Hyperosmolarity-induced suppression of group B1 Raf-like protein kinases modulates drought-growth trade-off in Arabidopsis

Protein phosphorylation/dephosphorylation has significant roles in drought stress signaling in plants(15–17). We performed a label-free quantitative phosphoproteomic analysis of Arabidopsis wild-type (WT) seedlings under drought treatment for 0, 3, 5, and 9 d. The result showed that several phosphopeptides from B3-Raf (Raf4/AtARK1), B4-Rafs (Raf18 and Raf20), SnRK2s (SRK2B/SnRK2.10, SRK2D/SnRK2.2, SRK2E/OST1/SnRK2.6, and SRK2I/SnRK2.3), or SnRK2 substrates (VARICOSE, AREB/ABFs, SLAC1, AKS1, and FREE1), were upregulated, consistent with previous reports (10, 13, 18–23) (Fig. 1 A and B and Dataset S1). We focused on a phosphopeptide from the B1-Raf Raf13. The peptide is phosphorylated on two serine residues (Ser-310 and Ser-313, KLEGYPNApSGSpSLR), and was significantly downregulated at 5 and 9 d (Fig. 1B and Dataset S1). This suggested that Raf13 could be dephosphorylated in response to drought stress. This result was further confirmed by immunoblotting of protein extracts from Arabidopsis transgenic plants expressing Raf13-CFP-HA or Raf4-HA. Raf13-CFP-HA showed electrophoretic mobility shifts with lower molecular mass after dehydration (DH) treatment for 30 min, whereas Raf4-HA protein shifted to higher molecular mass (Fig. 1C). Such mobility shifts were also observed under treatment of 400 mM NaCl or 800 mM sorbitol, but not observed during ABA treatment (SI Appendix, Fig. S1 A and B), suggesting that Raf13 is specifically dephosphorylated in response to osmotic stress. An additional LC–MS/MS analysis from WT seedlings treated with or without DH stress detected six peptides containing nine phosphosites (Ser-10, Ser-38, Ser-310, Ser-313, Ser-335, Ser-390, Ser-465, Ser-468, and Ser-469) in Raf13 (SI Appendix, Fig. S2), and four phosphopeptides containing six phosphosites (Ser-38, Ser-310, Ser-313, Ser-335, Ser-468, and Ser-469) showed a significant decrease after DH for 30 min (Fig. 1 D and E and Dataset S2). These results indicated that Raf13 is multiply dephosphorylated in planta during osmotic stress responses.

We next used immunoprecipitation-kinase (IP-kinase) assays to investigate possible functions of Raf13 dephosphorylation. As shown in Fig. 1F, autophosphorylation and histone transphosphorylation activities of Raf13-CFP-HA were reduced when Raf13-CFP-HA was immunoprecipitated from proteins extracted from seedlings treated with DH stress for 30 min. Consistent with these results, the kinase activities of recombinant maltose-binding protein (MBP)-tagged Raf13 (MBP-Raf13) were enhanced or reduced by Asp (D) or Ala (A) substitutions, respectively, at seven Ser residues that correspond to detected phosphorylation sites, including Ser-38, Ser-310, Ser-313, Ser-335, Ser-465, Ser-468, and Ser-469 (Fig. 1G). To evaluate the impact of osmotic stress-induced Raf13 dephosphorylation on its stability, Raf13 protein levels were measured using 35S:Raf13-CFP-HA transgenic plants following treatment with 400 mM sorbitol treatment for 0, 0.5, 3, and 6 h. The Raf13 protein levels in plants subjected to both cycloheximide (CHX) and sorbitol treatment were significantly lower at 3 h compared to those in plants treated with CHX alone (Fig. 1 H and I). Intriguingly, a decrease in Raf13 protein levels under osmotic stress was also observed in the Raf13pro: Raf13-3xFLAG line; however, this decrease was largely inhibited in the phospho-mimetic Raf13^7D (SI Appendix, Fig. S1C). This indicates that osmotic stress reduces Raf13 protein stability through dephosphorylation in planta. Together, these data suggested that hyperosmolarity-induced dephosphorylation downregulates Raf13 kinase catalytic activity and protein stability.

To gain insight into the physiological roles of three B1-Rafs, we next obtained mutants with individual transfer DNA (T-DNA) insertions in Raf13, Raf14, and Raf15 (SI Appendix, Fig. S3A), and generated a double mutant (raf13-1raf15-1) and a triple mutant (raf13-1raf14-1raf15-1; B1-TKO). These mutants showed reduced growth relative to WT under optimal growth conditions. In this regard, significantly shorter primary roots were observed in raf13-1raf15-1 and B1-TKO compared with WT under control conditions. Primary roots were slightly shortened in raf13-1 and raf13-2, but not in raf14-1, raf15-1, or raf15-2 (Fig. 2 A and B). In the presence of ABA, primary root elongation was inhibited by 39.8% in the WT and by approximately 60% in the double/triple mutants. On the other hand, 400 mM sorbitol inhibited root elongation similarly in WT and mutants (Fig. 2 A and B). Some ABA- and stress-responsive genes, KIN1, RD29A, and DREB1A, were hyperinduced in B1-TKO seedlings in response to 50 µM ABA treatment, but the expression level was similar between WT and B1-TKO in response to 400 mM sorbitol (Fig. 2C).

B1-TKO showed a significantly lower cotyledon greening rate relative to WT plants in the presence of ABA, but WT and B1-TKO were similar in the absence of ABA (Fig. 2 D and E and SI Appendix, Fig. S3 B and C). Conversely, 35S:Raf13-CFP-HA plants displayed an ABA-insensitive phenotype (SI Appendix, Fig. S3 D and E). Such a phenotype in B1-TKO is opposite to that of B3-Raf triple knockout mutant (raf4/5/6; B3-TKO) (Fig. 2 D and E). No significant change in seed germination rates was observed in the mutants (SI Appendix, Fig. S3F). We further measured water loss from detached leaves, and found that the lack of both Raf13 and Raf15 genes resulted in reduced water loss (Fig. 2F), which is probably attributed to reduced stomatal aperture even before ABA treatment (SI Appendix, Fig. S3G). Notably, the β-glucuronidase (GUS) activity was observed in young leaves, guard cells, and roots in Arabidopsis Raf13pro:GUS plants (SI Appendix, Fig. S3H). Collectively, these results indicated that B1-Rafs are required for some of the aspects of plant growth and appear to negatively regulate responses to exogenous ABA.

To test the significance of Raf13 phosphorylation in planta, we generated stable transgenic lines by introducing 3xFLAG-tagged Raf13 with WT, phosphor-null (7A), or phosphor-mimetic (7D) forms into raf13-1raf15-1 mutants under the control of the native promoter (SI Appendix, Fig. S4A). Both Raf13^WT and Raf13^7D partially complemented the reduced primary root length observed in raf13-1raf15-1, whereas Raf13^7A did not (SI Appendix, Fig. S4B). In addition, the accumulation of Raf13^7A was significantly lower than that of Raf13^WT or Raf13^7D (SI Appendix, Fig. S4C). These results suggested that osmotic stress-dependent dephosphorylation of Raf13 reduces its stability in planta.

Phenotyping tests suggested that B1-Rafs might have different functions from B2-, B3-, and B4-Rafs, some of which are upstream activators of SnRK2s (9–13). To check the biochemical properties of Raf13, we prepared the recombinant MBP-tagged Raf13 with autophosphorylation and histone transphosphorylation activities. A K546N substitution in Raf13 abolished all kinase activity, indicating that Raf13 is a canonical protein kinase (SI Appendix, Fig. S5A). Raf4 (B3) and Raf10 (B2) strongly phosphorylated SRK2E^K50N in the kinase activation loop (Fig. 3A). However, Raf13 did not phosphorylate SRK2E (Fig. 3A) and SRK2G/SnRK2.1 (SI Appendix, Fig. S5B).

Coimmunoprecipitation (Co-IP) assays showed no or negligible interaction between Raf13 and SRK2I, but both Raf4 and Raf10 interacted with SRK2I (Fig. 3B). Furthermore, in-gel kinase assays demonstrated that 800 mM sorbitol- or 50 µM ABA-induced SnRK2 activation was not affected in B1-TKO or 35S:Raf13-CFP-HA plants, but 800 mM sorbitol-induced SnRK2 activation was reduced in B3-TKO as previously reported (9) (Fig. 3 C and D and SI Appendix, Fig. S5 C and D). Collectively, these results indicate that B1-Rafs function independently of SnRK2s.

To identify signaling factors associated with Raf13, we performed immunoprecipitation followed by LC–MS/MS analyses (IP–MS). Raf13-CFP-HA protein was immunoprecipitated from Arabidopsis 35S:Raf13-CFP-HA plants with or without DH treatment for 30 min. As a result, a total of 37 proteins were identified as Raf13-CFP-HA-interacting candidates under the control and/or DH conditions (Fig. 4A and Dataset S3). In Arabidopsis, the B55 family of type 2A protein phosphatase regulatory B subunits (PP2A-B55s) comprises only two genes: ATBα and ATBβ, also referred to as B55α and B55β, respectively (24). Our dataset includes both ATBα and ATBβ, indicating a potential role for PP2A-B55s in the regulation of Raf13. To further confirm the physical interactions between Raf13 and ATBα/β, we performed Co-IP assays in N. benthamiana leaves, and found that FLAG-ATBα and -ATBβ were coimmunoprecipitated with Raf13-CFP-HA under both control and DH conditions (Fig. 4B). Raf13 and ATBα were localized in the cytosol, and ATBβ was localized in both cytosol and nuclei (SI Appendix, Fig. S6A), suggesting possible colocalization in the cytosol.

CT is a widely utilized PP2A inhibitor that preferentially inhibits the activities of Ser/Thr protein phosphatases type 1 (PP1) or type 2A (PP2A) (25). We tested electrophoretic mobility shifts of Raf13-CFP-HA with or without CT treatment. Raf13-CFP-HA did not show any mobility shifts, but its abundance significantly increased in CT-treated plants (Fig. 4 C and D), indicating one or more CT-sensitive protein phosphatases dephosphorylate Raf13 in vivo. Furthermore, in amiRNA-based ATBα/β-knockdown plants, the hyperosmolarity-induced mobility shift of Raf13-CFP-HA was abolished and was proved as increased protein amounts (Fig. 4E). Note that expressions of both ATBα and ATBβ mRNA were suppressed by about 55 to 60% in the two ATBα/β-knockdown plants (Fig. 4F). Moreover, E.coli-expressed MBP-Raf13 protein migrated faster in SDS–PAGE after being incubated with immunoprecipitates from Arabidopsis 35S:FLAG-ATBα transgenic seedlings, regardless of DH treatment (SI Appendix, Fig. S6B). This suggests a potential capability of ATBα-containing protein complexes for the dephosphorylation of Raf13.

We then obtained T-DNA insertion mutants for ATBα or ATBβ (SI Appendix, Fig. S6C). These mutants are less sensitive to ABA in cotyledon greening as well as increased water loss from detached leaves (SI Appendix, Fig. S6 D and E). To determine whether the reduced ABA sensitivity and increased water loss of atbβ require B1-Raf signaling, we generated a triple mutant, raf13-1raf15-1atbβ. Our observations revealed that this triple mutant exhibited greater sensitivity to ABA and demonstrated reduced water loss compared to atbβ (Fig. 4 G and H). These results indicate that Raf13 and Raf15 serve as genetic modifiers, at least in part, of atbβ-dependent hyposensitivity to ABA or DH stress. Notably, the mRNA abundances of ATBα, but not ATBβ, were upregulated after DH treatment (SI Appendix, Fig. S6F). These results indicated that ATBα and ATBβ are bona fide regulators to promote ABA signal transduction along with the acquisition of osmotolerance.

Collectively, these results strongly suggested the involvement of PP2A holoenzymes, which harbor ATBα or ATBβ as regulatory B subunits, in the hyperosmolarity-induced Raf13 dephosphorylation.

In addition to PP2A, IP-MS analyses identified Raf15 and the AGC kinase IREH1 as Raf13-interacting proteins, both exhibiting high PSM scores (Fig. 4A and Dataset S3). We confirmed that IREH1 can interact with Raf13 and Raf15, but not with Raf14, in yeast two-hybrid (Y2H) analyses (Fig. 5A) and bimolecular fluorescence complementation (BiFC) assays in N. benthamiana epidermal cells (Fig. 5B).

We next obtained two IREH1 T-DNA insertion lines (SI Appendix, Fig. S7A). Both ireh1-1 and ireh1-2 mutants exhibited ABA-hypersensitive phenotype in the greening response (SI Appendix, Fig. S7 B and C), and displayed shorter primary roots which were similar to B1-Raf knockout mutants (Fig. 5 C and D). To evaluate the genetic relationship between B1-Raf and IREH1, we generated a triple mutant, raf13-2raf15-2ireh1-2, and observed that it exhibits phenotypes similar to those of the raf13raf15 double mutants in terms of primary root length (Fig. 5 C and D) and cotyledon greening (SI Appendix, Fig. S7 B and C). These results indicate that Raf13/Raf15 are epistatic to IREH1, suggesting that Raf13/Raf15 and IREH1 function within the same pathway to positively regulate plant growth while negatively regulating ABA responses.

To gain insight into their signaling pathways, we performed a comparative phosphoproteomic analysis of WT, raf13-1raf15-1, and ireh1-2 seedlings grown under optimal growth conditions. As a result, phosphoproteomic profiles in ireh1-2 were significantly correlated with those in raf13-1raf15-1 (Fig. 5E). Importantly, of the 177 phosphopeptides that were downregulated in raf13-1raf15-1, 83.1% (147/177) overlapped with ireh1-2 (Fig. 5F and Dataset S4). This finding suggests that B1-Rafs and IREH1 share common phosphorylation targets, or that B1-Rafs and IREH1 phosphorylate their respective downstream substrates when they are bound together. Notably, gene ontology (GO) analysis reported terms related to growth and development such as cell division, carbon fixation, and flower development from 147 phosphopeptides downregulated in both raf13-1raf15-1 and ireh1-2 (SI Appendix, Fig. S8A and Dataset S5). Three phosphorylation motifs, [-pS-P-], [-R-x-x-pS-], and [-pS-M-], were enriched in the phosphopeptides downregulated in both raf13-1raf15-1 and ireh1-2 (SI Appendix, Fig. S8B). These results suggest that Raf13, Raf15, and/or IREH1 preferentially recognize the motifs [-pS-P-], [-R-x-x-pS-], and [-pS-M-]. Alternatively, Raf13, Raf15, and/or IREH1 may regulate kinases that phosphorylate these motifs in planta. Specifically, [-pS-P-] and [-R-x-x-pS-] are recognized as target motifs for MAPK and SnRK2s/calcium-dependent protein kinases, respectively (26).

Finally, we performed in vitro phosphorylation assays using Raf13 and IREH1. Recombinant Raf13 and IREH1 directly phosphorylated kinase-dead forms of IREH1^K911N and Raf13^K546N, respectively, indicating reciprocal phosphorylation between Raf13 and IREH1 (SI Appendix, Fig. S8C).

Arabidopsis thaliana ecotype Columbia (Col-0) was used as the WT plants. T-DNA insertion mutant lines, raf13-1 (SALK_137974C), raf13-2 (SAIL_104_B07), raf14-1 (SALK_126902C), raf15-1 (SALK_119918C), raf15-2 (SALK_020351C), atbα (SALK_095004C), atbβ (SALK_062514C), ireh1-1 (SALK_017861), and ireh1-2 (SALK_069962C) were obtained from the Arabidopsis Biological Resource Center (ABRC) (https://abrc.osu.edu/). The raf13-1raf15-1, raf13-2raf15-2, raf13-1raf14-1raf15-1 (B1-TKO), raf13-1raf15-1atbβ, or raf13-2raf15-2ireh1-2 mutant was generated by genetic crosses. The raf4/5/6 mutant (referred to as B3-TKO in this study) was established as previously described (9). Seeds of WT, mutants, or transgenic plants were sterilized, and sown on 0.8% (w/v) germination agar medium (GM) containing 1% (w/v) sucrose. After vernalization at 4 °C in the dark for 4 d, the plants were transferred to a growth chamber with continuous white light illumination of 90 µmol m⁻² s⁻¹ photon flux density and grown for indicated periods at 22 °C. For ABA sensitivity test, seeds were sown on GM agar medium with or without indicated concentrations of ABA (Sigma-Aldrich). Germination and greening rates were scored as any seed with root emergence or green cotyledons, respectively.

Full-length of the Raf4 cDNA was previously cloned into pENTR/D-TOPO vector (Thermo Fisher Scientific) (9). In addition, full-length of the Raf13, Raf14, Raf15, ATBα, ATBβ, and IREH1 cDNAs were cloned into pENTR1A vector (Thermo Fisher Scientific) and verified by sequencing. Amino acid substitutions were carried out by site-directed mutagenesis as previously described (18). Those cDNAs were transferred into destination vectors, such as pGreen0029-GFP, pGreen0029-HA, R4pGWB 501 (43), pEarleyGate 102, pEarleyGate 202 (44), pSITE-nEYFP-C1 (CD3-1648), pSITE-cEYFP-N1 (CD3-1651), pGBKT7, and pGADT7 (Takara Bio) by using Gateway LR Clonase II (Thermo Fisher Scientific). For the artificial microRNA to silence the expression of both ATBα and ATBβ, primer sequences were designed using a Web microRNA designer, WMD3 (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi), and the resulting PCR fragment was cloned into pENTR1A vector to generate pENTR1A harboring ATBα/β-ami. For the pDONR P4-P1R Raf13p or pDONR P4-P1R UBQ10p constructs, the putative Raf13 promoter (2,712 bp upstream sequence) and the UBQ10 promoter (636 bp upstream sequence) were amplified with specific primers with Gateway attB4/attB1r adaptor sequences and cloned into pDONR P4-P1R vector (Thermo Fisher Scientific) using Gateway BP Clonase II (Thermo Fisher Scientific). For the R4pGWB 501 Raf13p:GUS construct, pDONR P4-P1R Raf13p and pENTR-gus (Thermo Fisher Scientific) were reacted with R4pGWB 501 (43) using Gateway LR Clonase II. In addition, pDONR P4-P1R UBQ10p and pENTR1A ATBα/β-ami were used to establish the R4pGWB 501 UBQ10p:ATBα/β-ami.

The pGreen0029, pEarleyGate 102, or pEarleyGate 202 were used to express Raf4-HA, Raf13-CFP-HA, or FLAG-ATBα. R4pGWB 501 harboring Raf13p:GUS was prepared as described above. Each construct was transformed into Arabidopsis WT plants with Agrobacterium tumefaciens strain GV3101 or GV3101 (pSOUP). Transgenic plants were selected on GM agar medium containing 200 μg/ mL claforan with either 50 μg/mL kanamycin, 25 μg/ mL hygromycin or 10 μg/ mL Basta. For the 35S:Raf13-CFP-HA/ UBQ10p:ATBα/β-ami transgenic plants, the R4pGWB 501 vector harboring UBQ10p:ATBα/β-ami was transformed into the 35S:Raf13-CFP-HA transgenic plants and transformants were selected on GM agar medium containing 200 μg/ mL Claforan, 25 μg/ mL hygromycin and 10 μg/ mL Basta.

Arabidopsis Raf13p:GUS transgenic seedlings were pretreated with 90% acetone on ice for 15 min, then incubated in GUS staining solution [1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc), 0.5 mM K₃[Fe(CN)₆], 0.5 mM K₄[Fe(CN)₆], 0.1% (v/v) Triton X-100, 50 mM sodium phosphate (pH 7.2)] in the dark at 37 °C for 16 h. The samples were washed and bleached with 70% ethanol. Images of the stained samples were captured using an EPSON GT-X970 scanner (Epson) or using a Leica DMLB fluorescence microscope equipped with a Leica DFC420 camera (Leica).

Arabidopsis 7-d-old seedlings grown on 0.8% (w/v) GM agar plates under a 16 h/8 h (light/dark) photoperiod at 22 °C were transferred to soil, and the plants were grown under the same conditions for another 3- or 4-wk. The detached rosette leaves from 4- to 5-wk-old plants were placed on weighing dishes and left on the laboratory bench. Fresh weights were monitored as previously described (5).

Stomatal aperture was measured according to previous studies (6, 13). Epidermal strips were peeled from the rosette leaves of 4-wk-old Arabidopsis seedlings grown in soil under a 16 h/8 h (light/dark) photoperiod and incubated in MES buffer [10 mM MES-KOH (pH 6.2), 10 mM KCl, and 50 µM CaCl₂] under white light for 3 h to fully open the stomata. Then the strips were transferred to MES buffer containing 10 µM ABA for 2 h. Stomatal apertures were photographed using a BX53 fluorescence microscope (Olympus) and measured by quantifying pore width of stomata using ImageJ software.

Total RNA was extracted from 2-wk-old Arabidopsis seedlings as described previously (45), and 500 ng of total RNA was used for reverse transcription using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). qRT-PCR analysis was performed using LightCycler 480 SYBR Green I Master (Roche Life Science) with Light Cycler 96 (Roche Life Science). Each transcript was normalized by GAPDH and analyzed with three or four biological replicates. The gene-specific primers used for qRT-PCR are listed in Dataset S6.

pMAL-c5X vectors harboring SRK2E^K50N, SRK2E^{K50N S171A S175A}, SRK2G^K33N, or Raf4 cDNAs were previously generated (9). In addition, Raf10, Raf13, and IREH1 cDNAs were cloned in-frame to pMAL-c5X vector (New England Biolabs) by using the In-Fusion HD Cloning Kit (Takara Bio). Amino acid substitutions were introduced into pMAL-c5X Raf13 by site-directed mutagenesis to produce the kinase-dead Raf13^K546N, phospho-null Raf13^7A, or phospho-mimetic Raf13^7D. The recombinant proteins were expressed, and affinity purified from E. coli strain BL21 (DE3) using Amylose Resin (New England Biolabs) as previously described (5).

For the kinase assay using bacterially expressed proteins, proteins were mixed with the indicated pair(s) and incubated in a total volume of 10 μL of reaction buffer [50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 5 mM MnCl₂, 50 µM ATP, and 0.037 MBq of [γ-³²P] ATP (PerkinElmer)] at 30 °C for 30 min. To assess the kinase activity of the immunoprecipitated Raf13-CFP-HA protein, approximately 5 g of Arabidopsis 2-wk-old 35S:Raf13-CFP-HA transgenic seedlings grown on agar plates were treated with/without DH for 30 min on the filter paper, and Raf13-CFP-HA was immunoprecipitated using 20 µL of GFP-selector (NanoTag Biotechnologies, N0310, lot 03210303). The immunoprecipitates were washed three times and then incubated with 2 μg of histone in the same reaction buffer as described above. The protein samples were subsequently separated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), and phosphorylation levels were visualized by autoradiography using BAS-5000 (Fujifilm).

Crude proteins were extracted from 2-wk-old Arabidopsis seedlings in extraction buffer [50 mM HEPES-KOH (pH 7.5), 5 mM EDTA, 5 mM EGTA, 1 mM Na₃VO₄, 25 mM NaF, 50 mM β-glycerophosphate, 20% (v/v) glycerol and 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich)]. 40 µg of total protein was separated on a 10% SDS–PAGE gel containing histone IIIS as kinase substrate. After electrophoresis, the gel was washed four times at room temperature with wash buffer [25 mM Tris-HCl (pH 7.5), 5 mM NaF, 0.1 mM Na₃VO₄, 0.5 mg/mL BSA, 0.1% TritonX-100, and 0.5 mM dithiothreitol (DTT)] and incubated at 4 °C for 16 h with three changes of renaturation buffer [25 mM Tris-HCl (pH 7.5), 5 mM NaF, 0.1 mM Na₃VO₄, and 1 mM DTT]. The gel was then incubated in reaction buffer [40 mM HEPES-KOH (pH 7.5), 0.1 mM EGTA, 20 mM MgCl₂, and 1 mM DTT] with 30 µM ATP plus 25 mM [γ-³²P] ATP for 1 h at 30 °C. The reaction was stopped by transferring the gel to wash solution [5% (v/v) trichloroacetic acid (TCA) and 1% (w/v) sodium pyrophosphate], and the gel was washed with the same solution five times. Radioactivity was detected using a BAS -5000 (Fujifilm).

Proteins were extracted from plants in extraction buffer [20 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 5 mM MgCl₂, 20% glycerol, 0.5% TritonX-100, 1 mM Na₃VO₄, 25 mM NaF, and 1% Protease Inhibitor Cocktail (Sigma-Aldrich)] followed by two-step centrifugation at 4 °C for 10 min. The supernatants were separated by 8 or 10% SDS-PAGE. After electrophoresis, the proteins were transferred to the polyvinylidene fluoride membrane (0.45 μm, Millipore) and immunoblotted using the primary antibodies, anti-HA [16B12] (BioLegend, 901513, lot B274467, 1: 3,000 dilution), anti-FLAG (DYKDDDDK) (Wako, 014-22383, lot SAR0168, 1: 3,000 dilution) or Anti-GFP pAb (MBL, 598, lot 072, 1:3,000 dilution), using Horse anti-mouse IgG-horseradish peroxidase conjugates (Vector Laboratories, PI-2000, lot ZH0513, 1: 3,000 dilution) or Anti-IgG (H + L chain) (Rabbit) pAb-HRP (MBL, 458, lot 350, 1:5,000 dilution) as the secondary antibody.

pENTR entry clones harboring Raf13, Raf10, Raf4, ATBα, or ATBβ were subcloned into pEarleyGate 102 destination vector with C-terminal CFP-HA fusion or into pEarleyGate 202 with N-terminal FLAG epitope tag fusion (44). pEarleyGate 100 vector containing FLAG-SRK2I was generated as previously described (5). Constructs were introduced into N. benthamiana leaves with A. tumefaciens strain GV3101. After 3 d of incubation, the leaves were ground to a powder in liquid nitrogen, and total proteins were extracted in 10 mL of extraction buffer [20 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 5 mM MgCl₂, 20% glycerol, 0.5% TritonX-100, 1 mM Na₃VO₄, 25 mM NaF, and 1% Protease Inhibitor Cocktail (Sigma-Aldrich)], followed by centrifugation at 17,400×g for 10 min at 4 °C. The supernatant was incubated with 20 µL of GFP-selector (NanoTag Biotechnologies) for 3 h at 4 °C. The immunoprecipitates were washed four times with 1 mL of the extraction buffer without the protease inhibitor cocktail. After washing, the proteins were eluted in 2xSDS sample buffer [125 mM Tris-HCl (pH 6.8), 4% SDS, 10% (w/v) sucrose, 0.004% (w/v) bromophenol blue and 200 mM DTT]. Aliquots were separated by SDS-PAGE and analyzed by western blotting.

Arabidopsis WT or 35S:FLAG-ATBα transgenic seedlings were grown on GM agar medium for 2 wk, followed by DH stress on filter paper for 30 min. Crude proteins were extracted in PP2A IP buffer [50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.3 M Sucrose, 0.2% TritonX-100, and 1% Protease Inhibitor Cocktail (Sigma-Aldrich)], and then subjected to centrifugation at 4 °C for 10 min to remove cellular debris. The supernatant was incubated with 20 µL of anti-DYKDDDDK tag antibody beads (Wako) for 2 h at 4 °C. The immunoprecipitates were washed four times with 1 mL of the PP2A IP buffer. For dephosphorylation, 6.5 µL of the beads were incubated with 1 μg of MBP-Raf13 protein in a total volume of 10 μL of reaction buffer [50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, and 5 mM MnCl₂] at 30 °C for 30 min. Aliquots were separated by SDS-PAGE and visualized by western blotting.

For subcellular localization analysis, pGreen0029-GFP constructs harboring Raf13, ATBα, or ATBβ were introduced into N. benthamiana leaves using A. tumefaciens strain GV3101 (pSOUP). In addition, for BiFC assays, pSITE-nEYFP-C1 harboring Raf13, Raf14, or Raf15, and pSITE-cEYFP-N1 IREH1 were introduced into A. tumefaciens strain GV3101 (p19). The strains were mixed as indicated pairs and infiltrated into N. benthamiana leaves. 3 d after infiltration, GFP or complemented YFP fluorescence was observed using a BX53 fluorescence microscope (Olympus).

Y2H assays were performed with the MatchMaker GAL4 Two-Hybrid System 3 (Takara Bio). Saccharomyces cerevisiae strain AH109 was cotransformed with different pairs of pGADT7 and pGBKT7 harboring IREH1 or B1-Rafs. A single colony for each transformant grown on nonselective SD/-Leu (L)/-Trp (W) media was incubated in liquid media, and then tested on selective SD/-L/-W/-His (H)/-Ade (A) media at 30 °C for 5 d.

To assess primary root length, seeds were sown on 0.8% (w/v) GM agar plates and grown under continuous white light at 22 °C for 4 d. Seedlings were then transferred to 1.0% (w/v) GM agar plates supplemented with/without indicated concentrations of ABA (Sigma-Aldrich) or sorbitol and grown vertically for an additional 10 to 12 d. Primary root length was measured after the plants were transferred onto the black cloth, and then photographed.

For the identification of proteins potentially interacting with Raf13-CFP-HA, 2-wk-old Arabidopsis WT (Col-0) and 35S:Raf13-CFP-HA transgenic seedlings were grown on GM agar medium for 2 wk, followed by DH stress on filter paper for 30 min. Crude proteins were extracted in 5 mL of extraction buffer [20 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 5 mM MgCl₂, 20% glycerol, 0.5% TritonX-100, 1 mM Na₃VO₄, 25 mM NaF, and 1% Protease Inhibitor Cocktail (Sigma-Aldrich)], and then subjected to centrifugation at 4 °C for 10 min to remove cellular debris. The supernatant was incubated with 20 µL of GFP-selector (NanoTag Biotechnologies) for 3 h at 4 °C. After centrifugation at 4 °C for 20 s, immunoprecipitates were washed four times with 1 mL of the same extraction buffer. Tryptic digestion was performed according to the previous studies (46, 47) with some modifications. Briefly, the beads were resuspended in 12 µL of resuspension buffer [100 mM Tris-HCl (pH 9.0), 12 mM sodium N-lauroylsarcosinate (SLS), 12 mM sodium deoxycholate (SDC) and 50 mM ammonium bicarbonate], and then 68 µL of digestion buffer [50 mM ammonium bicarbonate, 10 μg/mL Trypsin (Promega), and 1 mM DTT] was added to digest protein samples on beads. After incubation at 37 °C for 12 h, peptides were alkylated by adding 20 µL of alkylation buffer [50 mM ammonium bicarbonate and 50 mM 2-iodoacetamide (IAM)]. Surfactants were removed by the addition of 100 µL of ethyl acetate containing 1% trifluoroacetic acid (TFA). The peptides were desalted using C18 Empore disks (GL sciences), dried in a centrifugal concentrator (CC-105, TOMY), and stored at −20 °C until the analysis using Orbitrap Exploris 480 (Thermo Fisher Scientific).

For the phosphoproteomic analysis under drought treatment, Arabidopsis WT seeds were sown and grown on GM agar plates under a 16 h/8 h (light/dark) photoperiod at 22 °C for 7 d. Seedlings were transferred from GM agar plates to soil, grown in soil for an additional 2 wk, and then treated with/without water supply for 3, 5, or 9 d. The aerial parts of seedlings were collected and subjected to analysis. For the phosphoproteome to identify the putative Raf13 dephosphorylation sites, Arabidopsis WT seeds were sown on GM agar plates and grown under continuous white light illumination at 22 °C for 2 wk. The seedlings were then transferred onto the filter paper and subjected to DH stress for 30 min. For the phosphoproteome to evaluate B1-Raf- or IREH1-mediated phosphorylation network, 2-wk-old Arabidopsis WT, raf13-1raf15-1, and ireh1-2 seedlings grown on GM agar plates under continuous white light at 22 °C were collected and subjected to the analysis.

Protein extraction and digestion were performed according to the previous studies (5, 10) with some modifications. Briefly, the collected plant tissues were ground to powder in liquid nitrogen, and total proteins were extracted in extraction buffer [100 mM Tris-HCl (pH 9.0), 8 M urea, 2% Phosphatase inhibitor cocktail 2 (Sigma-Aldrich) and 2% Phosphatase inhibitor cocktail 3 (Sigma-Aldrich)], followed by centrifugation at 17,400×g for 20 min at 4 °C. The supernatant was then precipitated by the methanol-chloroform precipitation method, and protein pellets were resuspended in digestion buffer [100 mM Tris-HCl (pH 9.0), 12 mM SLS, and 12 mM SDC]. Crude extracts containing 400 μg of protein were subjected to the following enzymatic digestion. The solution was reduced with 10 mM DTT for 30 min and then alkylated with 50 mM IAM for 20 min in the dark. After fivefold dilution with 50 mM ammonium bicarbonate in ultrapure water, proteins were digested with trypsin (Promega; 1:100 (w/w) enzyme-to-protein ratio) overnight at 37 °C. Surfactants were removed by addition of an equal volume of ethyl acetate containing 1% TFA, centrifugation at 15,000×g for 2 min, and the aqueous phase was collected for the phosphopeptide enrichment. Phosphopeptides were enriched by hydroxy acid-modified metal oxide chromatography method (48), and the enriched phosphopeptides were desalted using C18 Empore disks (GL sciences). After desalting, the peptides were dried using a centrifugal concentrator (CC-105, TOMY) and stored at −20 °C until use.

For the phosphoproteomic analysis or IP-MS analysis, the dried peptides were dissolved in 20 μL of 2% (v/v) acetonitrile (ACN) containing 0.1% (v/v) formic acid (FA) and injected into an Easy-nLC 1200 (Thermo Fisher Scientific). Peptides were separated on a C18 nano HPLC capillary column (NTCC-360/75-3, 75 µm ID×15 cm L, Nikkyo Technos) at 300 nL/min by a nonlinear gradient for 140 min for phosphoproteomic analysis, or 40 min for IP–MS analysis. The mobile phase buffer consisted of 0.1% FA in ultrapure water (Buffer A) with an elution buffer of 0.1% FA in 80% ACN (Buffer B). 140-min gradient was run under the following conditions: 0 to 5 min, B 6%; 5 to 79 min, B 6 to 23%; 79 to 107 min, B 23 to 35%; 107 to 125 min, B 35 to 50%; 125 to 130 min, B 50 to 90%; 130 to 140 min, B 90%, and 40-min gradient was performed as follows: 0 to 0.5 min, B 6%; 0.5 to 11.5 min, B 6 to 23%; 11.5 to 30.5 min, B 23 to 40%; 30.5 to 35 min, B 40 to 50%; 35 to 35.5 min, B 50 to 90%; 35.5 to 40 min, B 90%. The Easy-nLC 1200 was coupled to an Exploris 480 quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) with an FAIMS Pro high-field asymmetric waveform aerodynamic ion mobility spectrometry (FAIMS) device (Thermo Fisher Scientific). The mass spectrometer was operated in the data-dependent acquisition, positive ion mode in which MS1 spectra (375 to 1,500 m/z for phosphoproteomic analysis or 350 to 1,200 m/z for IP–MS, with a resolution of 60,000) were followed by MS2 spectra (over 120 m/z with the resolution of 30,000 for phosphoproteomics or 15,000 for IP-MS). For FAIMS, three conditions of compensation voltage values (−40/−50/−60 V, −40/−60 V, −40/−70 V, or −50/−70 V) were used with the following settings (standard resolution; inner temperature 100 °C / outer temperature 100 °C).

Peptide/Protein identification and MS1-based label-free quantification were carried out using Proteome Discoverer 2.5 (PD2.5) (Thermo Fisher Scientific) or Skyline version 22.2 (MacCoss lab software). MS2 spectra were searched with SEQUEST HT against the Arabidopsis protein database (Araport11_genes.201606.pep.fasta). The SEQUEST search parameters were set as follows: digestion enzyme trypsin, maximum missed cleavages 2, peptide length 6-144, precursor mass tolerance 10 ppm, fragment mass tolerance 0.02 Da, static modification: cysteine (C) carbamidomethylation, variable modification: methionine (M) oxidation/ N-terminal acetylation/ serine (S) threonine (T) tyrosine (Y) phosphorylation, maximum variable modifications 3. Peptide validation was performed using the Percolator, and only high-confidence peptides with a false discovery rate <1% were used for protein inference and quantification. Site localization probability of phosphorylation was calculated with IMP-ptmRS node implemented in PD2.5, and 75% was used as the cut-off for localization of phosphorylation site(s).

Y.K. and T.U. designed research; Y.K., S.K., Y.L., K.Y., and H.T. performed research; Y.K. contributed new reagents/analytic tools; Y.K., S.K., Y.L., K.Y., and H.T. analyzed data; and Y.K. and T.U. wrote the paper.

The authors declare no competing interest.