Arabidopsis group C Raf-like protein kinases negatively regulate abscisic acid signaling and are direct substrates of SnRK2

Abstract

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.

Keywords: Arabidopsis thaliana; Raf-like kinase; SnRK2; abscisic acid.

Fig. 1.

Raf36 interacts with subclass III SnRK2s. (A) AlphaScreen assay shows interaction of Raf36 and subclass III SnRK2s. Bars indicate means ± SE (n = 3), and asterisks indicate significant differences by two-tailed Student’s t test (P < 0.05). (B) Yeast two-hybrid (Y2H) assay shows interaction between Raf36 and subclass III SnRK2s. Yeast cells expressing GAL4AD:Raf36 and GAL4BD:SnRK2s fusion proteins were incubated on SD media supplemented with or without 3-amino-1,2,4-triazole (3-AT) and lacking combinations of amino acids leucine (L), tryptophan (W), and histidine (H), as follows (in order from low to high stringency): -LW, -LWH, -LWH +10 mM 3-AT, -LWH +50 mM 3-AT. Photographs were taken at 10 d (SRK2D and SRK2E) or 12 d (SRK2I) after incubation. (C) Subcellular localization of Raf36-GFP in leaf mesophyll cells. Chl indicates chlorophyll autofluorescence. (Scale bar, 20 µm.) (D) BiFC assays for Raf36 and subclass III SnRK2s. SnRK2 and Raf36 or Raf43 were transiently expressed in N. benthamiana leaves by Agrobacterium infiltration. nEYFP and cEYFP represent the N- and C-terminal fragments of the EYFP, respectively. BF indicates bright field images. (Scale bar, 50 µm.) (E) Coimmunoprecipitation assay of Raf36 and SRK2I. Proteins were extracted from 35S:Raf36-HA and 35S:Raf36-HA/ 35S:FLAG-SRK2I plants treated with/without 50 µM ABA for 30 min. Immunoblotting (IB) was performed with an anti-HA or anti-FLAG antibody. (F) Y2H assay for truncated versions of Raf36 and SRK2E. Yeast cells coexpressing GAL4AD:Raf36, Raf36 N, or Raf36 KD+C and GAL4BD:SRK2E fusion proteins were incubated on SD media lacking L, W, H, and adenine (A), as follows (in order from low to high stringency): -LW, -LWH, -LWHA.

Fig. 2.

Raf36 negatively regulates SnRK2-dependent ABA response phenotypes in Arabidopsis seedlings. (A) Abundance of Raf36 mRNA transcripts measured by qRT-PCR. Total RNA was extracted from 1-wk-old WT Col-0 Arabidopsis seedlings treated with 50 µM ABA for indicated periods. Bars indicate means ± SE (n = 3), and asterisks showed significant differences by Dunnett’s test between ΔCt (nontreated) and ΔCt (ABA-treated) (*P < 0.05, **P < 0.01). (B) Dwarf phenotype of raf36 plants under normal condition. WT (Col-0), raf36-1, and raf36-2 plants grown at 22 °C under 16/8 h photoperiod for 29 d. (Scale bar, 3 cm.) (C and D) Quantification of the cotyledon greening rates of WT (Col-0), raf36-1, and raf36-2 on germination agar medium (GM) with or without 0.5 µM ABA. Data are means ± SE (n = 3). Each replicate contains 36 seeds. Photographs were taken 6 d after vernalization. (E and F) Quantification of the cotyledon greening rates of WT (Col-0), raf36-1, srk2dsrk2e, and raf36-1srk2dsrk2e on GM agar medium in the presence or absence of 0.5 µM ABA. Data are means ± SE (n = 3). Each replicate contains 36 seeds. Photographs were taken 6 d after vernalization.

Fig. 3.

Subclass III SnRK2s directly phosphorylate Raf36. (A) In vitro phosphorylation assay using kinase-dead forms of GST-SRK2E (SRK2E K50N) or MBP-Raf36 (Raf36 K234N). Each kinase-dead form was coincubated with an active GST-SRK2E or MBP-Raf36 kinase as indicated. Assays were performed in the presence of 5 mM Mn²⁺ (Left three lanes) or 5 mM Mg²⁺ (Right three lanes) with [γ-³²P] ATP. (B) In vitro phosphorylation assay using truncated forms of MBP-tagged Raf36. Each MBP-Raf36 protein was incubated with MBP-SRK2E in the presence of 5 mM Mg²⁺ with [γ-³²P] ATP. N: N-terminal region, KD: kinase domain, C: C-terminal region. (C) Schematic representation of six Raf36 (134 to 163) tested as SRK2E substrates. Ser¹⁴¹, Ser¹⁴⁵, Ser¹⁵⁰, and Ser¹⁵⁵ are labeled in blue, with alanine substitutions shown in red. Ser¹⁵⁷, labeled in green, was replaced with alanine in Raf36 (134 to 163) #1 to #5. (D) In vitro phosphorylation of GST-Raf36 (134-163) #1 to #6 by MBP-SRK2E. (E) In vitro phosphorylation of GST-Raf36 (134-163) #3 by MBP-SRK2D or MBP-SRK2I. Autoradiography (³²P) and Coomassie brilliant blue (CBB) staining show protein phosphorylation and loading, respectively. (F and G) Phosphorylation of MBP-Raf36 Ser¹⁴⁵ identified by phosphopeptide mapping. The amino acid sequences with probable phosphorylated serine residue (pS), ion-current chromatograms (F), and MS/MS spectrum (G) derived from a phosphopeptide of m/z = 437.88+++ are shown. MBP-Raf36 protein was either phosphorylated by MBP-SRK2E (+ SRK2E) or autophosphorylation (Auto-P) for 30 min. (H) Phosphorylation levels of Raf36 Ser¹⁴⁵ (Upper) and Ser¹⁰¹ (Lower) after ABA treatment. Protein extracts were prepared from 2-wk-old Raf36pro:Raf36-3xFLAG/raf36-1 transgenic seedlings, which were treated with or without 50 µM ABA for 30 min, and total proteins were subjected to immunoprecipitation using anti-DYKDDDDK tag antibody beads followed by tryptic digestion. Peptides were analyzed by LC-MS/MS, and Raf36 phosphopeptides were identified and quantified based on the peak area of extracted ion chromatogram (XIC). Data are means ± SE from five independent biological replicates. Asterisks indicate significant differences (**P < 0.01, two-tailed Student’s t test). n.s., not significant.

Fig. 4.

Raf22, a C6 Raf-like kinase, functions redundantly with Raf36. (A) Phylogenetic tree of subfamily C5 and C6 Raf-like kinases in Arabidopsis. (B) Y2H assay for C5/C6 Raf kinases and SRK2I. Yeast cells expressing GAL4AD:Raf and GAL4BD:SRK2I fusion proteins were grown on nonselective SD-LW (synthetic defined media lacking leucine and tryptophan) or selective SD-LWHA (SD lacking leucine, tryptophan, histidine, and adenine) media, and LacZ reporter activity was determined by X-Gal overlay assay. (C) BiFC assay for C5/C6 Raf kinases and SnRK2s (SRK2E and SRK2I) in N. benthamiana leaves. nEYFP and cEYFP represent the N- and C-terminal fragments of the EYFP, respectively. BF indicates bright field images. (Scale bar, 50 µm.) (D) In vitro phosphorylation of C5/C6 Raf kinases by GST-tagged SRK2E. MBP-tagged kinase-dead forms of Raf43 (Raf43 K228N), Raf22 (Raf22 K157N), or Raf28 (Raf28 K158N) were used as substrates. (E) In vitro phosphorylation of kinase-dead Raf22 (K157N) and Raf22 (S81A K157N) proteins by GST-SRK2E. (F and G) Quantification of the cotyledon greening rates of WT (Col-0), raf36-1, raf22, and raf22raf36-1 on GM agar medium with or without 0.5 µM ABA. Data are means ± SE (n = 4). Each replicate contains 36 seeds. Photographs were taken 9 d after vernalization. (H and I) Relative gene expression of ABA-responsive genes. Total RNA was extracted from 1-wk-old plants including WT, raf36-1, raf22, and raf22raf36-1 treated with 50 µM ABA for indicated periods. Bars indicate means ± SE (n = 3), and asterisks showed significant differences by Dunnett’s test between ΔCt (Col-0) and ΔCt (mutant) for each time point (*P < 0.05, **P < 0.01).

Fig. 5.

Phosphoproteomic analysis of WT and raf22raf36-1 identifies ABA signaling components downstream of Raf kinases. (A and B) Functional complementation of raf36-1 by Raf36pro:Raf36-3xFLAG or Raf36pro:Raf36 K234N-3xFLAG. Shown is photograph of seedlings grown for 7 d on GM agar medium in the presence or absence of 0.5 µM ABA. The Western blot image shows the expression level of Raf36-3xFLAG protein in 1-wk-old seedlings grown on normal GM agar medium. Greening rates were scored 7 d after vernalization. Data are means ± SE (n = 3), and each replicate contains 54 seeds. Different letters indicate significant differences (Tukey’s test, P < 0.01). (C and D) Western blot analysis of Raf36-3xFLAG after ABA treatment. Protein extracts were prepared from 2-wk-old WT (Col-0) or 2x35S:Raf36-3xFLAG transgenic plants, which were treated with 50 µM CHX in the presence or absence of 50 µM ABA for the indicated time periods. The Raf36 protein was detected by Western blotting using anti-FLAG antibody. Ponceau-S staining was used as loading control. Raf36 protein level at 0 h was set to 1.0 as a reference for calculating the relative band intensities at the various time points. The values are presented as the means ± SE from five independent biological replicates. Asterisks indicate significant differences for each time point (*P < 0.05, **P < 0.01, two-tailed Student’s t test). (E) PCA of quantitative data of phosphopeptides from WT (Col-0) and raf22raf36-1. (F) Venn diagram of up- or down-regulated phosphopeptides in WT seedlings after 50 µM ABA treatment, and up- or down-regulated phosphopeptides in raf22raf36-1 compared with WT. (G) In vitro phosphorylation of GST-tagged peptides from AT5G04740.1 (124 to 154 aa, No. 4), AT5G16260.1 (141 to 171 aa, No. 5), AT3G23920.1 (42 to 72 aa, No. 6), AT1G70770.1 (72 to 102 aa, No. 7), AT3G54610.1 (69 to 99 aa, No. 8), AT2G24050.1 (18 to 48 aa, No. 9), and AT1G37130.1 (48 to 78 aa, No. 10) by MBP-Raf22. GST-OLE1 fragment (AT4G25140.1, 143 to 173 aa, No. 1) was used as a positive control. GST-tagged peptides from AT3G62800.1 (60 to 90 aa, No. 2) and AT2G02070.1 (56 to 86 aa, No. 3) were included as negative controls. The autoradiography (³²P) and Coomassie brilliant blue (CBB) staining show protein phosphorylation and loading, respectively.