Differential Function of Arabidopsis SERK Family Receptor-like Kinases in Stomatal Patterning

Abstract

Plants use cell-surface-resident receptor-like kinases (RLKs) to sense diverse extrinsic and intrinsic cues and elicit distinct biological responses. In Arabidopsis, ERECTA family RLKs recognize EPIDERMAL PATTERNING FACTORS (EPFs) to specify stomatal patterning. However, little is known about the molecular link between ERECTA activation and intracellular signaling. We report here that the SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) family RLKs regulate stomatal patterning downstream of EPF ligands and upstream of a MAP kinase cascade. EPF ligands induce the heteromerization of ERECTA and SERK family RLKs. SERK and ERECTA family RLKs transphosphorylate each other. In addition, SERKs associate with the receptor-like protein (RLP) TMM, a signal modulator of stomata development, in a ligand-independent manner, suggesting that ERECTA, SERKs, and TMM form a multiprotein receptorsome consisting of different RLKs and RLP perceiving peptide ligands to regulate stomatal patterning. In contrast to the differential requirement of individual SERK members in plant immunity, cell-death control, and brassinosteroid (BR) signaling, all four functional SERKs are essential but have unequal genetic contributions to stomatal patterning, with descending order of importance from SERK3/BAK1 to SERK2 to SERK1 to SERK4. Although BR signaling connects stomatal development via multiple components, the function of SERKs in stomatal patterning is uncoupled from their involvement in BR signaling. Our results reveal that the SERK family is a shared key module in diverse Arabidopsis signaling receptorsomes and that different combinatorial codes of individual SERK members regulate distinct functions.

Figure 1. Ectopic expression of effector protein AvrPto or AvrPtoB impairs stomatal patterning

(A) Dex-induced expression of AvrPto or AvrPtoB but not AvrRpt2 or AvrRpm1 in Arabidopsis transgenic plants leads to severe stomatal clustering phenotypes. (B) Expression of YDAac rescues the AvrPto-induced stomatal patterning defects. Confocal images were taken on the abaxial cotyledon epidermis of 10-day-old seedlings grown on ½ MS medium with (A and B, bottom panels) or without (A and B, top panels) 100 μM Dex. Cell outlines were visualized with propidium iodide staining. The representative images in A and B were selected from at least five replicates. (C) Expression of AvrPto does not affect YDAac-mediated activation of MPK3 and MPK6 in Arabidopsis protoplasts. The HA-tagged MPK3/MPK6 and FLAG-tagged YDAac were co-expressed with or without AvrPto in protoplasts. The MPK3/MPK6 proteins were immunoprecipitated with α-HA agarose beads for an in vitro kinase assay using myelin basic protein as the substrate. The phosphorylation of myelin basic protein by MPK3/MPK6 is shown with autoradiograph (top panel), and the protein expression is shown with immunoblotting (bottom three panels). The experiments were repeated three times with similar results. (see also Figure S1).

Figure 2. Redundant function of SERK family RLKs in stomatal patterning

(A–C) The serk1-1/serk2-1/bak1-4 mutant but not other serk mutants shows stomatal patterning defects. Confocal images of indicated genotypes were taken on the abaxial cotyledon epidermis of 10-day-old seedlings grown on ½ MS plates. The representative images were selected from at least five replicates. Brackets indicate clustered stomata (C). (D) The seedling phenotypes of two-week-old serk1-1/serk2-1/bak1-4 and er105/erl1-2/erl2-1 mutants grown on soil. (E) Abaxial cotyledon stomatal index of 10-day-old seedlings, expressed as percentage of the number of stomata to the total number of epidermal cells. The data are shown as mean + SD (n=8). Asterisks above the columns indicate significant difference compared with the data from WT plants (*** P<0.0001, Student’s t-test). The experiments were repeated three times with similar results. (see also Figures S2 and S3).

Figure 3. Differential contributions of SERK family RLKs in stomatal patterning

(A, B) The stomatal clustering phenotypes of serk higher-order mutants in the bak1-5 background. Confocal images were taken on the abaxial cotyledon epidermis at 10 days after germination on ½ MS medium. Brackets indicate clustered stomata. (C) Abaxial cotyledon stomatal indexes of indicated genotypes. The data are shown as mean + SD (n=8). The mean values marked with different letters are significantly different from each other (P<0.05, Student’s t-test). The experiments were repeated three times with similar results. (D) The phenotypes of two-week-old seedlings grown on soil. (E, F) The serk1-1^−/−/serk2-1^−/+/bak1-5^−/− plants phenocopy the er105 mutant in inflorescence architecture (E) and pedicel length (F). (see also Figures S4 and S5).

Figure 4. Uncoupled functions of SERK family RLKs in stomatal patterning and BR signaling

(A, B) The serk2-1/bak1-5 and serk1-1/serk2-1/bak1-5 mutants show normal hypocotyl elongation in response to brassinolide (BL) treatment. The seedlings were grown under the light for 10 days on ½ MS plates with or without 100 nM BL (A), and hypocotyl lengths were quantified (B). Brackets indicate hypocotyl (A). The data are shown as mean + SD (n=15) (B). (C) BL treatment induces the dephosphorylation of BES1 in serk2-1/bak1-5 and serk1-1/serk2-1/bak1-5 mutants. Ten-day-old seedlings grown in liquid ½ MS medium were treated with 0 or 1 μM BL for 2 hr, and the total proteins were analyzed by immunoblotting with α-BES1 antibody (Top panel). The protein loading is shown by Coomassie Brilliant Blue (CBB) staining for RuBisCO (RBC) (bottom panel). (D, E) The serk1-8/bak1-4/serk4-1 mutant exhibits normal stomatal patterning and index. Confocal images were taken on the abaxial cotyledon epidermis of 10-day-old seedlings (D), and the stomatal indexes were quantified (E). The experiments were repeated twice with similar results.

Figure 5. Interactions between SERK and ER family RLKs

(A) BAK1 associates with ER and ERL1 in pBAK1∷BAK1-GFP/pER∷ER-FLAG and pBAK1∷BAK1-GFP/pERL1∷ERL1-FLAG transgenic plants. Protein extracts from transgenic plants were immunoprecipitated with α-GFP antibody (IP: α-GFP), and immunoblotted with α-FLAG (IB: α-FLAG) or α-GFP antibody (IB: α-GFP) (top two panels). The protein inputs are shown with immunoblotting before immunoprecipitation (bottom two panels). The pER∷ER-FLAG and pERL1∷ERL1-FLAG plants were used as controls here. (B) EPF2 induces the association of ER with SERKs in Arabidopsis protoplasts. SERK-GFP and ER-HA were transiently co-expressed in Arabidopsis protoplasts. After protoplasts were treated with or without 1 μM EPF2 for 5 min, protein extracts were immunoprecipitated with α-GFP antibody (IP: α-GFP), and immunoblotted with α-HA (IB: α-HA) or α-GFP antibody (IB: α-GFP) (top two panels). The protein inputs are shown with immunoblotting before immunoprecipitation (bottom two panels). (C) EPF1 induces the association of ERL1 with SERKs in Arabidopsis protoplasts. (D) SERKs associate with TMM in Arabidopsis protoplasts. Protoplasts were co-transfected with SERK-GFP and TMM-HA, and then treated with or without 1 μM EPF2 for 5 min. The experiments were repeated three times with similar results. (see also Figure S6A).

Figure 6. Transphosphorylation between the cytosolic kinase domains of BAK1 and ER

(A) BAK1_CD interacts with ER_CD in vitro. MBP-BAK1_CD-HA proteins were incubated with GST or GST-ER_CD glutathione beads, and the pull-down (PD) proteins were immunoblotted with α-HA antibody (top panel). The CBB staining of input proteins is shown on the bottom panel. (B) The phosphorylation of ER_CD by BAK1_CD (top panel). (C) The phosphorylation of BAK1_CD by ER_CD (top panel). The kinase assays were performed using ER_CD and BAK1_CD kinase mutant (BAK1_CDKm) proteins as substrates in (B) and (C) respectively. The CBB staining of input proteins is shown on the bottom panels. The experiments were repeated three times with similar results. (see also Figure S6B).

Figure 7. SERKs function downstream of EPFs and upstream of YDA in regulating stomatal development

(A, B) SERKs are required for EPF1- and EPF2-mediated stomatal development. Confocal images were taken on the abaxial cotyledon epidermis of 6-day-old Col-0 and serk1-1/serk2-1/bak1-5 seedlings grown in ½ MS liquid medium containing 2.5 μM EPF1 or EPF2 (A) and 10-day-old transgenic seedlings of Est∷EPF1 or Est∷EPF2 grown on ½ MS plates with or without 10 μM estradiol (B). (C) Expression of YDAac driven by its native promoter rescues the growth and stomatal patterning defects of serk1-1/serk2-1/bak1-5. The images were taken on 4-week-old plants (top panels) or 10-day-old cotyledon epidermis (bottom panels). (D) Ectopic expression of MKK5^DD eliminates stomata in serk1-1/serk2-1/bak1-5 mutant. Confocal images were taken on the abaxial cotyledon epidermis of 10-day-old transgenic seedlings of Dex∷MKK5^DD with or without 0.02 μM Dex treatment. Brackets indicate clustered stomata. At least two transgenic lines for each construct in B-D were used, and the similar results were obtained. The representative images were selected from at least five replicates. (see also Figure S7).