the GhVQ22 gene in regulating pigment gland (PG) size and secondary metabolic accumulation in cotton. The researchers found that silencing GhVQ22 resulted in significantly enlarged PGs and altered composition and content of secondary metabolites. The study also revealed that GhVQ22 is a downstream target of Gl2/Gl3 genes and negatively regulates PG size. These findings provide insights into the genetic mechanisms involved in PG development and suggest potential applications for anti-pest strategies in cotton. [Extracted from the article]

Reactive oxygen species (ROS) are produced as undesirable by‐products of metabolism in various cellular compartments, especially in response to unfavorable environmental conditions, throughout the life cycle of plants. Stress‐induced ROS production disrupts normal cellular function and leads to oxidative damage. To cope with excessive ROS, plants are equipped with a sophisticated antioxidative defense system consisting of enzymatic and non‐enzymatic components that scavenge ROS or inhibit their harmful effects on biomolecules. Nonetheless, when maintained at relatively low levels, ROS act as signaling molecules that regulate plant growth, development, and adaptation to adverse conditions. Here, we provide an overview of current approaches for detecting ROS. We also discuss recent advances in understanding ROS signaling, ROS metabolism, and the roles of ROS in plant growth and responses to various abiotic stresses. [ABSTRACT FROM AUTHOR]

Plants have evolved complex physical and chemical defense systems that allow them to withstand herbivory infestation. Composed of a complex mixture of very‐long‐chain fatty acids (VLCFAs) and their derivatives, cuticular wax constitutes the first physical line of defense against herbivores. Here, we report the function of Glossy 8 (ZmGL8), which encodes a 3‐ketoacyl reductase belonging to the fatty acid elongase complex, in orchestrating wax production and jasmonic acid (JA)‐mediated defenses against herbivores in maize (Zea mays). The mutation of GL8 enhanced chemical defenses by activating the JA‐dependent pathway. We observed a trade‐off between wax accumulation and JA levels across maize glossy mutants and 24 globally collected maize inbred lines. In addition, we demonstrated that mutants defective in cuticular wax biosynthesis in Arabidopsis thaliana and maize exhibit enhanced chemical defenses. Comprehensive transcriptomic and lipidomic analyses indicated that the gl8 mutant confers chemical resistance to herbivores by remodeling VLCFA‐related lipid metabolism and subsequent JA biosynthesis and signaling. These results suggest that VLCFA‐related lipid metabolism has a critical role in regulating the trade‐offs between cuticular wax and JA‐mediated chemical defenses. [ABSTRACT FROM AUTHOR]

Summary: Gossypol and the related terpenoids are stored in the pigment gland to protect cotton plants from biotic stresses, but little is known about the synthetic sites of these metabolites.Here, we showed that GoPGF, a key gene regulating gland formation, was expressed in gland cells and roots. The chromatin immunoprecipitation sequencing (ChIP‐seq) analysis demonstrated that GoPGF targets GhJUB1 to regulate gland morphogenesis.RNA‐sequencing (RNA‐seq) showed high accumulation of gossypol biosynthetic genes in gland cells. Moreover, integrated analysis of the ChIP‐seq and RNA‐seq data revealed that GoPGF binds to the promoter of several gossypol biosynthetic genes. The cotton callus overexpressing GoPGF had dramatically increased the gossypol levels, indicating that GoPGF can directly activate the biosynthesis of gossypol. In addition, the gopgf mutant analysis revealed the existence of both GoPGF‐dependent and ‐independent regulation of gossypol production in cotton roots.Our study revealed that the pigment glands are synthetic sites of gossypol in aerial parts of cotton and that GoPGF plays a dual role in regulating gland morphogenesis and gossypol biosynthesis. The study provides new insights for exploring the complex relationship between glands and the metabolites they store in cotton and other plant species. [ABSTRACT FROM AUTHOR]

Chromosomal rearrangements play essential roles in crop evolution, and different types of rearrangements have been identified in various cultivars belonging to Triticeae. However, it has not been reported whether chromosome rearrangements also occur within the same cultivar. We screened 33 plants of a Chinese modern cultivar Aikang58 (AK58) using non‐denaturing fluorescence in situ hybridization technique and found two with reciprocal translocation 3BL‐6BL and 3BS‐6BS present, thereafter designated as AK58T. The breakpoint positions of AK58T were different from previous 3B‐6B translocation, indicating newly reported translocations in common wheat. The genetic similarity between AK58 and AK58T was greater than 99.8% by wheat 55K chip array analysis. Our results showed no significant phenotype difference in five developmental periods or seven seed traits between the two lines under speed breeding conditions. However, the F1 hybrids between AK58 and AK58T severely suffered from low seedset due to the documented occurrence of multivalent formation during meiosis. Screening of 41 F2 progenies identified eight types of chromosomal variations that affected agronomic traits and decreased the fertility. Therefore, we reported that chromosomal rearrangements within the same cultivar could be hidden by high phenotype and genotype similarity, and concluded that these types of hidden variations may lead to severe fertility penalty once outcross occurred. Core Ideas: Chromosomal translocation could be hidden by high phenotype and genotype similarity in modern wheat cultivar.Cytogenetic‐based techniques are valuable to diagnose hidden chromosomal translocation.Outcrossing between hidden chromosomal translocation and normal lines can result in severe fertility penalty. [ABSTRACT FROM AUTHOR]

Multicellular organisms such as plants contain various cell types with specialized functions. Analyzing the characteristics of each cell type reveals specific cell functions and enhances our understanding of organization and function at the organismal level. Guard cells (GCs) are specialized epidermal cells that regulate the movement of the stomata and gaseous exchange, and provide a model genetic system for analyzing cell fate, signaling, and function. Several proteomics analyses of GC are available, but these are limited in depth. Here we used enzymatic isolation and flow cytometry to enrich GC and mesophyll cell protoplasts and perform in‐depth proteomics in these two major cell types in Arabidopsis leaves. We identified approximately 3,000 proteins not previously found in the GC proteome and more than 600 proteins that may be specific to GC. The depth of our proteomics enabled us to uncover a guard cell‐specific kinase cascade whereby Raf15 and Snf1‐related kinase2.6 (SnRK2.6)/OST1(open stomata 1) mediate abscisic acid (ABA)‐induced stomatal closure. RAF15 directly phosphorylated SnRK2.6/OST1 at the conserved Ser175 residue in its activation loop and was sufficient to reactivate the inactive form of SnRK2.6/OST1. ABA‐triggered SnRK2.6/OST1 activation and stomatal closure was impaired in raf15 mutants. We also showed enrichment of enzymes and flavone metabolism in GC, and consistent, dramatic accumulation of flavone metabolites. Our study answers the long‐standing question of how ABA activates SnRK2.6/OST1 in GCs and represents a resource potentially providing further insights into the molecular basis of GC and mesophyll cell development, metabolism, structure, and function. [ABSTRACT FROM AUTHOR]

Reversible protein S-nitrosylation regulates a wide range of biological functions and physiological activities in plants. However, it is challenging to quantitively determine the S-nitrosylation targets and dynamics in vivo. In this study, we develop a highly sensitive and efficient fluorous affinity tag-switch (FAT-switch) chemical proteomics approach for S-nitrosylation peptide enrichment and detection. We quantitatively compare the global S-nitrosylation profiles in wild-type Arabidopsis and gsnor1/hot5/par2 mutant using this approach, and identify 2,121 S-nitrosylation peptides in 1,595 protein groups, including many previously unrevealed S-nitrosylated proteins. These are 408 S-nitrosylated sites in 360 protein groups showing an accumulation in hot5-4 mutant when compared to wild type. Biochemical and genetic validation reveal that S-nitrosylation at Cys337 in ER OXIDOREDUCTASE 1 (ERO1) causes the rearrangement of disulfide, resulting in enhanced ERO1 activity. This study offers a powerful and applicable tool for S-nitrosylation research, which provides valuable resources for studies on S-nitrosylation-regulated ER functions in plants. This study developed a highly sensitive method for detecting S-nitrosylation peptides, which allows quantitative identification of S-nitrosylated proteins and reveals a key role of GSNOR1 in regulating endoplasmic reticulum functions in Arabidopsis. [ABSTRACT FROM AUTHOR]

Sphingolipids are the structural components of membrane lipid bilayers and act as signaling molecules in many cellular processes. Serine palmitoyltransferase (SPT) is the first committed and rate‐limiting enzyme in the de novo sphingolipids biosynthetic pathway. The core SPT enzyme is a heterodimer consisting of LONG‐CHAIN BASE1 (LCB1) and LCB2 subunits. SPT activity is inhibited by orosomucoid proteins and stimulated by small subunits of SPT (ssSPTs). However, whether LCB1 is modified and how such modification might regulate SPT activity have to date been unclear. Here, we show that activation of MITOGEN‐ACTIVATED PROTEIN KINASE 3 (MPK3) and MPK6 by upstream MKK9 and treatment with Flg22 (a pathogen‐associated molecular pattern) increases SPT activity and induces the accumulation of sphingosine long‐chain base t18:0 in Arabidopsis thaliana, with activated MPK3 and MPK6 phosphorylating AtLCB1. Phosphorylation of AtLCB1 strengthened its binding with AtLCB2b, promoted its binding with ssSPTs, and stimulated the formation of higher order oligomeric and active SPT complexes. Our findings therefore suggest a novel regulatory mechanism for SPT activity. [ABSTRACT FROM AUTHOR]

Summary: Fatty acid (FA) β‐oxidation provides energy for oil seed germination but also produces massive byproduct reactive oxygen species (ROS), posing potential oxidative damage to plant cells. How plants overcome the contradiction between energy supply and ROS production during seed germination remains unclear.In this study, we identified an Arabidopsis mvs1 (methylviologen‐sensitive) mutant that was hypersensitive to ROS and caused by a missense mutation (G1349 substituted as A) of a cytochrome P450 gene, CYP77A4.CYP77A4 was highly expressed in germinating seedling cotyledons, and its protein is localized in the endoplasmic reticulum. As CYP77A4 catalyzes the epoxidation of unsaturated FA, disruption of CYP77A4 resulted in increased unsaturated FA abundance and over accumulated ROS in the mvs1 mutant. Consistently, scavenging excess ROS or blocking FA β‐oxidation could repress the ROS overaccumulation and hypersensitivity in the mvs1 mutant. Furthermore, H2O2 transcriptionally upregulated CYP77A4 expression and post‐translationally modified CYP77A4 by sulfenylating its Cysteine‐456, which is necessary for CYP77A4's role in modulating FA abundance and ROS production.Together, our study illustrates that CYP77A4 mediates direct balancing of lipid mobilization and ROS production by the epoxidation of FA during seed germination. [ABSTRACT FROM AUTHOR]

As the most abundant RNA modification, pseudouridylation has been shown to play critical roles in Escherichia coli , yeast and humans. However, its function in plants is still unclear. Here, we characterized leaf curly and small 1 (FCS1), which encodes a pseudouridine synthase in Arabidopsis. fcs1 mutants exhibited severe defects in plant growth, such as delayed development and reduced fertility, and were significantly smaller than the wild type at different developmental stages. FCS1 protein is localized in the mitochondrion. The absence of FCS1 significantly reduces pseudouridylation of mitochondrial 26S ribosomal RNA (rRNA) at the U1692 site, which sits in the peptidyl transferase center. This affection of mitochondrial 26S rRNA may lead to the disruption of mitochondrial translation in the fcs1-1 mutant, causing high accumulation of transcripts but low production of proteins. Dysfunctional mitochondria with abnormal structures were also observed in the fcs1-1 mutant. Overall, our results suggest that FCS1-mediated pseudouridylation of mitochondrial 26S rRNA is required for mitochondrial translation, which is critical for maintaining mitochondrial function and plant development. [ABSTRACT FROM AUTHOR]

Keywords: Gossypium; cell differentiation; specialized structure; transcription factors; terpenoids EN Gossypium cell differentiation specialized structure transcription factors terpenoids 1100 1102 3 05/30/23 20230601 NES 230601 Cotton ( I Gossypium i spp.) is a leading economic crop that is grown in more than 50 countries. Single-cell transcriptome atlas identified novel regulators for pigment gland morphogenesis in cotton Cluster 9 was identified in the cotyledons of gland cotton "CCRI12" but not glandless cotton "CCRI12gl", and I GoPGF i was specifically detected in the cells of cluster 9. [Extracted from the article]

Seed germination is a physiological process regulated by multiple factors. Abscisic acid (ABA) can inhibit seed germination to improve seedling survival under conditions of abiotic stress, and this process is often regulated by light signals. Constitutive photomorphogenic 1 (COP1) is an upstream core repressor of light signals and is involved in several ABA responses. Here, we demonstrate that COP1 is a negative regulator of the ABA‐mediated inhibition of seed germination. Disruption of COP1 enhanced Arabidopsis seed sensitivity to ABA and increased reactive oxygen species (ROS) levels. In seeds, ABA induced the translocation of COP1 to the cytoplasm, resulting in enhanced ABA‐induced ROS levels. Genetic evidence indicated that HY5 and ABI5 act downstream of COP1 in the ABA‐mediated inhibition of seed germination. ABA‐induced COP1 cytoplasmic localization increased HY5 and ABI5 protein levels in the nucleus, leading to increased expression of ABI5 target genes and ROS levels in seeds. Together, our results reveal that ABA‐induced cytoplasmic translocation of COP1 activates the HY5‐ABI5 pathway to promote the expression of ABA‐responsive genes and the accumulation of ROS during ABA‐mediated inhibition of seed germination. These findings enhance the role of COP1 in the ABA signal transduction pathway. [ABSTRACT FROM AUTHOR]

How horizontal gene transfer (HGT) has contributed to the evolution of animals and plants remains a major puzzle. Despite recent progress, defining the overall scale and pattern of HGT events in land plants has been largely elusive. In this study, we performed systematic analyses for acquired genes in different plant groups and throughout land plant evolution. We found that relatively recent HGT events occurred in charophytes and all major land plant groups, but their frequency declined rapidly in seed plants. Two major episodes of HGT events occurred in land plant evolution, corresponding to the early evolution of streptophytes and the origin of land plants, respectively. Importantly, a vast majority of the genes acquired in the two episodes have been retained in descendant groups, affecting numerous activities and processes of land plants. We analyzed some of the acquired genes involved in stress responses, ion and metabolite transport, growth and development, and specialized metabolism, and further assessed the cumulative effects of HGT in land plants. The role of horizontal gene transfer in the evolution of multicellular eukaryotes such as plants and animals has long been elusive. This study identifies two major episodes of horizontal gene transfer in the evolution of land plants. Genes acquired from environmental organisms have fundamentally shaped the structure, physiology, and development of land plants. [ABSTRACT FROM AUTHOR]

Leaf senescence is an evolutionarily acquired process and it is critical for plant fitness. During senescence, macromolecules and nutrients are disassembled and relocated to actively growing organs. Plant leaf senescence process can be triggered by developmental cues and environmental factors, proper regulation of this process is essential to improve crop yield. Protein kinases are enzymes that modify their substrates activities by changing the conformation, stability, and localization of those proteins, to play a crucial role in the leaf senescence process. Impressive progress has been made in understanding the role of different protein kinases in leaf senescence recently. This review focuses on the recent progresses in plant leaf senescence-related kinases. We summarize the current understanding of the function of kinases on senescence signal perception and transduction, to help us better understand how the orderly senescence degeneration process is regulated by kinases, and how the kinase functions in the intricate integration of environmental signals and leaf age information. [ABSTRACT FROM AUTHOR]

Heterotrimeric G protein Gβ‐deficient mutants in rice and maize display constitutive immune responses, whereas Arabidopsis Gβ mutants show impaired defense, suggesting the existence of functional differences between monocots and dicots. Using CRISPR/Cas9, we produced one hemizygous tomato line with a mutated SlGB1 Gβ gene. Homozygous slgb1 knockout mutants exhibit all the hallmarks of autoimmune mutants, including development of necrotic lesions, constitutive expression of defense‐related genes, and high endogenous levels of salicylic acid (SA) and reactive oxygen species, resulting in early seedling lethality. Virus‐induced silencing of Gβ in cotton reproduced the symptoms observed in tomato mutants, confirming that the autoimmune phenotype is not limited to monocot species but is also shared by dicots. Even though multiple genes involved in SA and ethylene signaling are highly induced by Gβ silencing in tomato and cotton, co‐silencing of SA or ethylene signaling components in cotton failed to suppress the lethal phenotype, whereas co‐silencing of the oxidative burst oxidase RbohD can repress lethality. Despite the autoimmune response observed in slgb1 mutants, we show that SlGB1 is a positive regulator of the pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) response in tomato. We speculate that the phenotypic differences observed between Arabidopsis and tomato/cotton/rice/maize Gβ knockouts do not necessarily reflect divergences in G protein‐mediated defense mechanisms. [ABSTRACT FROM AUTHOR]

A fundamental challenge for cells is how to coordinate various biochemical reactions in space and time. To achieve spatiotemporal control, cells have developed organelles that are surrounded by lipid bilayer membranes. Further, membraneless compartmentalization, a process induced by dynamic physical association of biomolecules through phase transition offers another efficient mechanism for intracellular organization. While our understanding of phase separation was predominantly dependent on yeast and animal models, recent findings have provided compelling evidence for emerging roles of phase separation in plants. In this review, we first provide an overview of the current knowledge of phase separation, including its definition, biophysical principles, molecular features and regulatory mechanisms. Then we summarize plant‐specific phase separation phenomena and describe their functions in plant biological processes in great detail. Moreover, we propose that phase separation is an evolutionarily conserved and efficient mechanism for cellular compartmentalization which allows for distinct metabolic processes and signaling pathways, and is especially beneficial for the sessile lifestyle of plants to quickly and efficiently respond to the changing environment. [ABSTRACT FROM AUTHOR]

As the diploid progenitor of the D genome of common wheat, Aegilops tauschii contains abundant genetic resources for potential economic traits, including yield, biotic and abiotic stress tolerance. In this study, phenotypic variation for 4 grain shapes in 221 Ae. tauschii accessions was evaluated in 5 environments for 2 consecutive crop seasons, and best linear unbiased estimates (BLUEs) for grain shapes were calculated across the seasons. To decrease false discovery rate, a total of 139 loci were only identified based on the BLUEs and each environment by genome-wide association study utilizing 4282 single nucleotide polymorphism (SNP) markers from D genome of wheat 55 K genotyping assay. These loci could explain 5.86–17.71% of the phenotypic variation of grain length (GL), 6.83–8.42% of grain width, 5.99–17.71% of grain perimeter, and 5.80–19.61% of grain length–width ratio (GLWR), respectively. Further, 26 significant loci were validated in the wheat mini-core collections population by two-tailed t test. Moreover, the physical positions of some loci for GL and GLWR were found to correspond with previously mapped loci that affect these traits. Based on the obtained SNP loci, 6 putative candidate genes related to grain shapes were identified. This study could provide valuable information for cloning genes related to grain shapes in Ae. tauschii. [ABSTRACT FROM AUTHOR]

The phytohormone abscisic acid (ABA) is crucial for plant responses to environmental challenges. The SNF1-regulated protein kinase 2s (SnRK2s) are key components in ABA-receptor coupled core signaling, and are rapidly phosphorylated and activated by ABA. Recent studies have suggested that Raf-like protein kinases (RAFs) participate in ABA-triggered SnRK2 activation. In vitro kinase assays also suggest the existence of autophosphorylation of SnRK2s. Thus, how SnRK2 kinases are quickly activated during ABA signaling still needs to be clarified. Here, we show that both B2 and B3 RAFs directly phosphorylate SnRK2.6 in the kinase activation loop. This transphosphorylation by RAFs is essential for SnRK2 activation. The activated SnRK2s then intermolecularly trans-phosphorylate other SnRK2s that are not yet activated to amplify the response. High-order Arabidopsis mutants lacking multiple B2 and B3 RAFs show ABA hyposensitivity. Our findings reveal a unique initiation and amplification mechanism of SnRK2 activation in ABA signaling in higher plants. SnRK2 kinase activity is rapidly activated in response to ABA. Here the authors show that initial activation of SnRK2s is achieved by B2 and B3 RAF kinase-mediated phosphorylation of the activation loop and that transphosphorylation between SnRK2s then amplifies the response. [ABSTRACT FROM AUTHOR]

Background and aims: As drought threatens the yield and quality of maize (Zea mays L.), it is important to dissect the molecular basis of maize drought tolerance. Flavonoids, participate in the scavenging of oxygen free radicals and alleviate stress-induced oxidative damages. This study aims to dissect the function of flavonoids in the improvement of maize drought tolerance. Methods: Using far-infrared imaging screening, we previously isolated a drought overly insensitivity (doi) mutant from an ethyl methanesulfonate (EMS)-mutagenized maize library and designated it as doi57. In this study, we performed a physiological characterization and transcriptome profiling of doi57 in comparison to corresponding wild-type B73 under drought stress. Results: Under drought stress, doi57 seedlings displayed lower leaf-surface temperature (LST), faster water loss, and better performance in growth than B73. Transcriptome analysis reveals that key genes involved in flavonoid biosynthesis are enriched among differentially expressed genes in doi57. In line with these results, more flavonols and less hydrogen peroxide (H2O2) were accumulated in guard cells of doi57 than in those of B73 with the decrease of soil water content (SWC). Moreover, the capacity determined from doi57 seedling extracts to scavenge oxygen free radicals was more effective than that of B73 under the drought treatment. Additionally, doi57 seedlings had higher photosynthetic rates, stomatal conductance, transpiration rates, and water use efficiency than B73 exposed to drought stress, resulting in high biomass and greater root/shoot ratios in doi57 mutant plants. Conclusion: Flavonoids may facilitate maize seedling drought tolerance by lowering drought-induced oxidative damage as well regulating stomatal movement. [ABSTRACT FROM AUTHOR]

Background: Plants are naturally associated with root microbiota, which are microbial communities influential to host fitness. Thus, it is important to understand how plants control root microbiota. Epigenetic factors regulate the readouts of genetic information and consequently many essential biological processes. However, it has been elusive whether RNA-directed DNA methylation (RdDM) affects root microbiota assembly. Results: By applying 16S rRNA gene sequencing, we investigated root microbiota of Arabidopsis mutants defective in the canonical RdDM pathway, including dcl234 that harbors triple mutation in the Dicer-like proteins DCL3, DCL2, and DCL4, which produce small RNAs for RdDM. Alpha diversity analysis showed reductions in microbe richness from the soil to roots, reflecting the selectivity of plants on root-associated bacteria. The dcl234 triple mutation significantly decreases the levels of Aeromonadaceae and Pseudomonadaceae, while it increases the abundance of many other bacteria families in the root microbiota. However, mutants of the other examined key players in the canonical RdDM pathway showed similar microbiota as Col-0, indicating that the DCL proteins affect root microbiota in an RdDM-independent manner. Subsequently gene analysis by shotgun sequencing of root microbiome indicated a selective pressure on microbial resistance to plant defense in the dcl234 mutant. Consistent with the altered plant-microbe interactions, dcl234 displayed altered characters, including the mRNA and sRNA transcriptomes that jointly highlighted altered cell wall organization and up-regulated defense, the decreased cellulose and callose deposition in root xylem, and the restructured profile of root exudates that supported the alterations in gene expression and cell wall modifications. Conclusion: Our findings demonstrate an important role of the DCL proteins in influencing root microbiota through integrated regulation of plant defense, cell wall compositions, and root exudates. Our results also demonstrate that the canonical RdDM is dispensable for Arabidopsis root microbiota. These findings not only establish a connection between root microbiota and plant epigenetic factors but also highlight the complexity of plant regulation of root microbiota. 9t4aP5wZAjZ4XcwBxRVNPd Video abstract [ABSTRACT FROM AUTHOR]

Summary: Plant stomata play a crucial role in leaf function, controlling water transpiration in response to environmental stresses and modulating the gas exchange necessary for photosynthesis. The phytohormone abscisic acid (ABA) promotes stomatal closure and inhibits light‐induced stomatal opening. The Arabidopsis thaliana E3 ubiquitin ligase COP1 functions in ABA‐mediated stomatal closure. However, the underlying molecular mechanisms are still not fully understood.Yeast two‐hybrid assays were used to identify ABA signaling components that interact with COP1, and biochemical, molecular and genetic studies were carried out to elucidate the regulatory role of COP1 in ABA signaling.The cop1 mutants are hyposensitive to ABA‐triggered stomatal closure under light and dark conditions. COP1 interacts with and ubiquitinates the Arabidopsis clade A type 2C phosphatases (PP2Cs) ABI/HAB group and AHG3, thus triggering their degradation. Abscisic acid enhances the COP1‐mediated degradation of these PP2Cs. Mutations in ABI1 and AHG3 partly rescue the cop1 stomatal phenotype and the phosphorylation level of OST1, a crucial SnRK2‐type kinase in ABA signaling.Our data indicate that COP1 is part of a novel signaling pathway promoting ABA‐mediated stomatal closure by regulating the stability of a subset of the Clade A PP2Cs. These findings provide novel insights into the interplay between ABA and the light signaling component in the modulation of stomatal movement. [ABSTRACT FROM AUTHOR]

Summary: As abscisic acid (ABA) receptors, PYR1/PYL/RCAR (PYLs) play important roles in ABA‐mediated seed germination, but the regulation of PYLs in this process, especially at the transcriptional level, remains unclear.In this study, we found that expression of 11 of 14 PYLs changes significantly during seed germination and is affected by exogenous ABA. Two PYLs, PYL11 and PYL12, both of which are expressed specifically in mature seeds, positively modulate ABA‐mediated seed germination.However, ABI5 was found to modulate the PYL11‐ and PYL12‐mediated ABA response. In the abi5‐7 mutant, ABA hypersensitivity caused by PYL11 and PYL12 overexpression was totally or partially blocked. By contrast, ABI5 regulates the expression of PYL11 and PYL12 by directly binding to their promoters. Moreover, the expression of eight other PYLs is also affected during the germination of abi5 mutants. Promoter analysis revealed that an ABI5‐binding region is present next to the TATA box or initiator box.Together, our data demonstrate the role of PYL11 and PYL12 in seed germination. In addition, the identification of PYLs as targets of ABI5 reveals a role of ABI5 in the feedback regulation of ABA‐mediated seed germination. [ABSTRACT FROM AUTHOR]

Trehalose plays important roles in plant growth and stress responses and is synthesized from trehalose‐6‐phosphate by trehalose‐6‐phosphate phosphatase (TPP). Here, we show that trehalose and abscisic acid (ABA) have synergistic effects on root growth and stomatal closure. The Arabidopsis thaliana genome contains ten genes encoding TPPs and the expression level of one, TPPE, and trehalose contents increased in response to ABA. In the presence of ABA, the ABA‐responsive transcription factor ABA RESPONSE ELEMENT BINDING FACTOR2 (ABF2) directly binds to the TPPE promoter to activate its expression. Genetic analysis revealed that TPPE acts downstream of ABF2, which is supported by the findings that TPPE expression and trehalose content are reduced in the abf2 mutant and that a mutation in TPPE abolished the ABA‐sensitive root elongation phenotype of 35S:ABF2 plants. Reactive oxygen species (ROS) accumulation in response to ABA failed to occur in tppe mutant plants, suggesting that TPPE is involved in ABA‐controlled root elongation and stomatal movement by inducing ROS accumulation. This study uncovers a new branch of the ABA signaling pathway and provides a molecular basis for the role of trehalose in plant responses to abiotic stress. [ABSTRACT FROM AUTHOR]

The plant hormone abscisic acid (ABA) regulates many processes of plant growth and development. ABA receptors have been identified in studies of the ABA response of stomatal movement, but the underlying mechanisms of ABA-regulated root growth and development are unclear. To address these questions, we screened for Arabidopsis thaliana mutants based on the sensitivity of root growth to exogenous ABA, using ethyl methanesulfonate-mutagenized (EMS) and T-DNA insertion mutant libraries. We identified 11 mutants, termed roa1–roa11, with Root growth Overly sensitive to ABA (ROA) phenotypes, and cloned two of the ROA genes, one by thermal asymmetric interlaced PCR technology (ROA3) and the other by map-based cloning (ROA9). The roa mutants were also found to have defects in other major ABA responses, including ABA-mediated seed germination and drought resistance. The roa mutants provide crucial genetic material for further studies of ABA signaling and regulatory mechanisms in root growth and development. [ABSTRACT FROM AUTHOR]

Cotton (Gossypium hirsutum L.) is a major crop and the main source of natural fiber worldwide. Because various abiotic and biotic stresses strongly influence cotton fiber yield and quality, improved stress resistance of this crop plant is urgently needed. In this study, we used Gateway technology to construct a normalized full‐length cDNA overexpressing (FOX) library from upland cotton cultivar ZM12 under various stress conditions. The library was transformed into Arabidopsis to produce a cotton‐FOX‐Arabidopsis library. Screening of this library yielded 6,830 transgenic Arabidopsis lines, of which 757 were selected for sequencing to ultimately obtain 659 cotton ESTs. GO and KEGG analyses mapped most of the cotton ESTs to plant biological process, cellular component, and molecular function categories. Next, 156 potential stress‐responsive cotton genes were identified from the cotton‐FOX‐Arabidopsis library under drought, salt, ABA, and other stress conditions. Four stress‐related genes identified from the library, designated as GhCAS, GhAPX, GhSDH, and GhPOD, were cloned from cotton complementary DNA, and their expression patterns under stress were analyzed. Phenotypic experiments indicated that overexpression of these cotton genes in Arabidopsis affected the response to abiotic stress. The method developed in this study lays a foundation for high‐throughput cloning and rapid identification of cotton functional genes. [ABSTRACT FROM AUTHOR]

Summary: Pigment glands, also known as black glands or gossypol glands, are specific for Gossypium spp. These glands strictly confine large amounts of secondary metabolites to the lysigenous cavity, leading to the glands' intense colour and providing defence against pests and pathogens. This study performed a comparative transcriptome analysis of glanded versus glandless cotton cultivars. Twenty‐two transcription factors showed expression patterns associated with pigment glands and were characterized. Phenotypic screening of the genes, via virus‐induced gene silencing, showed an apparent disappearance of pigmented glands after the silencing of a pair of homologous MYB‐encoding genes in the A and D genomes (designated as CGP1). Further study showed that CGP1a encodes an active transcription factor, which is specifically expressed in the gland structure, while CGP1d encodes a non‐functional protein due to a fragment deletion, which causes premature termination. RNAi‐mediated silencing and CRISPR knockout of CGP1 in glanded cotton cultivars generated a glandless‐like phenotype, similar to the dominant glandless mutant Gl2e. Microscopic analysis showed that CGP1 knockout did not affect gland structure or density, but affected gland pigmentation. The levels of gossypol and related terpenoids were significantly decreased in cgp1 mutants, and a number of gossypol biosynthetic genes were strongly down‐regulated. CGP1 is located in the nucleus where it interacts with GoPGF, a critical transcription factor for gland development and gossypol synthesis. Our data suggest that CGP1 and GoPGF form heterodimers to control the synthesis of gossypol and other secondary metabolites in cotton. [ABSTRACT FROM AUTHOR]

Background: ABC1K (Activity of BC1 complex Kinase) is an evolutionarily primitive atypical kinase family widely distributed among prokaryotes and eukaryotes. The ABC1K protein kinases in Arabidopsis are predicted to localize either to the mitochondria or chloroplasts, in which plastid-located ABC1K proteins are involved in the response against photo-oxidative stress and cadmium-induced oxidative stress. Results: Here, we report that the mitochondria-localized ABC1K10a functions in plant salt stress tolerance by regulating reactive oxygen species (ROS). Our results show that the ABC1K10a expression is induced by salt stress, and the mutations in this gene result in overaccumulation of ROS and hypersensitivity to salt stress. Exogenous application of the ROS-scavenger GSH significantly represses ROS accumulation and rescues the salt hypersensitive phenotype of abc1k10a. ROS overaccumulation in abc1k10a mutants under salt stress is likely due to the defect in mitochondria electron transport chain. Furthermore, defects of several other mitochondria-localized ABC1K genes also result in salt hypersensitivity. Conclusions: Taken together, our results reveal that the mitochondria-located ABC1K10a regulates mitochondrial ROS production and is a positive regulator of salt tolerance in Arabidopsis. [ABSTRACT FROM AUTHOR]

Background: The increase in atmospheric CO2 is causing a number of changes in plant growth such as increases in leaf area and number, branching, plant size and biomass, and growth rate. Despite the importance of stomatal responses to CO2, little is known about the genetic and molecular mechanisms that mediate stomatal development and movement in response to CO2 levels. Deciphering the mechanisms that sense changes in CO2 and/or HCO3− concentration is critical for unraveling the role of CO2 in stomatal development movement. In Arabidopsis, CO2-induced stomatal closure is strongly Ca2+-dependent. To further dissect this signaling pathway and identify new components in the CO2 response pathway, we recorded [Ca2+]cyt changes in mutagenized Arabidopsis leaves and screened for mutants with abnormal guard cell behavior in response to CO2/HCO3−. Results: We observed that 1 mM HCO3− induces [Ca2+]cys transient changes in guard cells and stomatal closure both in light and darkness. The changes in [Ca2+]cys induced by HCO3− could be detected by an aequorin-based calcium imaging system. Using this system, we identified a number of Arabidopsis mutants defective in both [Ca2+]cyt changes and the stomatal response to CO2/HCO3−. Conclusions: We provide a sensitive method for isolating stomatal CO2/HCO3− response genes that function early in stomatal closure and that have a role in regulating [Ca2+]cyt. This method will be helpful in elucidating the Ca2+-dependent regulation of guard cell behavior in response to CO2/HCO3−. [ABSTRACT FROM AUTHOR]

In plants, the spatiotemporal expression of circadian oscillators provides adaptive advantages in diverse species. However, the molecular basis of circadian clock in soybean is not known. In this study, we used soybean hairy roots expression system to monitor endogenous circadian rhythms and the sensitivity of circadian clock to environmental stimuli. We discovered in experiments with constant light and temperature conditions that the promoters of clock genes GmLCLb2 and GmPRR9b1 drive a self‐sustained, robust oscillation of about 24‐h in soybean hairy roots. Moreover, we demonstrate that circadian clock is entrainable by ambient light/dark or temperature cycles. Specifically, we show that light and cold temperature pulses can induce phase shifts of circadian rhythm, and we found that the magnitude and direction of phase responses depends on the specific time of these two zeitgeber stimuli. We obtained a quadruple mutant lacking the soybean gene GmLCLa1, LCLa2, LCLb1, and LCLb2 using CRISPR, and found that loss‐of‐function of these four GmLCL orthologs leads to an extreme short‐period circadian rhythm and late‐flowering phenotype in transgenic soybean. Our study establishes that the morning‐phased GmLCLs genes act constitutively to maintain circadian rhythmicity and demonstrates that their absence delays the transition from vegetative growth to reproductive development. We investigated the molecular characteristics and the physiological function of circadian oscillators in soybean. We found that soybean circadian clock is very sensitive to ambient light/temperature signals, and GmLCL genes are required for self‐sustained circadian period length and the timing of flowering. [ABSTRACT FROM AUTHOR]

PHYTOCHROME-INTERACTING FACTORS (PIFs) are a group of basic helix-loop-helix transcription factors that can physically interact with photoreceptors, including phytochromes and cryptochromes. It was previously demonstrated that PIFs accumulated in darkness and repressed seedling photomorphogenesis, and that PIFs linked different photosensory and hormonal pathways to control plant growth and development. In this study, we show that PIFs positively regulate the ABA signaling pathway during the seedling stage specifically in darkness. We found that PIFs positively regulate ABI5 transcript and protein levels in darkness in response to exogenous ABA treatment by binding directly to the G-box motifs in the ABI5 promoter. Consistently, PIFs and the G-box motifs in the ABI5 promoter determine ABI5 expression in darkness, and overexpression of ABI5 could rescue the ABA-insensitive phenotypes of pifq mutants in the dark. Moreover, we discovered that PIFs can physically interact with the ABA receptors PYL8 and PYL9, and that this interaction is not regulated by ABA. Further analyses showed that PYL8 and PYL9 promote PIF4 protein accumulation in the dark and enhance PIF4 binding to the ABI5 promoter, but negatively regulate PIF4-mediated ABI5 activation. Taken together, our data demonstrate that PIFs interact with ABA receptors to orchestrate ABA signaling in darkness by controlling ABI5 expression, providing new insights into the pivotal roles of PIFs as signal integrators in regulating plant growth and development. This study reveals that PIFs positively regulate the ABA signaling pathway in darkness by binding directly to the G-box motifs in the ABI5 promoter. Moreover, the ABA receptors PYL8 and PYL9 physically interact with PIFs and regulate the protein abundance and transcriptional activity of PIF4. Thus, PIFs function as dark-specific ABA signaling components by linking ABA receptors with ABI5 expression. [ABSTRACT FROM AUTHOR]

Osmoregulation is important for plant growth, development and response to environmental changes. SNF1-related protein kinase 2s (SnRK2s) are quickly activated by osmotic stress and are central components in osmotic stress and abscisic acid (ABA) signaling pathways; however, the upstream components required for SnRK2 activation and early osmotic stress signaling are still unknown. Here, we report a critical role for B2, B3 and B4 subfamilies of Raf-like kinases (RAFs) in early osmotic stress as well as ABA signaling in Arabidopsis thaliana. B2, B3 and B4 RAFs are quickly activated by osmotic stress and are required for phosphorylation and activation of SnRK2s. Analyses of high-order mutants of RAFs reveal critical roles of the RAFs in osmotic stress tolerance and ABA responses as well as in growth and development. Our findings uncover a kinase cascade mediating osmoregulation in higher plants. Rapid activation of SnRK2 kinases is central to plant responses to osmotic stress and abscisic acid. Here the authors show that a group of Raf-like kinases are very quickly activated by osmotic stress, and then phosphorylate and activate SnRK2s. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) is an important phytohormone regulating plant growth, development, and stress responses. It has an essential role in multiple physiological processes of plants, such as stomatal closure, cuticular wax accumulation, leaf senescence, bud dormancy, seed germination, osmotic regulation, and growth inhibition among many others. Abscisic acid controls downstream responses to abiotic and biotic environmental changes through both transcriptional and posttranscriptional mechanisms. During the past 20 years, ABA biosynthesis and many of its signaling pathways have been well characterized. Here we review the dynamics of ABA metabolic pools and signaling that affects many of its physiological functions. [ABSTRACT FROM AUTHOR]

Key message: Su1-Ph1, which we previously introgressed into wheat from Aegilops speltoides, is a potent suppressor of Ph1 and a valuable tool for gene introgression in tetraploid wheat. We previously introgressed Su1-Ph1, a suppressor of the wheat Ph1 gene, from Aegilops speltoides into durum wheat cv Langdon (LDN). Here, we evaluated the utility of the introgressed suppressor for inducing introgression of alien germplasm into durum wheat. We built LDN plants heterozygous for Su1-Ph1 that simultaneously contained a single LDN chromosome 5B and a single Ae. searsii chromosome 5Sse, which targeted them for recombination. We genotyped 28 BC1F1 and 84 F2 progeny with the wheat 90-K Illumina single-nucleotide polymorphism assay and detected extensive recombination between the two chromosomes, which we confirmed by non-denaturing fluorescence in situ hybridization (ND-FISH). We constructed BC1F1 and F2 genetic maps that were 65.31 and 63.71 cM long, respectively. Recombination rates between the 5B and 5Sse chromosomes were double the expected rate computed from their meiotic pairing, which we attributed to selection against aneuploid gametes. Recombination rate between 5B and 5Sse was depressed compared to that between 5B chromosomes in the proximal region of the long arm. We integrated ND-FISH signals into the genetic map and constructed a physical map, which we used to map a 172,188,453-bp Ph1 region. Despite the location of the region in a low-recombination region of the 5B chromosome, we detected three crossovers in it. Our data show that Su1-Ph1 is a valuable tool for gene introgression and gene mapping based on recombination between homoeologous chromosomes in wheat. [ABSTRACT FROM AUTHOR]

Pseudouridine (Ψ) is widely distributed in mRNA and various non-coding RNAs in yeast and mammals, and the specificity of its distribution has been determined. However, knowledge about Ψs in the RNAs of plants, particularly in mRNA, is lacking. In this study, we performed genome-wide pseudouridine-sequencing in Arabidopsis and for the first time identified hundreds of Ψ sites in mRNA and multiple Ψ sites in non-coding RNAs. Many predicted and novel Ψ sites in rRNA and tRNA were detected. mRNA was extensively pseudouridylated, but with Ψs being under-represented in 3′-untranslated regions and enriched at position 1 of triple codons. The phenylalanine codon UUC was the most frequently pseudouridylated site. Some Ψs present in chloroplast 23S, 16S, and 4.5S rRNAs in wild-type Col-0 were absent in plants with a mutation of SVR1 (Suppressor of variegation 1), a chloroplast pseudouridine synthase gene. Many plastid ribosomal proteins and photosynthesis-related proteins were significantly reduced in svr1 relative to the wild-type, indicating the roles of SVR1 in chloroplast protein biosynthesis in Arabidopsis. Our results provide new insights into the occurrence of pseudouridine in Arabidopsis RNAs and the biological functions of SVR1, and will pave the way for further exploiting the mechanisms underlying Ψ modifications in controlling gene expression and protein biosynthesis in plants. [ABSTRACT FROM AUTHOR]

Intercellular communication in adjacent cell layers determines cell fate and polarity, thus orchestrating tissue specification and differentiation. Here we use the maize stomatal apparatus as a model to investigate cell fate determination. Mutations in ZmBZU2 (ii2, bzu2) confer a complete absence of subsidiary cells (SCs) and normal guard cells (GCs), leading to failure of formation of mature stomatal complexes. Nuclear polarization and actin accumulation at the interface between subsidiary mother cells (SMCs) and guard mother cells (GMCs), an essential pre-requisite for asymmetric cell division, did not occur in Zmbzu2 mutants. ZmBZU2 encodes a basic helix-loop-helix (bHLH) transcription factor, which is an ortholog of AtMUTE in Arabidopsis (BZU2/ZmMUTE). We found that a number of genes implicated in stomatal development are transcriptionally regulated by BZU2/ZmMUTE. In particular, BZU2/ZmMUTE directly binds to the promoters of PAN1 and PAN2, two early regulators of protodermal cell fate and SMC polarization, consistent with the low levels of transcription of these genes observed in bzu2-1 mutants. BZU2/ZmMUTE has the cell-to-cell mobility characteristic similar to that of BdMUTE in Brachypodium distachyon. Unexpectedly, BZU2/ZmMUTE is expressed in GMC from the asymmetric division stage to the GMC division stage, and especially in the SMC establishment stage. Taken together, these data imply that BZU2/ZmMUTE is required for early events in SMC polarization and differentiation as well as for the last symmetrical division of GMCs to produce the two GCs, and is a master determinant of the cell fate of its neighbors through cell-to-cell communication. [ABSTRACT FROM AUTHOR]

Anthocyanins are secondary metabolites that play important roles in plant adaption to adverse environments. The anthocyanin biosynthetic pathway is conserved in high plants. Previous studies revealed the significant role of anthocyanins in natural-colorized cotton. However, little is known about the involvement of anthocyanins in the interaction of cotton and pathogen. In this study, a pathogen-induced gene was isolated from Gossypium barbadense that encodes an anthocyanidin synthase protein (GbANS) with dioxygenase structures. GbANS was preferentially expressed in colored tissue. Silencing of GbANS significantly reduced the production of anthocyanins, as well as the cotton’s resistance to Verticillium dahliae. Biochemical studies revealed that GbANS-silenced cotton accumulated more hydrogen peroxide compared to control plants during the V. dahliae invasion process. This accumulation of hydrogen peroxide corresponded with increased cell death around the invasion sites, which in turn accelerated the V. dahliae infection. Taken together, we found that GbANS contributes to the biosynthesis of anthocyanins in cotton and anthocyanins positively regulate cotton’s resistance to V. dahliae. [ABSTRACT FROM AUTHOR]

Abstract In plants, stomatal movements are tightly controlled by changes in cellular turgor pressure. Carbohydrates produced by glycolysis and the tricarboxylic acid cycle play an important role in regulating turgor pressure. Here, we describe an Arabidopsis mutant, bzu1 , isolated in a screen for elevated leaf temperature in response to drought stress, which displays smaller stomatal pores and higher drought resistance than wild-type plants. BZU1 encodes a known acetyl-coenzyme A synthetase, ACN1, which acts in the first step of a metabolic pathway converting acetate to malate in peroxisomes. We showed that BZU1/ACN1-mediated acetate-to-malate conversion provides a shunt that plays an important role in osmoregulation of stomatal turgor. We found that the smaller stomatal pores in the bzu1 mutant are a consequence of reduced accumulation of malate, which acts as an osmoticum and/or a signaling molecule in the control of turgor pressure within guard cells, and these results provided new genetic evidence for malate-regulated stomatal movement. Collectively, our results indicate that a peroxisomal BZU1/ACN1-mediated acetate–malate shunt regulates drought resistance by controlling the turgor pressure of guard cells in Arabidopsis. This study reveals that a peroxisomal BZU1/ACN1-mediated acetate–malate shunt produces malate, an osmotically active solute and/or signaling molecule, to regulate the turgor pressure of guard cells, thereby controlling guard cell opening and regulating drought resistance in Arabidopsis. [ABSTRACT FROM AUTHOR]

Abstract: Stomata, the pores formed by a pair of guard cells, are the main gateways for water transpiration and photosynthetic CO2 exchange, as well as pathogen invasion in land plants. Guard cell movement is regulated by a combination of environmental factors, including water status, light, CO2 levels and pathogen attack, as well as endogenous signals, such as abscisic acid and apoplastic reactive oxygen species (ROS). Under abiotic and biotic stress conditions, extracellular ROS are mainly produced by plasma membrane‐localized NADPH oxidases, whereas intracellular ROS are produced in multiple organelles. These ROS form a sophisticated cellular signaling network, with the accumulation of apoplastic ROS an early hallmark of stomatal movement. Here, we review recent progress in understanding the molecular mechanisms of the ROS signaling network, primarily during drought stress and pathogen attack. We summarize the roles of apoplastic ROS in regulating stomatal movement, ABA and CO2 signaling, and immunity responses. Finally, we discuss ROS accumulation and communication between organelles and cells. This information provides a conceptual framework for understanding how ROS signaling is integrated with various signaling pathways during plant responses to abiotic and biotic stress stimuli. [ABSTRACT FROM AUTHOR]