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

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.

DREB2A, the expression of which is not induced by ABA, plays key roles in osmotic-and heat-stress responses in Arabidopsis 19,20 . On the basis of the extensive studies on these transcription factors, AREB/ABF and DREB2A signalling pathways are thought to be pivotal in plant responses to osmotic stress 21 .
However, as demonstrated in the transcriptional change in response to ABA 22 , the gene expression under osmotic stress conditions is dynamically regulated by a myriad of cis-elements and transcription factors, and not these pathways alone. Epigenetic regulations, including DNA methylation, histone modifications, and nucleosome assembly by chaperones, also play important roles in osmotic-stress responses in plants and are well summarized in other reviews [23][24][25] .
Osmotic stress generated by drought and high-salinity conditions promotes the accumulation of ABA in plant cells and the activation of the ABA signalling pathway. The core molecular components of ABA perception and signal transduction have been well studied [1][2][3] . Following prior studies of sucrose nonfermenting-1-related protein kinases 2 (SnRK2s) in several species, including Arabidopsis 26-28 , Vicia faba 29 , and barley 30 , three Arabidopsis SnRK2s activated by ABA were shown to be important components of the ABA signalling pathway [31][32][33] . Under control conditions, when plants are growing in wellwatered environments, the ABA-dependent SnRK2s are inhibited by clade A protein phosphatases of type 2C (PP2C) 34,35 , keeping the ABA signalling pathway inactive. Under specific water-deficit stress conditions, ABA levels increase and it binds to the receptor proteins, designated as PYRABACTIN RESISTANCE 1 (PYR1), PYR1-LIKE (PYL), or REGULATORY COMPONENTS OF ABA RECEPTOR (RCAR) 36,37 . The receptor complexes of PYR/PYL/ RCARs and PP2Cs, which serve as coreceptors, are formed, allowing the activation of the SnRK2s proteins. Once released from PP2C, SnRK2s auto-phosphorylate and self-activate 38,39 , subsequently activating or inhibiting a group of downstream transcription factors, including AREB/ABFs, and membrane proteins, including SLOW ANION CHANNEL-ASSOCIATED (SLAC1), via phosphorylation. Given that a large majority of the downstream genes of the SnRK2s were downregulated in the multiple areb/abf-knockout mutant 40 , AREB/ABFs have been suggested to be the key transcription factors in the ABA core signalling. Consistently, AREB/ABFs are among the top contributors in the hierarchical gene regulatory network in response to ABA 22 .
Although ABA is the key signalling molecule in response to osmotic stress, osmotic stress additionally activates signalling pathways even if ABA signalling is off. For example, the DRE/Crepeat cis-element has been shown to be crucial for the gene induction independently of ABA, as evidenced by the dehydration-responsive expression of RESPONSIVE TO DESIC-CATION 29A (RD29A) in the aba insensitive 1 (abi1) mutant 41,42 , the genetically isolated dominant mutant of clade A PP2C. Taking advantage of the genetic resources, in which ABA metabolism, signalling, or transport are impaired, the signalling pathway has been well studied. In contrast to the well-described ABA signalling pathway, an ABA-independent signalling pathway is activated in response to osmotic stress in plants as well, yet the molecular components of this osmotic stress-signalling pathway and the early osmosensing mechanisms used by plants are still not well understood. A plasma membrane protein that forms a calcium-permeable channel sensitive to osmotic stress named OSCA1 (REDUCED HYPEROSMOLARITY INDUCED Ca 2+ INCREASE 1) was proposed as a plausible sensor of rapid calcium signalling activated by osmotic stress 43 . However, whether OSCA1 is the responsible protein for directly sensing the osmotic stress in the plant cells has not been fully demonstrated.
Given that ARABIDOPSIS HISTIDINE KINASE 1 (AHK1) complemented the high-salinity-sensitive phenotype of the Saccharomyces cerevisiae (yeast) mutant lacking osmosensors, AHK1 was also proposed to sense and transduce osmotic-stress signals 44,45 . By contrast, AHK1 was reported to be not involved in a rapid stomatal closure or ABA biosynthesis in response to decrease of air humidity 46 and thus further experimental validations in plants are required. Similarly, the upstream molecular components of the signalling pathways regulating the DREB2A transcription factor in response to osmotic stress remain unknown. Even in the absence of osmotic stress, DREB2A is expressed and the DREB2A protein is degraded via ubiquitin-proteasome pathways, preventing activation of the stress signal 47,48 , implying that stress-responsive stabilization and activation mechanisms exist. In heat-stress responses, DNA POLYMERASE II SUBUNIT B3-1 (DPB3-1)/NUCLEAR FAC-TOR Y, SUBUNIT C10 (NF-YC10) was isolated as an interacting protein of DREB2A and the NF-Y protein complex has been suggested to regulate the target selectivity of DREB2A. However, DPB3-1/NF-YC10 was found to be unlikely involved in drought or high-salinity stresses 49 . Despite this insight, our current knowledge on the molecular mechanisms of osmotic stress signalling pathways in plants is still limited. In particular, little is known about key components in the early events and, therefore, the global osmotic stress signalling network remains to be elucidated.
Genetic approaches have been playing a fundamental role in exploring key genes of osmotic stress and ABA signalling pathways. Indeed, a chemical genetic screening has identified the ABA receptor proteins 36 . Alongside developments of genomics and transcriptomics in the early 2000s, the focus of reverse-genetic studies was mainly on transcriptional changes in response to stresses. However, the cellular responses to stress occur more quickly through post-transcriptional and posttranslational modifications, including phosphorylation. Nowadays, high-throughput proteomic analyses based on mass spectrometry (MS) are available, enabling us to investigate phosphoproteomic changes in response to short-term osmotic stress and to explore the interaction of proteins with known key regulators. Here, we review the most recent advances in the identification of new molecular components with essential roles in the ABA-dependent and -independent signalling pathways in response to osmotic stress. Five independent studies have recently described different families of Raf-like protein kinases, which are activated early in response to osmotic stress and operate upstream of both the ABA-dependent and -independent signalling pathways in Physcomitrella patens (P. patens) 50 and Arabidopsis 51-54 . We aim to connect these novel findings concerning the different families of Raf-like protein kinases with state-of-the-art knowledge in the field to obtain a general schematic picture of the osmotic regulation of ABA-dependent and -independent pathways. We further discuss the stressresponsive kinase network in plants from an evolutionary perspective.
SnRK2s contributes to ABA-dependent and -independent signalling pathways. The SnRK2 family of proteins contains ten members in Arabidopsis, which can be divided into three subclasses I, II, and III on the basis of their sequence homology and responsiveness to osmotic stress and ABA 17,55 . For the clarity of the readers, the different nomenclatures used by the different studies reviewed herein are summarized and classified in Supplementary Table 1. In this review, we mainly use the alphabetical nomenclature rather than the numerical one (e.g., SRK2G instead of SnRK2.1).
Given that they are activated by ABA, subclass II and III are classified as ABA-dependent SnRK2s (Supplementary Table 1). In Arabidopsis, SRK2E (also known as OST1) was first functionally characterized as a key regulator of ABA signalling in guard cells by genetic screening 26 and biochemical analyses 28 . Consistent with the strong activation by ABA 55 , the knockout mutants of three subclass III SnRK2 members, SRK2D, SRK2E, and SRK2I, showed extreme insensitivity to ABA, and it has been revealed that they play an essential role in the ABA signal transduction pathway [31][32][33] . Among varieties of their substrates, AREB/ABFs are key transcription factors phosphorylated and activated by the SnRK2s in response to ABA 33,56 ( Fig. 1) with the SnRK2-AREB/ ABF signalling pathway also being reported in rice 57 . By contrast, ABA-RESPONSIVE KINASE SUBSTRATE 1, also known as FLOWERING bHLH 3, is phosphorylated and inactivated by SRK2E in guard cells 58 (Fig. 1). SRK2E additionally phosphorylates membrane proteins essential for ABA-mediated stomatal closure response following water-deficit stress, namely SLAC1 and POTASSIUM CHANNEL IN ARABIDOPSIS THALIANA 1 (KAT1) [59][60][61] (Fig. 1). SLAC1 channel activity was shown to be tightly controlled via phosphorylation and dephosphorylation. In the absence of ABA, SLAC1 is inhibited by phosphatases, including ABI1 and ABI2 62 , whereas SLAC1 is activated in the presence of ABA via phosphorylation by SRK2E and the calciumdependent protein kinases (CPKs), such as CPK6, CPK21, and CPK23 59,60,62 . Moreover, the NADPH oxidase, RESPIRATORY BURST OXIDASE HOMOLOG F (RBOHF), which is responsible for ABA-induced ROS production and subsequent stomatal closure, as well as RBOHD 63 , has been shown to be phosphorylated by SRK2E 64 . Collectively, SRK2E promotes stomatal closure through activation of SLAC1, inhibition of KAT1, and activation of RBOHF to induce ROS production ( Fig. 1). Furthermore, phosphoproteomic analyses identified putative targets of subclass III SnRK2s proteins, which are involved in transcription, RNA processing, epigenetic modifications, chloroplast processes, and control of flowering 65,66 . SNRK2-SUBSTRATE 1 was suggested to function as a negative regulator of ABA signalling 66 , whereas SERRATE and HYPONASTIC LEAVES, which are involved in microRNA biogenesis, were shown to be phosphorylated by the SnRK2s 67 . The SWI2/SNF2 chromatin remodelling ATPase BRAHMA was also reported to be inhibited via phosphorylation by the SnRK2s in response to ABA (Fig. 1) and activated via dephosphorylation by the clade A PP2Cs 68 . For an extensive review on previously described substrates and functions of the subclass III SnRK2s and their functional relationship with calcium signalling, please see refs. 56  Subclass II members, SRK2C and SRK2F, are activated by osmotic stress (Supplementary Table 1) and show a weaker activation by ABA than the subclass III SnRK2s 55 . This result appears to be consistent with the fact that ABA-responsive SnRK2s of subclasses II and III harbour the acidic Asp-rich domain at the C terminus 17 , which nearly corresponds to the ABA box essential for the interaction with PP2C 38 . However, as the triple mutant of subclass III SnRK2s shows extreme ABA insensitivity 33 , the role of subclass II SnRK2s in ABA signalling is thought to be minor. Notably, rice subclass III SnRK2s, but not subclass II SnRK2s, were shown to be activated by ABA 71 . SRK2C mainly expressed in root tips has been shown to be involved in enhancement of drought tolerance 72 and vegetative growth 73 in Arabidopsis. By contrast to the phenotypic changes exhibited by the overexpressing lines 72,73 , the single and double mutants of SRK2C and SRK2F did not show altered growth phenotypes 74 . The downstream substrates of SRK2C has been suggested to include AREB/ABF transcription factors, such as ABF3 74 (Fig. 1). Furthermore, a phosphoproteomic approach identified candidate target proteins of SRK2C associated with metabolic processes, such as 14-3-3 proteins, glyoxalase 1, adenosine kinase 1, and ribose 5-phosphate isomerase 73 (Fig. 1). However, further investigation of the candidate substrates of subclass II SnRK2s will be required to reveal their biological functions under osmotic stress conditions. Given that SnRK2s from subclass I, with the exception of SRK2J, are activated by osmotic stress but not ABA 55 , SRK2A, SRK2B, SRK2G, and SRK2H (Supplementary Table 1) are potentially involved in the ABA-independent osmotic-stress signalling pathway. Compared to subclass III SnRK2s, until recently little was known about the activation mechanisms, targets and interactors of subclass I SnRK2s [75][76][77] . SRK2A and SRK2B are involved in maintenance of root growth and development 76 and ROS homoeostasis 78 in response to salt stress. In addition to their activation within 30 s of salt treatment 76 , SRK2A localized at the primary root tip displayed a punctuate pattern under salt stress conditions. However, a similar pattern was not observed for SRK2B localized in the vascular tissues at the site of the lateral roots 76 . Furthermore, fractionation experiments of SRK2A and SRK2B indicated that these cytosolic kinases are associated to the membrane in salt stress conditions and interact with phosphatidic acid 76,79 (Fig. 1). By contrast to ABA-dependent SnRK2s, no transcription factors have been characterized to be substrates of subclass I SnRK2s. However, co-immunoprecipitation analyses detected different components of the mRNA decapping complex, such as VARICOSE (VCS) and DECAPPING 2 (DCP2), as candidate interactors of SRK2G 77 . The mRNA decapping protein complex is responsible for removal of the 5′-cap, a critical step for exonuclease-mediated mRNA decay 80 . The core decapping complex is formed by DCP2, the active enzyme of the decapping complex, DCP1, and the scaffold protein VCS, and it assembles in processing bodies (P-bodies) in the cytoplasm 80 . Bimolecular fluorescence complementation and phosphorylation assays confirmed that the subclass I SnRK2s directly interact and phosphorylate VCS 77 (Fig. 1). As well as SRK2A 76 , SRK2G also showed the translocation from cytosol to P-body in response to osmotic stress but not ABA, corroborating the interactions of VCS with SRK2A and SRK2G in P-bodies 77 . Moreover, mRNA decay was shown to be impaired in the srk2a srk2b srk2g srk2h quadruple mutant and the VCS artificial microRNA (amiRNA) line, both of which showed growth retardation under osmotic stress conditions, indicating that subclass I SnRK2s are involved in mRNA metabolism via phosphorylation of VCS, and that this process appears to be important for shifting mRNA population in response to osmotic stress. As no interactions were detected between VCS and ABA-responsive SnRK2s 77 , ABA-unresponsive SnRK2s might exclusively regulate VCS, yet it has been recently reported that SRK2E was able to phosphorylate VCS in vitro 81 . Another recent phosphoproteomic study suggested that SRK2B phosphorylates two LATE EMBRYOGENESIS ABUNDANT/ dehydrin proteins, EARLY RESPONSIVE TO DEHYDRATION 10 and 14, in response to osmotic stress 82 (Fig. 1). Further in vivo studies need to be carried out to identify the molecular components regulated by the subclass I of SnRK2s. In conclusion, different studies have supported that osmotic stress-activated subclass I SnRK2s play different roles in plants than ABAresponsive SnRK2s.
Novel roles for Raf-like kinases in osmotic stress pathways upstream of the SnRK2s. With the exception of a few examples, such as Arabidopsis SRK2J, SnRK2 protein kinases were reported to be activated by osmotic stress in Arabidopsis 55 , rice 71 , soybean 83 , tobacco 84 , and P. patens 85 . Although a subset of them grouped into subclass II and III were also activated in response to ABA 55,71,85 , the activation by osmotic stress still occurred in ABA signalling mutants where the ABA-induced activation is abolished [86][87][88] , implying that both ABA-dependent and -independent signal transduction cascades are involved. SnRK2s are suggested to be auto phosphorylated 38,39 , although structural analysis 39 revealed that the activation loop of SRK2E is more stable than that of SRK2D and SRK2I, implying that the level of autophosphorylation might differ among the SnRK2s. By contrast, previous pharmacological studies demonstrated that the general kinase inhibitor staurosporine inhibits the SnRK2s' kinase activities in vitro but not their activation by osmotic stress or ABA treatment 84,86 , suggesting that the osmotic-stress responsive SnRK2 activation requires upstream protein kinases that are insensitive to staurosporine. A pioneering study in P. patens 50 and more recent four independent ones in Arabidopsis 51-54 have identified a family of protein kinases that are activated in response to osmotic stress and subsequently phosphorylate and activate SnRK2s.
A mutagenesis screening for ABA-insensitive P. patens mutants revealed that a mutation in a Raf-like protein kinase cause reduced ABA sensitivity and osmotic stress tolerance 50 . The authors named this Raf-like protein ARK (ABA and abiotic stress-responsive Raf-like kinase). ARK is a single gene from the B3 Raf-like kinases family group of mitogen-activated protein kinase kinase kinases (B3-MAPKKKs) in P. patens and ark lossof-function mutants showed decreased ABA sensitivity and cold acclimation capacity, yet increased sensitivity to osmotic stress. Moreover, ABA-responsive genes were differentially expressed in the ark mutant. Given that the P. patens SnRK2-like protein was shown to be activated by ABA in a prior study 89 , the kinase activity of the corresponding protein was examined in the ark mutant, in which ABA, cold, desiccation and osmotic stress treatments did not fully activate the kinase. The ark mutants identified in the study were not defective in the amount of ARK protein per se but rather by an impaired activity of the ARK protein due to a single C to T mutation (Ser532Phe) that impairs the ARK protein phosphorylation and causes ABA insensitivity 50 . Furthermore, ARK was shown to directly phosphorylate and activate PpSnRK2B (Fig. 2a). Specifically, ARK phosphorylated the PpSnRK2B protein on the Ser165 and Ser169 residues, the latter is corresponding to the critical residue for the activation of SnRK2s in Arabidopsis 86 . To be active, ARK itself needs to be phosphorylated on the Ser1029 residue 50 . However, no upstream kinase phosphorylating ARK has been identified to date.
Recently, four independent studies have also identified different groups of Raf-like protein kinases activated by osmotic stress [51][52][53][54] . All four studies converge in a common finding: the Raf-like MAPKKKs are activated in response to osmotic stress and function upstream of the SnRK2 kinases in Arabidopsis, as previously reported in P. patens 50 . The B2/B3 family of Raf-like MAPKKKs regulate the ABA signalling pathway by phosphorylation of the ABA-responsive SnRK2s from subclass III, consistent with the findings in P. patens (Fig. 2). By contrast, B4 Raf-like MAPKKKs regulate the ABA-independent signalling pathway by phosphorylation of the ABA-unresponsive SnRK2s from subclass I (Fig. 2). It is noteworthy that the studies discussed below use different nomenclatures, which can be confusing for the reader (Raf-like MAPKKKs, M3PK∂ kinases, OK 100 90,91 . There are 48 Raf-like kinase grouped into three families, A, B, and C, in the Arabidopsis genome, yet the function of most members has not been uncovered. The family B contains 22 members and is divided in 4 subfamilies: B1 subfamily includes 3 members, B2 subfamily includes 6 members, B3 subfamily includes 6 members, and the B4 subfamily includes 7 members (Supplementary Table 2). Within the B3 subfamily, RAF1, also named constitutive triple response 1 (CTR1, Supplementary Table 2), is one of the bestcharacterized MAPKKKs in plants and constitutes the MAPK pathway in ethylene signalling 92 . RAF1 has been shown to play essential roles both in ethylene and ABA signalling pathways 93 as well as in response to hypoxia 94 . The B3 family member RAF2, also named enhanced resistance (EDR. Supplementary Table 2), negatively regulates the biotic stress immune response 95 . In Arabidopsis, loss-of-function mutants of members of the B2 and B3 families of the Raf-like protein kinases such as RAF1, RAF10, and RAF11, are insensitive to ABA 96,97 and the raf5 mutant is hypersensitive to salt stress 98 . In addition, a member of the B4 subfamily, RAF18 is phosphorylated during the early stages of the osmotic stress response 99 . Although all these studies revealed important roles of Raf-like protein kinases in plants, their function in osmotic stress and ABA signalling pathways in higher plants remained unknown.
Takahashi et al. 51 performed genetic screenings for ABAinsensitive seed germination and identified two independent amiRNA lines targeting five or seven genes of B1 and B3 Raf-like MAPKKKs. Among the overlapped targets, B3 Raf-like MAPKKKs, RAF3, RAF4, and RAF5 phosphorylated in vitro dephosphorylated-SRK2E, which had lost its autophosphorylation activity 51 . Notably, in the presence of the PYR1 receptor and HYPERSENSITIVE TO ABA 1 (HAB1) PP2C phosphatase, SRK2E was inhibited and was not reactivated by ABA addition, whereas RAF5 reactivated the SRK2E inhibited by HAB1 in response to ABA. Further reconstitution experiments in Xenopus oocytes confirmed that SRK2E is activated by upstream RAF3, RAF4, and RAF5 kinases (Fig. 2). The same conclusion has been reached from the analyses of Arabidopsis RAF4, RAF5 and RAF6 54 (Fig. 2), which were able to restore the ABA insensitivity of the P. patens ark mutant 50 . Consistent with their ability of complementation, the Ser residue corresponding to the point mutation of the ark mutant is conserved in RAF4, RAF5, and RAF6 but not in other B3 Raf-like MAPKKKs 54 . In agreement with the ABA-insensitive seed germination in the series of triple knockout and knockdown mutants of RAF3, RAF4, and RAF5 51 , the triple mutant of RAF4, RAF5, and RAF6 showed moderate ABA insensitivities in seedling establishment, stomatal closure, and gene expression yet greatly impaired responses to osmotic stress and desiccation 54 . These different sensitivities to ABA and osmotic stress appeared to be associated with the fact that the activation of subclass III SnRK2s by ABA was reduced in the triple mutant but that by osmotic stress was abolished 54 . In addition to RAF3, RAF4, and RAF5 51 , RAF6 phosphorylated SRK2E weakly compared to RAF4 and RAF5 54 (Fig. 2). Noticeably, SRK2E was not able to phosphorylate RAF4 51,54 , whereas the SRK2E incubated with ABI1, which had reduced autophosphorylation activity, was activated by RAF4 54 . Collectively, B3 Raf-like MAPKKKs, RAF3, RAF4, RAF5, and RAF6, were shown to be the upstream kinases phosphorylating and activating ABA-responsive SnRK2s in response to ABA and osmotic stress.
Another study 53 shed new light on the subclass I ABAindependent SnRK2s (Supplementary Table 1), which are little studied compared to the subclass III ABA-dependent SnRK2s. In vivo protein co-immunoprecipitation of subclass I SnRK2s followed by liquid chromatography-MS/MS identified candidate interacting proteins of SRK2A and SRK2G under osmotic stress conditions 53 . The common candidate interactors included three B4 Raf-like MAPKKKs (RAF18, RAF20, and RAF24), which have been shown to be phosphorylated in 5 min, in response to osmotic stress 99 . Importantly, previous phosphoproteomic analysis 99 identified B4 Raf-like MAPKKKs, subclass I SnRK2, VCS, and DCP2 proteins to be rapidly phosphorylated by osmotic stress. In addition to the in vivo interaction of these three Raf-like MAPKKKs with SRK2A and SRK2G, further microscopic analyses revealed that RAF18, and SRK2A and SRK2G colocalized to P-bodies in response to osmotic stress, showing a punctuate pattern as previously reported for subclass I SnRK2s 76,77 . Furthermore, RAF18, RAF20, and RAF24 were shown to activate four subclass I SnRK2 members (SRK2A, SRK2B, SRK2G, and SRK2H) by phosphorylation in vitro (Fig. 2). Among several phosphorylation sites, Ser154 in SRK2G, the residue conserved in subclass I SnRK2s, was suggested to be directly phosphorylated by Raf-like MAPKKKs, consistent with the previous report that the residue in SRK2B was phosphorylated by osmotic stress 88 . In fact, the phosphorylation of SRK2G by itself or RAF20 was decreased by the substitution of Ser154 to Ala 53 . The in vivo kinase activity of subclass I SnRK2s in response to dehydration was significantly decreased in the raf18 raf20 raf24 triple mutant plants, but not in the single or double mutant plants. By contrast, the activity of subclass III SnRK2s was not altered in the triple mutants, suggesting that the three RAF kinases have the redundant role in regulating specifically subclass I SnRK2s under osmotic stress conditions (Fig. 2). Consistent with previous reports showing that subclass I SnRK2s are involved in growth and mRNA decay under osmotic stress conditions 77 , the raf18 raf20 raf24 triple mutants display growth retardation and altered gene expression similar to the srk2a srk2b srk2g srk2h quadruple mutants under stress conditions 53 , suggesting that the Raf-like MAPKKKs regulate the mRNA population under osmotic stress by activating subclass I SnRK2s. However, the raf18 raf20 raf24 mutants showed slightly retarded growth compared with the srk2a srk2b srk2g srk2h mutants, implying that subclass I SnRK2s are main but not exclusive substrates of the three Raf-like MAPKKKs. In summary, B4 Raflike MAPKKKs, RAF18, RAF20, and RAF24, were shown to regulate subclass I SnRK2-VCS signalling pathway under osmotic stress conditions but not ABA-responsive subclass III SnRK2s.
These new findings on B3 and B4 Raf-like MAPKKKs are supported by another study, in which two groups of protein kinases of~130 and 100 kDa showed strong activation in response to short-term osmotic stress, but not ABA, even in the multiple mutants of SnRK2s 52 . The authors named these protein kinases OKs (osmotic stress-activated protein kinases). Further phosphoproteomic and mutational analyses revealed that the OK 130 and OK 100 groups correspond to B4 and B2/B3 Raf-like MAPKKKs, respectively (Supplementary Table 2). Consistent with the functional redundancy among RAF18, RAF20, and RAF24 53 , the single mutants of the B4 family of Raf-like MAPKKKs did not show significant changes on the kinase activity 52 . However, the high-order mutants generated by using CRISPR/Cas9-mediated genome editing for all members of B4 Raf-like MAPKKKs (Supplementary Table 2) in the raf16 T-DNA background (OK 130 -weak and OK 130 -null) showed greatly reduced activities of the B4 Raf-like MAPKKKs and subclass I SnRK2s 52 . In particular, the activity of subclass I SnRK2s in response to osmotic stress was completely abolished in the B4 null mutant. The B4 null mutants displayed severe growth and developmental effects, including seed production, and were hypersensitive to salt and osmotic stresses, similar to the raf18 raf20 raf24 triple mutants 53 . Altogether, these results support a redundant upstream regulation of subclass I SnRK2s by the B4 family of Raf-like MAPKKKs. By contrast, the activation of subclass III SnRK2s by ABA and osmotic stress was not altered in the B4 null mutant, suggesting that ABA-responsive SnRK2s are regulated independently of the B4 Raf-like MAPKKKs (Fig. 2). The high-order mutant for the B2/B3 families of Raf-like MAPKKKs, raf2 raf4 raf5 raf10 raf11 quintuple mutant, showed reduced activity of subclass III SRK2D, SRK2E and SRK2I members under osmotic stress and strong plant growth inhibition phenotypes as well 52 . Furthermore, in the quindec mutant of B2/ B3 Raf-like MAPKKKs generated in the B4 weak mutant background (raf16 raf40 raf24 raf18 raf20 raf35 raf42 raf2 raf3 raf4 raf5 raf10 raf11), the kinase activity of subclass III SnRK2 was greatly reduced or abolished by ABA and osmotic stress, respectively. Consistently, the quatdec mutant in the B4 weak mutant (raf16 raf40 raf24 raf18 raf20 raf35 raf42 raf3 raf4 raf5 raf10 raf11), which has no mutations in RAF2 and shows minor growth defects under control conditions unlike the quindec mutant, resulted in increased water loss and decreased ABA sensitivities in seed germination, seedling establishment, and gene expression compared to wild-type plants and other OK mutants lacking either B2/B3 or B4 Raf-like MAPKKKs. Although B4 Raflike MAPKKKs RAF24 and RAF40 phosphorylated and activated subclass I SRK2A and SRK2B, several B2 and B3 family members, including RAF6, RAF7, and RAF10, phosphorylated subclass III SRK2E 52 (Fig. 2). Previously, RAF10 from B2 Raf-like MAPKKKs was reported to be involved in ABA signalling by phosphorylating subclass III SnRK2s and their substrates, including ABI5 100 . Although the role of B3 Raf-like MAPKKKs was repeatedly reported 51,52,54 , further studies are needed to reveal how B2 and B3 Raf-like MAPKKKs function together in ABA and osmoticstress signalling.
In conclusion, these four studies 51-54 elucidated novel and specific roles for B2/B3 family of Raf-like MAPKKKs in regulating the ABA signalling pathway by phosphorylation of the SnRK2s from subclass III and, for B4 Raf-like kinases, regulating the ABA-independent signalling pathway by phosphorylation of the SnRK2s from subclass I (Fig. 3).
Self-activation of SnRK2s. Although SnRK2s were reported to be phosphorylated by RAF10 and BRASSINOSTEROID INSENSI-TIVE 2 kinases 100,101 , it has been accepted that once released from PP2Cs in response to ABA, subclass III SnRK2s are selfactivated by autophosphorylation 38,39 . The studies reviewed herein suggest that Raf-like MAPKKKs mainly phosphorylate SnRK2s. Two serine residues in the activation loop, Ser171 and Ser175, are crucial for activation of SRK2E kinase 88 . MS analyses further revealed that RAF3 phosphorylates Ser171 of SRK2E 51 . Consistently, the substitutions Ser171Ala and/or Ser175Ala in SRK2E resulted in the loss of phosphorylation by ABA 51 and by RAF4 54 , indicating that the conserved Ser residues in the activation loop of SRK2E are phosphorylated by RAF-like MAPKKKs. However, the ABA-responsive activation of SnRK2s was decreased but not abolished in the multiple mutants of RAF3, RAF4, RAF5, or RAF6 51,54 or the quintuple mutants of RAF2, RAF4, RAF5, RAF10, and RAF11 B2/B3 Raf-like MAPKKKs 52 . Moreover, quintuple raf2 raf4 raf5 raf10 raf11 mutants showed moderate insensitivities to ABA compared to the triple srk2d srk2e srk2i mutants 51,52,54 , implying that the ABA signalling pathway is still active to some extent, presumably because of the activation of SnRK2s by dissociation from PP2Cs, remaining members of MAPKKKs, and/or any other kinases. A hint to address the kinetic mechanisms of the inhibition and activation of SnRK2 by PP2Cs and Raf-like MAPKKKs, respectively, was provided by a recent report showing that group-A PP2Cs inhibit both subclass I and III SnRK2s 102 . Although group-A PP2C were thought to interact and dephosphorylate ABA-responsive SnRK2s, ABA-unresponsive SRK2A was shown to be inhibited by ABI1-type PP2Cs but not PP2CA-types, both of which inhibited SRK2E 102 , consistent with another report 103 . Further, fluorescence life-time imaging (Förster resonance energy transfer) in tobacco leaves revealed that RCAR receptors did not disrupt the interaction of ABI1 with SRK2E but SRK2A in the absence or low concentration of ABA, implying that the PP2Cs have different affinities to subclass I and III SnRK2s. Taken together, subclass III SnRK2s may require both dissociation from PP2Cs in response to ABA and activation by B2/B3 Raf-like kinases for full activation, whereas subclass I SnRK2 may be fully activated by B4 Raf-like kinases even before the accumulation of ABA.
Crosstalk mechanisms of ABA-dependent and -independent pathways. Osmotic stress can be divided into the ABA-dependent and the ABA-independent responses. However, many experimental evidences indicate that these pathways are not lineal and independent but functionally interact with one another. The crosstalk between ABA-dependent and -independent pathways can occur at different levels. Even though the B2/B3 and B4 Raflike MAPKKKs activate different subclasses of SnRK2s, the activation of Raf-like MAPKKKs is unlikely independent. In the B4 mutants, the activation of B2/B3 Raf-like MAPKKKs were partially impaired 52 , implying that B4 Raf-like MAPKKKs are involved in the regulation of the B2/B3 Raf-like MAPKKKs in response to osmotic stress. It still remains to be understood whether Raf-like MAPKKKs directly regulate each other or upstream proteins are involved in the control of the MAPKKKs in a connected way. B4 and B2/B3 Raf-like MAPKKKs showed preferences to phosphorylate subclass I and III SnRK2s, respectively. However, weak phosphorylation events of SRK2E by B4 Raf-like MAPKKKs and those of subclass I SnRK2s by B2/3 Raflike MAPKKKs have been reported [51][52][53][54] (Fig. 2a), supporting that these signalling pathways are indeed not lineal.
Evidences for crosstalk were also reported at the level of SnRK2s. As proven by the crystal structure 38 , subclass III SnRK2s interact with PP2Cs in a similar manner to the RCAR-PP2C ABA coreceptors. By contrast, ABI1-type PP2Cs were reported to be functional phosphatases of SnRK2 regardless of subclasses 102,103 . Moreover, the substrates of SnRK2s might be regulated in response to both ABA and osmotic stress. Although the large overlap of the downstream genes indicated that subclass III SnRK2s are the main activators of AREB/ABFs 33 , AREB/ABFs were also activated by subclass I and II SnRK2s 74,102 . Furthermore, the phosphorylation of VCS appeared to be downregulated in the srk2d srk2e srk2i mutants 66 as well as in srk2a srk2b srk2g srk2h mutants 77 . Unlike subclass I SnRK2s, SRK2D did not show interaction with VCS in planta, whereas the slight in vivo phosphorylation of VCS by SRK2D in response to dehydration was reported 77 . The transcription factors downstream of the SnRK2s, including AREB/ABFs and DREB2A, also function interdependently 21 . In summary, many evidences indicate that ABA-dependent and -independent pathways might converge at the SnRK2s, as well as in their upstream and downstream factors.
Evolution of osmotic stress signalling pathways. Plants have acquired molecular and physiological mechanisms to adapt osmotic and drought tolerance traits, which were critical in the evolution of plants from water to land. Recently reported genomes of charophyte algae, members of Zygnematophyceae as the sister linage to land plant, provided insights into the terrestrial adaptation 104,105 . In particular, RCAR/PYR/PYL ABA receptors were suggested to be acquired by horizontal gene transfer from soil bacteria 104 , likely consistent with the presence of a gene corresponding to RCAR/PYR/ PYL in streptophyte algae 106 . Moreover, the ancestral type of the receptor in Zygnema circumcarinatum alga inhibited the phosphatase activity of PP2C independently of ligand, i.e. ABA, suggesting that the property of ABA binding was acquired during evolution from algae to land plants 107 . By contrast, Klebsormidium nitens (K. nitens) a semi-terrestrial green alga, have all the components of ABA signalling, except for the ABA receptors 108 . Nonetheless, the K. nitens SnRK2 kinase, which is closer to subclass III SnRK2s in Arabidopsis, restored the downstream pathways in response to ABA in Arabidopsis 109 and P. patens 85 . In addition to the K. nitens SnRK2 kinase, Arabidopsis SRK2C (subclass II) and SRK2E (subclass III) kinases restored the ABA-and osmotic stress-responses in the null mutant of PpSnRK2s, whereas SRK2H (subclass I) kinase did not 85  Altogether, these studies suggest that subclass III is an ancient form of SnRK2s and the most recent form is subclass I emerged before angiosperms. Vegetative desiccation tolerance is a typical trait of bryophytes to survive in extreme water-deficit conditions that is not present in vascular plants. Both ABA treatment 110 and downregulation of clade A PP2Cs 89 promote P. patens survival in extreme desiccation conditions. Clade A PP2C act downstream of ABAactivated SnRK2s in P. patens and it has been hypothesized that they emerged to repress ABA-mediated desiccation tolerance, facilitating the vegetative propagation of land plants 89 . Although two genes encode for clade A PP2C in P. patens, which mainly regulate ABA-induced genes 89 , nine genes in the seed plant Arabidopsis genome may play different roles in different tissues and organs. Later in evolution, PYR/PYL/RCAR ABA receptors might be able to recruit PP2Cs, thus activating SnRK2s and improving growth and stress survival on land plants 89 .
Although ARK is the single gene representing the B3 Raf-like MAPKKKs in P. patens, this clade might be later expanded in angiosperms, as six B3-MAPKKK genes are encoded in Arabidopsis (Supplementary Table 2 and Fig. 2a). In particular, RAF4, RAF5, and RAF6, in which the Ser residue of the ark point mutation is conserved, appeared to be the functional orthologs of ARK 50,54 . The current hypothesis is that ABA-B3 Raf-like MAPKKK-subclass III SnRK2 signalling module existing in P. patens has been conserved to protect plants from drought during evolution. Later, bryophytes plants evolved by acquiring a novel subclass I SnRK2 system in vascular plants that conferred further protection to osmotic stress independently from the ancient system 54,85 . Similarly, B4 Raf-like kinases might have been specifically acquired by vascular plants as well, suggesting that the signalling module B4 Raf-like MAPKKK-subclass I SnRK2s-VCS emerged to control post-transcriptional functions and improve the stress-adaptive response of plants against osmotic stress 77 . As some B4 Raf-like kinases family members are not conserved in P. patens, specific members of the B4 Raf-like kinases and subclass I SnRK2s might have been specifically acquired by seed plants to improve the adaptation mechanisms of these plants in osmotic stress conditions. Consistently, the RAF18/20/24-subclass I SnRK2-VCS signalling cascade is activated in response to osmotic stress and not ABA 53 . In summary, land plants have developed new molecular mechanisms for perception and signal transduction in response to osmotic stress, which have been crucial to confer protection to higher plants against drought.
Outlook and future perspectives. Although the ABA activation of SnRK2s and the ABA-core signalling pathway were well studied, Raf-like MAPKKKs have been just recently identified to be crucial for the osmotic stress-mediated activation of SnRK2s [50][51][52][53][54] . Importantly, these studies report the role of Raf-like MAPKKKs in early osmotic stress signalling controlling both ABA-dependent and ABA-independent pathways in higher plants [51][52][53][54] . Thus, Raf-like kinases are likely connecting the osmosensing machinery of plants to SnRK2s (Fig. 3). However, the osmosensor components upstream Raf-like MAPKKKs still need to be identified. The structural comparison with well-studied Raf-like MAPKKK, such as CTR1 interacting with ethylene receptors 111 , may provide insights into the upstream sensory proteins and the substrate specificities for SnRK2s. Importantly, Raf-like MAPKKKs seem to function independently from the OSCA1 osmosensor 52 . Besides the Raf-like MAPKKKs, an increasing number of proteins have been reported to regulate SnRK2s post-translationally [112][113][114][115][116][117] . Future studies addressing how Raf-like MAPKKs function together with OSCA1 and other upstream factors of SnRK2s in response to osmotic stress should shed new light on this direction.
Here we connect the Raf-like MAPKKKs with the state-of-theart knowledge offering general schematic picture of the osmotic regulation of ABA-dependent and -independent pathways (Fig. 3). Still, many remaining questions need to be addressed: (i) How plants sense osmotic stress? (ii) What is the signal that leads to the triggering of the osmotic stress signalling pathways? (iii) What are the molecular and physiological functions of ABAindependent pathways in response to osmotic stress? Further studies in this direction are needed to continue deciphering the osmotic stress signalling pathways and improving our understanding on how plants cope with water-deficit stress.