The phytohormone abscisic acid (ABA) plays a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) kinases mediate ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in Arabidopsis, as a protein that interacts with multiple SnRK2s. A series of reverse genetic and biochemical analyses revealed that 1) Raf36 negatively regulates ABA responses during postgermination growth, 2) the N terminus of Raf36 is directly phosphorylated by SnRK2s, and 3) Raf36 degradation is enhanced in response to ABA. In addition, Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. A comparative phosphoproteomic analysis of ABA-induced responses of wild-type and raf22raf36-1 plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results support a model in which Raf36/Raf22 function mainly under optimal conditions to suppress ABA responses, whereas in response to ABA, the SnRK2 module promotes Raf36 degradation as a means of alleviating Raf36-dependent inhibition and allowing for heightened ABA signaling to occur.

Reversible phosphorylation is a major mechanism for regulating protein function and controls a wide range of cellular functions including responses to external stimuli. The plant-specific SNF1-related protein kinase 2s (SnRK2s) function as central regulators of plant growth and development, as well as tolerance to multiple abiotic stresses. Although the activity of SnRK2s is tightly regulated in a phytohormone abscisic acid (ABA)-dependent manner, recent investigations have revealed that SnRK2s can be activated by group B Raf-like protein kinases independently of ABA. Furthermore, evidence is accumulating that SnRK2s modulate plant growth through regulation of target of rapamycin (TOR) signaling. Here, we summarize recent advances in knowledge of how SnRK2s mediate plant growth and osmotic stress signaling and discuss future challenges in this research field.

The Raf-like protein kinase abscisic acid (ABA) and abiotic stress-responsive Raf-like kinase (ARK) previously identified in the moss Physcomitrium (Physcomitrella) patens acts as an upstream regulator of subgroup III SNF1-related protein kinase2 (SnRK2), the key regulator of ABA and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2, including the role of ABA receptor-associated group A PP2C (PP2C-A), are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic P. patens ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance, and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in the wild type (WT). The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking protein phosphatase 2C-A (PP2C-A) was similar to that in the WT, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense mutant lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. Therefore, ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototype mechanism for ABA and abiotic stress responses in plants.

When plants are exposed to drought stress, there is a trade-off between plant growth and stress responses. Here, we identified a signaling mechanism for the initial steps of the drought-growth trade-off. Phosphoproteomic profiling revealed that Raf13, a B1 subgroup Raf-like kinase, is dephosphorylated under drought conditions. Raf13 and the related B1-Raf Raf15 are required for growth rather than the acquisition of osmotolerance. We also found that Raf13 interacts with B55-family regulatory subunits of protein phosphatase 2A (PP2A), which mediates hyperosmolarity-induced dephosphorylation of Raf13. In addition, Raf13 interacts with an AGC kinase INCOMPLETE ROOT HAIR ELONGATION HOMOLOG 1 (IREH1), and Raf13 and IREH1 have similar functions in regulating cellular responses that promote plant growth. Overall, our results support a model in which Raf13-IREH1 activity promotes growth under nonstressed conditions, whereas PP2A activity suppresses Raf13-IREH1 during osmotic stress to modulate the physiological trade-off between plant growth and stress responses.

33 34 ABSTRUCT 35 A phytohormone abscisic acid (ABA) has a major role in abiotic stress 36 responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) 37 mediates ABA signaling. In this study, we identified Raf36, a group C Raf-like 38 protein kinase in Arabidopsis, as an interacting protein with SnRK2. A series 39 of reverse genetic and biochemical analyses revealed that Raf36 negatively 40 regulates ABA responses and is directly phosphorylated by SnRK2s. In 41 addition, we found that Raf22, another C-type Raf-like kinase, functions 42 3 partially redundantly with Raf36 to regulate ABA responses. Comparative 43 phosphoproteomic analysis using Arabidopsis wild-type and raf22raf36-1 44 plants identified proteins that are phosphorylated downstream of Raf36 and 45 Raf22 in planta. Together, these results reveal a novel subsection of 46 ABA-responsive phosphosignaling pathways branching from SnRK2. 47 48 93 Rafs, Raf22 and Raf36, functions as a "brake" of ABA response downstream of 94 SnRK2s. 95 96 97 6 RESULTS 98 Raf36 interacts with subclass III SnRK2 99To identify additional kinases that regulate ABA signaling pathways, we 100 used the AlphaScreen ® assay to screen a collection of Arabidopsis MAPKKK 101 proteins for their ability to physically interact with SRK2I (SnRK2.3), a subclass III 102 SnRK2. From a pilot experiment, several Raf-like protein kinases were identified as 103 candidate interactors with SRK2I (Supplemental Figure 1). Raf36, which belongs to 104 a C5 subgroup kinase (Supplemental Figure 2), was one of the SRK2I-interacting 105 proteins. Interaction between Raf36 and SRK2I, as well as between Raf36 and 106 additional subclass III SnRK2s, SRK2D (SnRK2.2) and SRK2E (OST1/SnRK2.6), 107 was confirmed by AlphaScreen ® assay (Figure 1A) and yeast two-hybrid assay 108 (Figure 1B). SnRK2s were previously found within the cytosol and nuclei of 109 Arabidopsis cells (Umezawa et al., 2009). We observed that Raf36-GFP is localized 110 mainly in the cytosol (Figure 1C), and bimolecular fluorescence complementation 111 (BiFC) assay confirmed that the interactions between SnRK2s and Raf36 take 112 place in cytosol (Figure 1D) Together, these results demonstrate that Raf36 113 physically interacts with ABA-responsive SnRK2s both in vitro and in vivo. 114 Next, we investigated which domain(s) of Raf36 may be responsible for the 115 interaction with SnRK2s. According to the PROSITE database 116 (https://prosite.expasy.org/), Raf36 contains an unknown N-terminal stretch (N, 1-117 206 aa), a predicted kinase catalytic domain (KD, 207-467 aa) and a short 118 7 C-terminal domain (C, 468-525 aa) ( Figure 1E). In yeast two-hybrid assay, SRK2E 119 strongly interacted with Raf36 full-length protein (FL), but just slightly or not 120 interacted with Raf36 N and KD+C alone, respectively ( Figure 1E). These results 121 indicated that the complete structure of Raf36 protein is required for the interaction 122 with SnRK2. 123 124 Raf36 negatively regulates ABA response at post-germination growth stage 125 To cha...