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Enabling rapid activations of B2 and B3 RAFs by SnRK2s in early osmotic stress signaling

ABSTRACT: Environmental water deficit imposes osmotic stress on plants, limiting plant growth and yield. The B2, B3, and B4 subgroup rapidly accelerated ﬁbrosarcoma-like kinases (RAFs) are rapidly activated within minutes by osmotic stress and function as upstream regulators of SNF1-related protein kinase 2s (SnRK2s), mediating early osmotic stress signaling in Arabidopsis thaliana and moss P. patens. However, little is known about how B RAF kinases are rapidly activated under osmotic stress. Here, we show that in addition to B4 RAFs initiating the activation of SnRK2s under osmotic stress, the SnRK2s also phosphorylate and activate B2/B3 RAFs to form a four-layer RAF-SnRK2 kinase cascade. The mannitol-triggered activation of B2/B3 RAFs was impaired in snrk2-dec and OK130-null mutants lacking all SnRK2s or B4 RAFs, respectively. SnRK2s, especially subclass I members, directly phosphorylate at a serine residue conserved in all B2/B3 RAFs and enhance their kinase activity. The non-phosphorylatable mutant of this serine in RAF3, RAF3S735A, could not rescue the hyposensitivity of the OK100-nonu mutant to both abscisic acid (ABA) and osmotic stresses.

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

SUBMITTER: wang pengcheng

PROVIDER: PXD057740 | JPOST Repository | Tue Nov 11 00:00:00 GMT 2025

REPOSITORIES: jPOST

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			Action	DRS
	20220219_F_WPC_ZCD_SNRK24_R3_18O.mgf	Mgf
	20220219_F_WPC_ZCD_SNRK24_R3_18O.msf	Msf
	20220219_F_WPC_ZCD_SNRK24_R3_18O.msfView	Msf
	20220219_F_WPC_ZCD_SNRK24_R3_18O.pdResult	Other
	20220219_F_WPC_ZCD_SNRK24_R3_18O.pdResult.lock	Other

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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.

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Enabling rapid activations of B2 and B3 RAFs by SnRK2s in early osmotic stress signaling

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