Dataset Information

OK-SnRK2 cascade mediates osmotic stress signaling in plant

ABSTRACT: Osmotic stress associated with drought, salinity and cold is a major factor limits plant productivity. A few signaling factors have been identified in the sensing or early signaling of osmotic stress. SNF1-related protein kinase 2 (SnRK2) are quickly activated by osmotic stress and the snrk2 decuple are sensitive to hyperosmolality and defective in osmotic stress induced cellular responses. Mechanically activated ion channels Hyperosmolality-induced Ca2+ increases (OSCA) may involve in the sorbitol-evoked Ca2+ increase. However, how plant senses osmotic stress and quickly activates SnRK2s is still unclear. Here we report osmotic stress active protein kinases (OKs), a subfamily of Raf protein kinases, involved in early signaling of osmotic stress in plant. OKs quickly and specifically activated by multiple hyperosmolality, but not by phytohormone abscisic acid or cold stress. The high order mutants of oks were hypertensive to hyperosmolality. In addition, OKs physically interact with and phosphorylate SnRK2s in vivo and in vitro, and this phosphorylation is required for activation of SnRK2s upon osmotic stress. Our results indicated the OKs-SnRK2s kinase cascade in plant osmotic stress responses, which provide a potential molecular machinery mediates osmotic stress signaling in plant.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Whole Plant

SUBMITTER: Chuan-Chih Hsu

LAB HEAD: Pengcheng Wang

PROVIDER: PXD014435 | Pride | 2020-01-17

REPOSITORIES: Pride

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A RAF-SnRK2 kinase cascade mediates early osmotic stress signaling in higher plants.

Lin Zhen Z Li Yuan Y Zhang Zhengjing Z Liu Xiaolei X Hsu Chuan-Chih CC Du Yanyan Y Sang Tian T Zhu Chen C Wang Yubei Y Satheesh Viswanathan V Pratibha Pritu P Zhao Yang Y Song Chun-Peng CP Tao W Andy WA Zhu Jian-Kang JK Wang Pengcheng P

Nature communications 20200130 1

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 w ...[more]

PMID: 32001690

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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.

Project description:Abscisic acid (ABA) is a key hormone for plant growth, development and stress adaptation. Perception of ABA through four types of receptors has been reported. We show here that impairment of ABA perception through the PYR/PYL/RCAR branch reduces vegetative growth and seed production, and leads to a severely open stomata and dramatic ABA insensitive phenotype, even though other branches for ABA perception remain functional. Arabidopsis sextuple mutant impaired in 6 PYR/PYL receptors, namely PYR1, PYL1, PYL2, PYL4, PYL5 and PYL8, was able to germinate and grow even on 100 mM ABA. Whole-rosette stomatal conductance (Gst) measurements revealed that leaf transpiration in the sextuple pyr/pyl mutant was higher than in the ABA-deficient aba3-1 or ABA-insensitive snrk2.6 mutants. The gradually increasing Gst values of plants lacking three, four, five and six PYR/PYLs indicate quantitative regulation of stomatal aperture by this family of receptors. The sextuple mutant lacked ABA-mediated activation of SnRK2s and ABA-responsive gene expression was dramatically impaired as was reported in snrk2.2/2.3/2.6. In summary, these results show that ABA perception by PYR/PYLs plays a major role to regulate seed germination and establishment, basal ABA signaling required for vegetative and reproductive growth, stomatal aperture and transcriptional response to the hormone. Four biological replicates were generated for the 3 genotypes, pyr1pyl1pyl2pyl4pyl5pyl8 sextuple mutant, snrk2.2/2.3/2.6 triple mutant, and Col-0. All the samples were treated with ABA for 3 hours before harvesting. The four Col-0 samples were mix to create an unique reference Col-0 sample. Two comparasion were made, fisrt one, PYR/PYL sextuple mutant versus Col-0 reference sample and second one, SnRK2 triple mutant versus Col-0 reference sample. In each comparasion four biological replicates were made. Replicas number 1 and 2 were labeled with Cy5 for the mutant sample and Cy3 for the Col-0 reference sample, while the other two replicas,#3 and #4, were reversed-labeled.

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