Role of Raf-like kinases in SnRK2 activation and osmotic stress response in plants

Abstract

Environmental drought and high salinity impose osmotic stress, which inhibits plant growth and yield. Thus, understanding how plants respond to osmotic stress is critical to improve crop productivity. Plants have multiple signalling pathways in response to osmotic stress in which the phytohormone abscisic acid (ABA) plays important roles. However, since little is known concerning key early components, the global osmotic stress-signalling network remains to be elucidated. Here, we review recent advances in the identification of osmotic-stress activated Raf-like protein kinases as regulators of ABA-dependent and -independent signalling pathways and discuss the plant stress-responsive kinase network from an evolutionary perspective.

Fig. 1. SnRK2s downstream targets in osmotic stress signalling pathways.

Schematic cartoon of the downstream targets described for the different subclasses of SnRK2s. ABA-unresponsive subclass I SnRK2s are activated by phosphorylation under osmotic stress and activate components of the mRNA decapping complex VCS through direct phosphorylation. Subclass I SnRK2s also interact with PA and phosphorylate two dehydrin proteins, ERD10 and ERD14, in response to osmotic stress. ABA-responsive subclass II SnRK2s are activated by phosphorylation under osmotic stress and phosphorylate GLX1, 14-3-3, ADK1 and R5P target proteins. Subclass II SnRK2s likely activate AREB/ABF transcription factors in response to osmotic stress. Subclass III SnRK2s, the core components of ABA signalling, are activated by phosphorylation under osmotic stress and they phosphorylate AREB/ABF, which in turn activate the expression of stress-responsive genes. Subclass III SnRK2s also phosphorylate a number of substrates, including AKS1 transcription factors, BRM, SNS1, HYL1, and SE1 proteins and SLAC1, RBOHF, and KAT1 membrane proteins. PA, phosphatidic acid; VCS, VARICOSE; ERD, EARLY RESPONSIVE TO DEHYDRATION; GLX1, GLYOXALASE 1; ADK1, ADENOSINE KINASE 1; R5P, Ribose 5-phosphate isomerase; AKS1, ABA-RESPONSIVE KINASE SUBSTRATE 1; BRM, BRAHMA; SNS1, SNRK2-SUBSTRATE 1; HYL1, HYPONASTIC LEAVES; SE, SERRATE; MAPKKK, mitogen-activated protein kinase kinase kinases; SLAC, SLOW ANION CHANNEL-ASSOCIATED; A⁻, anions; KAT1, POTASSIUM CHANNEL IN ARABIDOPSIS THALIANA 1; K⁺, potassium; RBOHF, RESPIRATORY BURST OXIDASE HOMOLOG F. Straight arrows indicate direct interaction and phosphorylation, dashed arrows (black) indicate in vitro interactions not fully experimentally validated in vivo, and dashed arrows (grey) depict ion flux through membrane channel proteins.

Fig. 2. Subgroup B Raf-like MAPKKKs and their substrate specificities for SnRK2 kinases.

a Heat map shows the in vitro phosphorylation of SnRK2 kinases by Raf-like MAPKKKs of Arabidopsis and P. patens. Arabidopsis SRK2J (subclass I) and SRK2F (subclass II) were excluded due to no data available. According to the phylogenetic tree, PpSnRK2B belongs to subclass III SnRK2s. 1, Takahashi et al.; 2, Katsuta et al.; 3, Lin et al.; 4, Soma et al.; 5, Saruhashi et al.. Please also refer to Supplementary Tables for the accession numbers and the gene names. The accession numbers of P. patens genes: ARK, Pp3c12_3550 (in the P. patens genome v.3.3), also referred to as Pp1s462_10V6 or Phypa_30352; PpSnRK2B, Pp3c6_16600, also referred to as Pp1s240_91V6 or Phypa_195464. ARK, ABA, and abiotic stress-responsive Raf-like kinase; M3K, MAP kinase kinase kinases. b Raf-like MAPKKKs are activated by phosphorylation under osmotic stress upstream of SnRK2s in Arabidopsis. B2/B3 family of Raf-like MAPKKKs regulate the ABA signalling pathway by upstream phosphorylation of the ABA-responsive subclass III SnRK2s and B4 Raf-like MAPKKKs regulate the ABA-independent signalling pathway by phosphorylation of the ABA-unresponsive subclass I SnRK2s. Schematic cartoon displays proposed signalling pathways based on in vitro assays (a) and detailed examinations, including in vivo phosphorylation, using the mutants of Raf-like MAPKKKs. Straight arrows indicate direct interaction and phosphorylation.

Fig. 3. Current model of osmotic stress kinases network and signalling pathways.

Osmotic stress evokes rapid increase of Ca²⁺, which is perceived by OSCA1 osmosensor proteins localized at plasma membrane. Question marks in violet objects depict possible downstream components activated by OSCA1 in response to osmotic stress. Raf-like kinases are activated in response to osmotic stress likely via other uncharacterized osmosensor proteins depicted as question mark in black box. B4 Raf-like MAPKKKs phosphorylate and activate subclass I SnRK2s, which are not responsive to ABA, and in turn SnRK2s phosphorylate mRNA binding proteins, such as VCS, which forms a protein complex with the decapping proteins DCP1 and DCP2 and are involved in post-transcriptional regulation of mRNA in p-bodies. B2/B3 Raf-like MAPKKKs phosphorylate and activate subclass III SnRK2s, which are ABA-responsive. In the presence of ABA, PYR/PYL/RCAR receptors form protein complexes with PP2C, resulting in the release of subclass III SnRK2s from inhibition by PP2Cs. Subclass III SnRK2s, phosphorylate SLAC1, KAT1 and RBOHF membrane proteins, which are essential for stomatal aperture regulation. Subclass III SnRK2s phosphorylate AREB/ABF transcription factors, which in turn activate the expression of stress-responsive genes. DREB2A transcription factor is activated through an ABA-independent signalling pathway. Question mark pink box depicts plausible upstream regulators of DREB2A still unknown. Straight arrows indicate direct interaction and phosphorylation, dashed arrows (black) indicate putative interactions not fully experimentally validated in vivo, and dashed arrows (grey) depict ion flux through membrane channel proteins. Double arrowhead dashed arrow indicates association–dissociation of PP2C with SnRK2s in the absence–presence of ABA.