Comparative phosphoproteomic analysis of raf22raf36 in Arabidopsis
Ontology highlight
ABSTRACT:
ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)
SUBMITTER: Taishi Umezawa
PROVIDER: PXD021372 | JPOST Repository | Sat May 15 00:00:00 BST 2021
REPOSITORIES: jPOST
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| 1_1.wiff.scan | Wiff | |||
| 1_2.wiff.scan | Wiff | |||
| 1_3.wiff.scan | Wiff | |||
| 24_2.wiff.scan | Wiff | |||
| 24_3.wiff.scan | Wiff |
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Project description:A phythormone abscisic acid (ABA) has a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) mediates ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in Arabidopsis, as an interacting protein with SnRK2. A series of reverse genetic and biochemical analyses revealed that Raf36 negatively regulates ABA responses and is directly phosphorylated by SnRK2s. In addition, we found that Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. Comparative phosphoproteomic analysis using Arabidopsis wild-type and raf22raf36-1 plants indicate proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results reveal a novel subsection of ABA-responsive phosphosignaling pathways branching from SnRK2.
2020-12-22 | PXD016894 | JPOST Repository
Project description:Resistance to drought stress is fundamental to plant survival and development. Abscisic acid (ABA) is one of the major hormones involved in different types of abiotic and biotic stress responses. ABA intracellular signaling has been extensively explored in Arabidopsis thaliana and occurs via a phosphorylation cascade mediated by three related protein kinases, denominated SnRK2s (SNF1-related protein kinases). However, the role of ABA signaling and the biochemistry of SnRK2 in crop plants remains underexplored. Considering the importance of the ABA hormone in abiotic stress tolerance, here we investigated the regulatory mechanism of sugarcane SnRK2s - known as SAPKs (Stress/ABA-activated Protein Kinases). The crystal structure of ScSAPK10 revealed the characteristic SnRK2 family architecture, in which the regulatory SnRK2-box interacts with the kinase domain αC helix. To study sugarcane SnRK2 regulation, we produced a series of mutants for the protein regulatory domains SnRK2-box and ABA-box. Surprisingly, mutations in the SnRK2-box did not drastically affect sugarcane SnRK2 activity, in contrast to previous observations for the homologous proteins in Arabidopsis. Also, we found that the ABA-box might have a role in SnRK2 activation in the absence of PP2C phosphatase. Taken together, our results demonstrate that both C-terminal domains of sugarcane SnRK2 proteins play a role in protein activation and activity.
2019-11-13 | PXD014298 | Pride
Project description:The rapid activation of RAF-SnRK2 cascades in response to hyperosmolarity is a crucial event in plants' adaptation to osmotic stress, yet the underlying mechanisms have remained elusive. In this study, we reveal that the calcium chelator EGTA, but not other calcium modulators, can potently activate both RAFs and SnRK2s. We demonstrate that EGTA induces the phosphorylation of RAF24, serving as an indicator of rapid RAF activation. Furthermore, we show that EGTA treatment results in increased phosphorylation of Ser171 and Ser175 in SnRK2.6, known markers of SnRK2 activation. In-gel kinase assays confirmed the activation of RAFs and SnRK2s. These findings collectively highlight that exogenous EGTA application mimics mannitol treatment, effectively triggering the activation of both RAF and SnRK2 subfamily kinases. Utilizing a DIA-based phosphoproteomics approach, we profiled global phosphorylation changes in response to EGTA and mannitol treatments. We identified 22,991 phosphorylated peptides corresponding to 6,515 phosphosites on 1,790 proteins. Notably, 5,067 sites were classified as class I phosphorylation sites, indicating high confidence in their localization. Principal Component Analysis confirmed the distinct phosphoproteomic profiles of the three conditions, with excellent reproducibility among biological replicates. EGTA treatment induced substantial changes in global phosphorylation, with 3,987 phosphosites increased and 964 decreased, signifying a profound impact on protein phosphorylation. The activation of RAFs and SnRK2s was supported by significant phosphorylation changes in these kinases. Additionally, EGTA-induced phosphoproteins included downstream effectors of RAF-SnRK2 cascades. GO analysis revealed distinct functional categories for EGTA and mannitol-responsive phosphoproteins, shedding light on the divergent roles of these treatments. Our study provides novel insights into the role of EGTA in the activation of RAF-SnRK2 cascades and its broader impact on the plant phosphoproteome, offering a valuable resource for understanding the downstream events related to exocellular calcium depletion in plants. It also highlights the potential involvement of MAP4Ks and resistosome-related kinases in osmotic stress signaling. Overall, our research enhances our understanding of osmotic stress response mechanisms and the power of DIA-based phosphoproteomics in unraveling complex phosphorylation events in plants.
2024-04-18 | PXD046802 | Pride
Project description:Gas exchange between plants and the atmosphere is critical for regulating plant growth or stress responses. When plants are exposed to drought stress, abscisic acid (ABA)-activated SnRK2 protein kinases play a central role for stomatal closure to prevent excessive water loss. To identify SnRK2 substrates in guard cells, a cell-type-specific phosphoproteomic analysis was performed, and MAP4K1 and MAP4K2 were identified as putative signaling factors in guard cells. MAP4K1 and MAP4K2 function redundantly in guard cells, as a map4k1map4k2 mutant impaired ABA-induced stomatal closure. MAP4K1 Ser-479 is directly phosphorylated by SRK2E (OST1/SnRK2.6), and it physiologically interacts with SRK2E in planta, suggesting that MAP4K1 is in vivo substrates of SnRK2. Mutational analysis revealed that phosphorylation of MAP4K1 Ser-479 is required for ABA-induced stomatal closure. Furthermore, an electrophysiological assay suggested that MAP4K1/2 are involved in the ABA-responsive activation of Ca2+-permeable channels. Collectively, our results propose that ABA-activated SnRK2 and MAP4K1/2 constitute a signaling pathway that positively regulates ABA-induced stomatal closure via Ca2+ signaling.
2025-01-30 | PXD049021 | JPOST Repository
Project description:Gas exchange between plants and the atmosphere is critical for regulating plant growth or stress responses. When plants are exposed to drought stress, abscisic acid (ABA)-activated SnRK2 protein kinases play a central role for stomatal closure to prevent excessive water loss. To identify SnRK2 substrates in guard cells, a cell-type-specific phosphoproteomic analysis was performed, and MAP4K1 and MAP4K2 were identified as putative signaling factors in guard cells. MAP4K1 and MAP4K2 function redundantly in guard cells, as a map4k1map4k2 mutant impaired ABA-induced stomatal closure. MAP4K1 Ser-479 is directly phosphorylated by SRK2E (OST1/SnRK2.6), and it physiologically interacts with SRK2E in planta, suggesting that MAP4K1 is in vivo substrates of SnRK2. Mutational analysis revealed that phosphorylation of MAP4K1 Ser-479 is required for ABA-induced stomatal closure. Furthermore, an electrophysiological assay suggested that MAP4K1/2 are involved in the ABA-responsive activation of Ca2+-permeable channels. Collectively, our results propose that ABA-activated SnRK2 and MAP4K1/2 constitute a signaling pathway that positively regulates ABA-induced stomatal closure via Ca2+ signaling.
2025-01-29 | PXD048986 | JPOST Repository
Project description:The signaling pathway of the phytohormone abscisic acid (ABA) regulates responses towards abiotic stress such as drought and high osmotic conditions. The multitude of functionally redundant components involved in ABA signaling, poses a major challenge for elucidating the largely unresolved response selectivity. We decided rebuilding single linear ABA signaling pathways in yeast for combinatoric permutation of ABA receptors and coreceptors, as well as the response-mediating SnRK2 protein kinases and their targeted transcription factors to drive luciferase expression in a heterologous host. We show that SnRK2s differ in the regulation by ABA receptor complexes, affect ABA responsiveness of the pathway, and differ in their transactivation activity but have similar preferences for ABA-responsive transcription factors. SnRK2s thought to act ABA-independently and known to be activated under osmotic stress in plants were regulated by ABA receptor complexes in yeast and competed with ABA receptor components in an ABA-dependent manner in plant tissue. The study reveals the suitability of the yeast system for analysis of ABA signaling factors and allowing the future dissection of ligand-receptor specificities in a functional response pathway. The analysis provides new insights into SnRK2 regulation indicating that four SnRK2 members of the osmotic stress response are tightly embedded into the ABA signaling pathway.
2019-07-30 | PXD013669 | Pride
Project description:Subfamily 2 of SNF1-related protein kinase (SnRK2) plays important roles in plant abiotic stress responses as a global positive regulator of abscisic acid signaling. In the genome of the model tree Populus trichocarpa, 12 SnRK2 genes have been identified, and some are upregulated by abiotic stresses. In this study, we heterologously overexpressed the PtSnRK2 genes in Arabidopsis thaliana and found that overexpression of PtSnRK2.5 and PtSnRK2.7 genes enhanced stress tolerance. In the PtSnRK2.5 and PtSnRK2.7 overexpressors, chlorophyll content and root elongation were maintained under salt stress conditions, leading to higher survival rates under salt stress compared with those in the wild type. Transcriptomic analysis revealed that PtSnRK2.7 overexpression affected stress-related metabolic genes, including lipid metabolism and flavonoid metabolism, even under normal growth conditions. However, the stress response genes reported to be upregulated in Arabidopsis SRK2C/SnRK2.6 and wheat SnRK2.8 overexpressors were not changed by PtSnRK2.7 overexpression. Instead, PtSnRK2.7 overexpression widely and largely influenced the transcriptome in response to salt stress; genes related to transport activity, including anion transport-related genes, were characteristically upregulated, and a variety of metabolic genes were specifically downregulated. We also found that the salt stress response genes were greatly upregulated in the PtSnRK2.7 overexpressor. Taken together, poplar subclass 2 PtSnRK2 genes can modulate salt stress tolerance in Arabidopsis, through the activation of cellular signaling pathways in a different manner from that by herbal subclass 2 SnRK2 genes. Total RNAs of the transgenic plants expressing 35S:PtSnRK2.7 treated with or without 200 mM NaCl. As a control experiment, the wild-type plants were treated similarly. The total RNAs were used for microarray analysis to reveal genes affected by the PtSnRK2.7 overexpression.
2016-08-01 | E-GEOD-79997 | biostudies-arrayexpress
Project description:Unveiling the signal transduction of phytohormone abscisic acid (ABA) will be greatly helpful to improve plant responses to stressful environment. ABA signaling is perceived and mediated by multi-member receptor PYR/PYLs, while whose post-translational modification, especially phosphorylation, is largely unknown. Here we demonstrated that Arabidopsis EL1-like (AEL), a casein kinase that regulates various physiological processes, phosphorylates PYR/PYLs and stimulates whose ubiquitination and degradation, leading to the suppressed ABA responses. Arabidopsis ael triple mutants display hypersensitive responses to ABA treatment, which is consistent with the suppressed degradation of PYR/PYL proteins. PYR/PYLs are phosphorylated in vivo and mutation of the conserved AEL-phosphorylation sites results in the reduced phosphorylation, ubiquitination, and degradation of PYR/PYLs, and hence the enhanced ABA responses. Our studies demonstrate the crucial roles of AEL-mediated phosphorylation in regulating the stability and function of PYR/PYLs and provide informative clues for elucidating the functional mechanism of ABA through regulating PYR/PYL receptors.
2022-02-23 | PXD008565 | Pride
Project description:Plants physiological mechanisms are affected by Abscisic acid (ABA) via changing gene expression and empowers plants to adapt in numerous environments. Plants have evolved their protection mechanisms for seed germination and abiotic environments to deal with critical harsh conditions. Here, we have explored those changes in Arabidopsis thaliana plants subjected to multiple abiotic stresses. AtBro1 transcripts showed up-regulation in the presence of salt, ABA and mannitol stress conditions. AtBRO1 over-expression lines exhibited robust tolerance to drought and salt stress. Further, ABA elicits resistance responses in loss-of-function bro1-1 mutant plants and AtBro1 positively regulates drought resistance in Arabidopsis. Promoter of AtBro1 fused with GUS showed GUS expression mainly in the rosette leaves and floral clusters, especially in anthers. AtBro1 protein was found to be localized in the plasma membrane of Arabidopsis at the subcellular level by using AtBro1::GFP fusion. Using a broad RNA-sequencing analysis, we observed that early transcriptional responses prompted by ABA induction exhibit specific quantitative differences at different time points, suggesting that ABA stimulates resistance responses in bro1-1 mutant plants. Additionally, transcripts levels of MOP9.5, MRD1, HEI10, and MIOX4 were altered in loss-of-function mutant plants which are involved in different stress conditions. Collectively, our results have shown that AtBro1 is involved in a significant role by regulating plant transcriptional response to ABA and induction of resistance response against abiotic stress.
2023-04-18 | GSE225062 | GEO
Project description:FERONIA (FER) receptor kinase has a versatile role in plant growth and development, biotic and abiotic stress responses, and reproduction. To gain new insights into the molecular interplay of these processes and to identify new functions of FER, we carried out quantitative transcriptome, proteome, and phosphoproteome profiling of wild type (WT) and a loss-of-function fer mutant in Arabidopsis plants. Gene Ontology terms for hormone signaling, abiotic stress, and biotic stress were significantly enriched among mis-expressed transcripts, proteins, and/or mis-phosphorylated proteins, in agreement with FER’s known roles in these processes. Analysis of multi-omics data and subsequent experimental evidence identified previously unknown functions of FER in ER (Endoplasmic Reticulum) body formation, glucosinolate biosynthesis. FER functions through transcription factor NAI1 to mediate ER body. FER also negatively regulates indole glucosinolates biosynthesis, partially through NAI1. Furthermore, we found that a group of abscisic acid (ABA)-induced transcription factors are hypo-phosphorylated in the fer mutant and demonstrated that FER acts through ABI5 to negatively regulate the ABA response during germination. Our integrated-omics study therefore reveals novel functions of FER and provides new insights into the underlying mechanisms of FER function.
2022-04-04 | GSE191303 | GEO