FERONIA regulates salt tolerance in Arabidopsis by controlling photorespiratory flux.

Photorespiration is an energetically costly metabolic pathway in plants that responds to environmental stresses. The molecular basis of the regulation of the photorespiratory cycle under stress conditions remains unclear. Here, we discovered that FERONIA (FER) regulates photorespiratory flow under salt stress in Arabidopsis (Arabidopsis thaliana). FER mutation results in hypersensitivity to salt stress, but disruption of ferredoxin-dependent glutamate synthase 1 (GLU1), an enzyme that participates in the photorespiratory pathway by producing glutamate, greatly suppresses fer-4 hypersensitivity to salt stress primarily due to reduced glycine yield. In contrast, disrupting mitochondrial serine hydroxymethyltransferase1 (SHM1), which is supposed to increase glycine levels by hampering the conversion of glycine to serine in the photorespiratory cycle, aggravates fer-4 hypersensitivity to salt stress. Biochemical data show that FER interacts with and phosphorylates SHM1, and this phosphorylation modulates SHM1 stability. Additionally, the production of proline and its intermediate △1-pyrroline-5-carboxylate (P5C), which are both synthesized from glutamate, also contributes to fer-4 hypersensitivity to salt stress. In conclusion, this study elucidates the functional mechanism of FER in regulating salt tolerance by modulating photorespiratory flux, which greatly broadens our understanding of how plants adapt to high salinity.

Simultaneous mutations in ITPK4 and MRP5 genes result in a low phytic acid level without compromising salt tolerance in Arabidopsis.

Generation of crops with low phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP6)) is an important breeding direction, but such plants often display less desirable agronomic traits. In this study, through ethyl methanesulfonate-mediated mutagenesis, we found that inositol 1,3,4-trisphosphate 5/6-kinase 4 (ITPK4), which is essential for producing InsP6, is a critical regulator of salt tolerance in Arabidopsis. Loss of function of ITPK4 gene leads to reduced root elongation under salt stress, which is primarily because of decreased root meristem length and reduced meristematic cell number. The itpk4 mutation also results in increased root hair density and increased accumulation of reactive oxygen species during salt exposure. RNA sequencing assay reveals that several auxin-responsive genes are down-regulated in the itpk4-1 mutant compared to the wild-type. Consistently, the itpk4-1 mutant exhibits a reduced auxin level in the root tip and displays compromised gravity response, indicating that ITPK4 is involved in the regulation of the auxin signaling pathway. Through suppressor screening, it was found that mutation of Multidrug Resistance Protein 5 (MRP5)5 gene, which encodes an ATP-binding cassette (ABC) transporter required for transporting InsP6 from the cytoplasm into the vacuole, fully rescues the salt hypersensitivity of the itpk4-1 mutant, but in the itpk4-1 mrp5 double mutant, InsP6 remains at a very low level. These results imply that InsP6 homeostasis rather than its overall amount is beneficial for stress tolerance in plants. Collectively, this study uncovers a pair of gene mutations that confer low InsP6 content without impacting stress tolerance, which offers a new strategy for creating "low-phytate" crops.

Cell wall-mediated root development is targeted by a soil-borne bacterial pathogen to promote infection.

Plant pathogens manipulate host development, facilitating colonization and proliferation. Ralstonia solanacearum is a soil-borne bacterial pathogen that penetrates roots and colonizes plants through the vascular system, causing wilting and death. Here, we find that RipAC, an effector protein from R. solanacearum, alters root development in Arabidopsis, promoting the formation of lateral roots and root hairs. RipAC interacts with CELLULOSE SYNTHASE (CESA)-INTERACTIVE PROTEIN 1 (CSI1), which regulates the activity of CESA complexes at the plasma membrane. RipAC disrupts CESA-CSI1 interaction, leading to a reduction in cellulose content, root developmental alterations, and a promotion of bacterial pathogenicity. We find that CSI1 also associates with the receptor kinase FERONIA, forming a complex that negatively regulates immunity in roots; this interaction, however, is not affected by RipAC. Our work reveals a bacterial virulence strategy that selectively affects the activities of a host target, promoting anatomical alterations that facilitate infection without causing activation of immunity.

Identification of major QTLs for drought tolerance in soybean, together with a novel candidate gene, GmUAA6.

Drought tolerance is a complex trait in soybean that is controlled by polygenetic quantitative trait loci (QTLs). In this study, wilting score, days-to-wilting, leaf relative water content, and leaf relative conductivity were used to identify QTLs associated with drought tolerance in recombinant inbred lines derived from a cross between a drought-sensitive variety, Lin, and a drought-tolerant variety, Meng. A total of 33 drought-tolerance QTLs were detected. Of these 17 were major QTLs. In addition, 15 were novel drought-tolerance QTLs. The most predominant QTL was on chromosome 11. This was detected in at least three environments. The overlapped mapping interval of the four measured traits was 0.2 cM in genetic distance (about 220 kb in physical length). Glyma.11g143500 (designated as GmUAA6), which encodes a UDP-N-acetylglucosamine transporter, was identified as the most likely candidate gene. The allele of GmUAA6 from Lin (GmUAA6Lin) was associated with improved soybean drought tolerance. Overexpression of GmUAA6Lin in Arabidopsis and soybean hairy roots enhanced drought tolerance. Furthermore, a 3-bp insertion/deletion (InDel) in the coding sequence of GmUAA6 explained up to 49.9% of the phenotypic variation in drought tolerance-related traits, suggesting that this InDel might be used in future marker-assisted selection of drought-tolerant lines in soybean breeding programs.

Liquid-liquid phase separation as a major mechanism of plant abiotic stress sensing and responses.

Identification of environmental stress sensors is one of the most important research topics in plant abiotic stress research. Traditional strategies to identify stress sensors or early signaling components based on the cell membrane as a primary site of sensing and calcium signal as a second messenger have had only limited successes. Therefore, the current theoretical framework underlying stress sensing in plants should be reconsidered and additional mechanisms need to be introduced. Recently, accumulating evidence has emerged to suggest that liquid-liquid phase separation (LLPS) is a major mechanism for environmental stress sensing and response in plants. In this review, we briefly introduce LLPS regarding its concept, compositions, and dynamics, and then summarize recent progress of LLPS research in plants, emphasizing the contribution of LLPS to the sensing of various environmental stresses, such as dehydration, osmotic stress, and low and high temperatures. Finally, we propose strategies to identify key proteins that sense and respond to environmental stimuli on the basis of LLPS, and discuss the research directions of LLPS in plant abiotic stress responses and its potential application in enhancing stress tolerance in crops.

FERONIA coordinates plant growth and salt tolerance via the phosphorylation of phyB.

Phosphorylation modification is required for the modulation of phytochrome B (phyB) thermal reversion, but the kinase(s) that phosphorylate(s) phyB and the biological significance of the phosphorylation are still unknown. Here we report that FERONIA (FER) phosphorylates phyB to regulate plant growth and salt tolerance, and the phosphorylation not only regulates dark-triggered photobody dissociation but also modulates phyB protein abundance in the nucleus. Further analysis indicates that phosphorylation of phyB by FER is sufficient to accelerate the conversion of phyB from the active form (Pfr) to the inactive form (Pr). Under salt stress, FER kinase activity is inhibited, leading to delayed photobody dissociation and increased phyB protein abundance in the nucleus. Our data also show that phyB mutation or overexpression of PIF5 attenuates growth inhibition and promotes plant survival under salt stress. Together, our study not only reveals a kinase that controls phyB turnover via a signature of phosphorylation, but also provides mechanistic insights into the role of the FER-phyB module in coordinating plant growth and stress tolerance.

The MEKK1-MKK1/2-MPK4 cascade phosphorylates and stabilizes STOP1 to confer aluminum resistance in Arabidopsis.

Aluminum (Al) toxicity can seriously restrict crop production on acidic soils, which comprise 40% of the world's potentially arable land. The zinc finger transcription factor STOP1 has a conserved and essential function in mediating plant Al resistance. Al stress induces STOP1 accumulation via post-transcriptional regulatory mechanisms. However, the upstream signaling pathway involved in Al-triggered STOP1 accumulation remains unclear. Here, we report that the MEKK1-MKK1/2-MPK4 cascade positively regulates STOP1 phosphorylation and stability. Mutations of MEKK1, MKK1/2, or MPK4 lead to decreased STOP1 stability and Al resistance. Al stress induces the kinase activity of MPK4, which interacts with and phosphorylates STOP1. The phosphorylation of STOP1 reduces its interaction with the F-box protein RAE1 that mediates STOP1 degradation, thereby leading to enhanced STOP1 stability and Al resistance. Taken together, our results suggest that the MEKK1-MKK1/2-MPK4 cascade is important for Al signaling and confers Al resistance through phosphorylation-mediated enhancement of STOP1 accumulation in Arabidopsis.

The cell biology of primary cell walls during salt stress.

Salt stress simultaneously causes ionic toxicity, osmotic stress, and oxidative stress, which directly impact plant growth and development. Plants have developed numerous strategies to adapt to saline environments. Whereas some of these strategies have been investigated and exploited for crop improvement, much remains to be understood, including how salt stress is perceived by plants and how plants coordinate effective responses to the stress. It is, however, clear that the plant cell wall is the first contact point between external salt and the plant. In this context, significant advances in our understanding of halotropism, cell wall synthesis, and integrity surveillance, as well as salt-related cytoskeletal rearrangements, have been achieved. Indeed, molecular mechanisms underpinning some of these processes have recently been elucidated. In this review, we aim to provide insights into how plants respond and adapt to salt stress, with a special focus on primary cell wall biology in the model plant Arabidopsis thaliana.

Functional analysis of CqPORB in the regulation of chlorophyll biosynthesis in Chenopodium quinoa.

Protochlorophyllide oxidoreductase (POR) plays a key role in catalyzing the light-dependent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), and thus promotes the transit from etiolated seedlings to green plants. In this study, by exploring ethyl methanesulfonate (EMS)-mediated mutagenesis in Chenopodium quinoa NL-6 variety, we identified a mutant nl6-35 that displays faded green leaf and reduced chlorophyll (Chl) and carotenoid contents. Bulk segregant analysis (BSA) revealed that a mutation in CqPORB gene is genetically associated with the faded green leaf of the nl6-35 mutant. Further study indicates that the nl6-35 mutant exhibits abnormal grana stacks and compromised conversion of Pchlide to Chlide upon illumination, suggesting the important role of CqPORB in producing photoactive Pchlide. Totally three CqPOR isoforms, including CqPORA, CqPORA-like, and CqPORB are identified in NL-6 variety. Transcriptional analysis shows that the expression of all these three CqPOR isoforms is regulated in light- and development-dependent manners, and in mature quinoa plants only CqPORB isoform is predominantly expressed. Subcellular localization analysis indicates that CqPORB is exclusively localized in chloroplast. Together, our study elucidates the important role of CqPORB in the regulation of Chl biosynthesis and chloroplast development in quinoa.

CRISPR/Cas9-mediated mutation in auxin efflux carrier OsPIN9 confers chilling tolerance by modulating reactive oxygen species homeostasis in rice.

Phytohormone auxin plays a vital role in plant development and responses to environmental stresses. The spatial and temporal distribution of auxin mainly relies on the polar distribution of the PIN-FORMED (PIN) auxin efflux carriers. In this study, we dissected the functions of OsPIN9, a monocot-specific auxin efflux carrier gene, in modulating chilling tolerance in rice. The results showed that OsPIN9 expression was dramatically and rapidly suppressed by chilling stress (4°C) in rice seedlings. The homozygous ospin9 mutants were generated by CRISPR/Cas9 technology and employed for further research. ospin9 mutant roots and shoots were less sensitive to 1-naphthaleneacetic acid (NAA) and N-1-naphthylphthalamic acid (NPA), indicating the disturbance of auxin homeostasis in the ospin9 mutants. The chilling tolerance assay showed that ospin9 mutants were more tolerant to chilling stress than wild-type (WT) plants, as evidenced by increased survival rate, decreased membrane permeability, and reduced lipid peroxidation. However, the expression of well-known C-REPEAT BINDING FACTOR (CBF)/DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 1 (DREB)-dependent transcriptional regulatory pathway and Ca2+ signaling genes was significantly induced only under normal conditions, implying that defense responses in ospin9 mutants have probably been triggered in advance under normal conditions. Histochemical staining of reactive oxygen species (ROS) by 3'3-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) showed that ospin9 mutants accumulated more ROS than WT at the early stage of chilling stress, while less ROS was observed at the later stage of chilling treatment in ospin9 mutants. Consistently, antioxidant enzyme activity, including catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), improved significantly during the early chilling treatments, while was kept similar to WT at the later stage of chilling treatment, implying that the enhanced chilling tolerance of ospin9 mutants is mainly attributed to the earlier induction of ROS and the improved ROS scavenging ability at the subsequent stages of chilling treatment. In summary, our results strongly suggest that the OsPIN9 gene regulates chilling tolerance by modulating ROS homeostasis in rice.

Genome-Wide Analysis of CqCrRLK1L and CqRALF Gene Families in Chenopodium quinoa and Their Roles in Salt Stress Response.

Chenopodium quinoa is a halophyte with exceptional nutritional qualities, and therefore it is potentially an ideal crop to grow in saline soils, not only addressing the problem of land salinization, but also providing nutrient food for the health of humans. Currently, the molecular mechanisms underlying salt tolerance in quinoa are still largely unknown. In Arabidopsis thaliana, Catharanthus roseus receptor-like kinase (CrRLK1Ls) FERONIA (FER) and its ligands rapid alkalinization factors (RALFs) have been reported that participate in the regulation of salt tolerance. Here, we performed a genome-wide analysis and identified 26 CqCrRLK1L and 18 CqRALF family genes in quinoa genome. Transcriptomic profiling of the leaf, root, stamen, and pistil tissues of quinoa reveals that different CqCrRLK1L and CqRALF genes exhibit tissue-specific expression patterns, which is consistent with that observed in other plant species. RNA-seq data show that three CqCrRLK1L genes are highly up-regulated after salt treatment, suggesting that some CqCrRLK1L family genes are transcriptionally responsive to salt stress in quinoa. Biochemical study indicates that CqRALF15, a paralog of Arabidopsis RALF22, is physically associated with CrRLK1L proteins CqFER and AtFER. CqRALF15 and AtRALF22 are functionally conserved in inducing the internalization of AtFER and in triggering root growth inhibition in both quinoa and Arabidopsis. Moreover, overexpression of CqRALF15 in Arabidopsis results in enhanced leaf bleaching under salt stress, indicating that CqRALF15 is involved in salt stress response. Together, our study characterizes CqCrRLK1L and CqRALF family genes in quinoa at genomic, transcriptional, and protein levels, and provides evidence to support their roles in salt stress response.

A novel mitochondrial protein is required for cell wall integrity, auxin accumulation and root elongation in Arabidopsis under salt stress.

Maintenance of root elongation is beneficial for the growth and survival of plants under salt stress, but currently the cellular components involved in the regulation of root growth under high salinity are not fully understood. In this study, we identified an Arabidopsis mutant, rres1, which exhibited reduced root elongation under treatment of a variety of salts, including NaCl, NaNO3, KCl, and KNO3. RRES1 encodes a novel mitochondrial protein and its molecular function is still unknown. Under salt stress, the root meristem length was shorter in the rres1 mutant compared to the wild type, which was correlated with a reduced auxin accumulation in the mutant. Reactive oxygen species (ROS), as important signals that regulate root elongation, were accumulated to higher levels in the rres1 mutant than the wild type after salt treatment. Measurement of monosaccharides in the cell wall showed that arabinose and xylose contents were decreased in the rres1 mutant under salt stress, and application of boric acid, which is required for the crosslinking of pectic polysaccharide rhamnogalacturonan-II (RG-II), largely rescued the root growth arrest of the rres1 mutant, suggesting that RRES1 participates in the maintenance of cell wall integrity under salt stress. GUS staining assay indicated that the RRES1 gene was expressed in leaves and weakly in root tip under normal conditions, but its expression was dramatically increased in leaves and roots after salt treatment. Together, our study reveals a novel mitochondrial protein that regulates root elongation under salt stress via the modulation of cell wall integrity, auxin accumulation, and ROS homeostasis.

The LRXs-RALFs-FER module controls plant growth and salt stress responses by modulating multiple plant hormones.

Salt stress is a major environmental factor limiting plant growth and productivity. We recently discovered an important new salt tolerance pathway, where the cell wall leucine-rich repeat extensins LRX3/4/5, the RAPID ALKALINIZATION FACTOR (RALF) peptides RALF22/23 and receptor-like kinase FERONIA (FER) function as a module to simultaneously regulate plant growth and salt stress tolerance. However, the intracellular signaling pathways that are regulated by the extracellular LRX3/4/5-RALF22/23-FER module to coordinate growth, cell wall integrity and salt stress responses are still unknown. Here, we report that the LRX3/4/5-RALF22/23-FER module negatively regulates the levels of jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA). Blocking JA pathway rescues the dwarf phenotype of the lrx345 and fer-4 mutants, while disruption of ABA biosynthesis suppresses the salt-hypersensitivity of these mutants. Many salt stress-responsive genes display abnormal expression patterns in the lrx345 and fer-4 mutants, as well as in the wild type plants treated with epigallocatechin gallate (EGCG), an inhibitor of pectin methylesterases, suggesting cell wall integrity as a critical factor that determines the expression pattern of stress-responsive genes. Production of reactive oxygen species (ROS) is constitutively increased in the lrx345 and fer-4 mutants, and inhibition of ROS accumulation suppresses the salt-hypersensitivity of these mutants. Together, our work provides strong evidence that the LRX3/4/5-RALF22/23-FER module controls plant growth and salt stress responses by regulating hormonal homeostasis and ROS accumulation.

Genome-wide identification of ZmSnRK2 genes and functional analysis of ZmSnRK2.10 in ABA signaling pathway in maize (Zea mays L).

BACKGROUND: Phytohormone abscisic acid (ABA) is involved in the regulation of a wide range of biological processes. In Arabidopsis, it has been well-known that SnRK2s are the central components of the ABA signaling pathway that control the balance between plant growth and stress response, but the functions of ZmSnRK2 in maize are rarely reported. Therefore, the study of ZmSnRK2 is of great importance to understand the ABA signaling pathways in maize. RESULTS: In this study, 14 ZmSnRK2 genes were identified in the latest version of maize genome database. Phylogenetic analysis revealed that ZmSnRK2s are divided into three subclasses based on their diversity of C-terminal domains. The exon-intron structures, phylogenetic, synteny and collinearity analysis indicated that SnRK2s, especially the subclass III of SnRK2, are evolutionally conserved in maize, rice and Arabidopsis. Subcellular localization showed that ZmSnRK2 proteins are localized in the nucleus and cytoplasm. The RNA-Seq datasets and qRT-PCR analysis showed that ZmSnRK2 genes exhibit spatial and temporal expression patterns during the growth and development of different maize tissues, and the transcript levels of some ZmSnRK2 genes in kernel are significantly induced by ABA and sucrose treatment. In addition, we found that ZmSnRK2.10, which belongs to subclass III, is highly expressed in kernel and activated by ABA. Overexpression of ZmSnRK2.10 partially rescued the ABA-insensitive phenotype of snrk2.2/2.3 double and snrk2.2/2.3/2.6 triple mutants and led to delaying plant flowering in Arabidopsis. CONCLUSION: The SnRK2 gene family exhibits a high evolutionary conservation and has expanded with whole-genome duplication events in plants. The ZmSnRK2s expanded in maize with whole-genome and segmental duplication, not tandem duplication. The expression pattern analysis of ZmSnRK2s in maize offers important information to study their functions. Study of the functions of ZmSnRK.10 in Arabidopsis suggests that the ABA-dependent members of SnRK2s are evolutionarily conserved in plants. Our study elucidated the structure and evolution of SnRK2 genes in plants and provided a basis for the functional study of ZmSnRK2s protein in maize.

Maintenance of Cell Wall Integrity under High Salinity.

Cell wall biosynthesis is a complex biological process in plants. In the rapidly growing cells or in the plants that encounter a variety of environmental stresses, the compositions and the structure of cell wall can be dynamically changed. To constantly monitor cell wall status, plants have evolved cell wall integrity (CWI) maintenance system, which allows rapid cell growth and improved adaptation of plants to adverse environmental conditions without the perturbation of cell wall organization. Salt stress is one of the abiotic stresses that can severely disrupt CWI, and studies have shown that the ability of plants to sense and maintain CWI is important for salt tolerance. In this review, we highlight the roles of CWI in salt tolerance and the mechanisms underlying the maintenance of CWI under salt stress. The unsolved questions regarding the association between the CWI and salt tolerance are discussed.

SWO1 modulates cell wall integrity under salt stress by interacting with importin ɑ in Arabidopsis.

Maintenance of cell wall integrity is of great importance not only for plant growth and development, but also for the adaptation of plants to adverse environments. However, how the cell wall integrity is modulated under salt stress is still poorly understood. Here, we report that a nuclear-localized Agenet domain-containing protein SWO1 (SWOLLEN 1) is required for the maintenance of cell wall integrity in Arabidopsis under salt stress. Mutation in SWO1 gene results in swollen root tips, disordered root cell morphology, and root elongation inhibition under salt stress. The swo1 mutant accumulates less cellulose and pectin but more lignin under high salinity. RNA-seq and ChIP-seq assays reveal that SWO1 binds to the promoter of several cell wall-related genes and regulates their expression under saline conditions. Further study indicates that SWO1 interacts with importin ɑ IMPA1 and IMPA2, which are required for the import of nuclear-localized proteins. The impa1 impa2 double mutant also exhibits root growth inhibition under salt stress and mutations of these two genes aggravate the salt-hypersensitive phenotype of the swo1 mutant. Taken together, our data suggest that SWO1 functions together with importin ɑ to regulate the expression of cell wall-related genes, which enables plants to maintain cell wall integrity under high salinity.

CDK8 is associated with RAP2.6 and SnRK2.6 and positively modulates abscisic acid signaling and drought response in Arabidopsis.

CDK8 is a key subunit of Mediator complex, a large multiprotein complex that is a fundamental part of the conserved eukaryotic transcriptional machinery. However, the biological functions of CDK8 in plant abiotic stress responses remain largely unexplored. Here, we demonstrated CDK8 as a critical regulator in the abscisic acid (ABA) signaling and drought response pathways in Arabidopsis. Compared to wild-type, cdk8 mutants showed reduced sensitivity to ABA, impaired stomatal apertures and hypersensitivity to drought stress. Transcriptomic and chromatin immunoprecipitation analysis revealed that CDK8 positively regulates the transcription of several ABA-responsive genes, probably through promoting the recruitment of RNA polymerase II to their promoters. We discovered that both CDK8 and SnRK2.6 interact physically with an ERF/AP2 transcription factor RAP2.6, which can directly bind to the promoters of RD29A and COLD-REGULATED 15A (COR15A) with GCC or DRE elements, thereby promoting their expression. Importantly, we also showed that CDK8 is essential for the ABA-induced expression of RAP2.6 and RAP2.6-mediated upregulation of ABA-responsive genes, indicating that CDK8 could link the SnRK2.6-mediated ABA signaling to RNA polymerase II to promote immediate transcriptional response to ABA and drought signals. Overall, our data provide new insights into the roles of CDK8 in modulating ABA signaling and drought responses.

The transcription factor ICE1 functions in cold stress response by binding to the promoters of CBF and COR genes.

A recent paper by Kidokoro et al. (2020) in The Plant Cell reported a transgene-dependent transcriptional silencing phenomenon in the dominant ice1-1 Arabidopsis mutant containing the CBF3-LUC reporter, and questioned whether ICE1 may regulate CBF genes and may be involved in plant cold response. Here, we evaluate available evidence supporting the involvement of ICE1 in plant cold response, and provide ChIP-seq data showing ICE1 binding to the promoters of CBF genes and other regulatory genes known to be critical for cold response as well as to the promoters of some COR genes.

Mapping proteome-wide targets of protein kinases in plant stress responses.

Protein kinases are major regulatory components in almost all cellular processes in eukaryotic cells. By adding phosphate groups, protein kinases regulate the activity, localization, protein-protein interactions, and other features of their target proteins. It is known that protein kinases are central components in plant responses to environmental stresses such as drought, high salinity, cold, and pathogen attack. However, only a few targets of these protein kinases have been identified. Moreover, how these protein kinases regulate downstream biological processes and mediate stress responses is still largely unknown. In this study, we introduce a strategy based on isotope-labeled in vitro phosphorylation reactions using in vivo phosphorylated peptides as substrate pools and apply this strategy to identify putative substrates of nine protein kinases that function in plant abiotic and biotic stress responses. As a result, we identified more than 5,000 putative target sites of osmotic stress-activated SnRK2.4 and SnRK2.6, abscisic acid-activated protein kinases SnRK2.6 and casein kinase 1-like 2 (CKL2), elicitor-activated protein kinase CDPK11 and MPK6, cold-activated protein kinase MPK6, H2O2-activated protein kinase OXI1 and MPK6, and salt-induced protein kinase SOS1 and MPK6, as well as the low-potassium-activated protein kinase CIPK23. These results provide comprehensive information on the role of these protein kinases in the control of cellular activities and could be a valuable resource for further studies on the mechanisms underlying plant responses to environmental stresses.