A receptor-like protein acts as a specificity switch for the regulation of stomatal development

Abstract

Stomata are microscopic openings that allow for the exchange of gases between plants and the environment. In Arabidopsis, stomatal patterning is specified by the ERECTA family (ERf) receptor kinases (RKs), the receptor-like protein (RLP) TOO MANY MOUTHS (TMM), and EPIDERMAL PATTERNING FACTOR (EPF) peptides. Here we show that TMM and ER or ER-LIKE1 (ERL1) form constitutive complexes, which recognize EPF1 and EPF2, but the single ERfs do not. TMM interaction with ERL1 creates a binding pocket for recognition of EPF1 and EPF2, indicating that the constitutive TMM-ERf complexes function as the receptors of EPF1 and EPF2. EPFL9 competes with EPF1 and EPF2 for binding to the ERf-TMM complex. EPFL4 and EPFL6, however, are recognized by the single ERfs without the requirement of TMM. In contrast to EPF1,2, the interaction of EPFL4,6 with an ERf is greatly reduced in the presence of TMM. Taken together, our data demonstrate that TMM dictates the specificity of ERfs for the perception of different EPFs, thus functioning as a specificity switch for the regulation of the activities of ERfs.

Keywords: biochemical interaction; peptide hormones; receptor kinases; receptor-like protein; stomatal development; structure.

Figure 1.

Overall structure of the ERL1^LRR–TMM^LRR complex. (A) Overall structure of the ERL1^LRR–TMM^LRR complex. “N” and “C” represent the N terminus and C terminus, respectively. Color codes are indicated. Schematic representations of the domain structures of ERL1^LRR and TMM^LRR are shown at the top. (SP) Signal peptide; (NC) N-terminal capping; (CC) C-terminal capping; (TM) transmembrane; (KD) kinase domain. (B) Surface representation of the electrostatic potential of ERL1^LRR–TMM^LRR. The electrostatic potential of TMM^LRR and a cartoon representation of ERL1^LRR in transparency are shown at the left. The right panel shows the electrostatic potential of ERL1^LRR and a cartoon representation of TMM^LRR in transparency. (White) Neutral surface; (blue) positive surface; (red) negative surface; (N) N terminus; (C) C terminus. Color codes are indicated.

Figure 2.

Mechanism of ERL1^LRR–TMM^LRR interaction. (A) Detailed interactions of ERL1^LRR with TMM^LRR. Interaction regions are indicated by red and blue squares in the top panel. Red dashed lines indicate hydrogen bonds or salt bridges. (B) Mutagenesis analysis of the ERL1–TMM interaction in vitro. Mutant ERL1 or TMM was coexpressed with its wild-type partners in insect cells. C-terminally 6xHis-tagged ERL1^LRR was pulled down by Ni⁺ beads. The bound proteins were eluted and visualized by Coomassie blue staining following SDS-PAGE. (C) The TMM E379R reduces the functionality of TMM in plants. The stomatal index (top) and stomatal clustering (middle) were analyzed on the abaxial side of 14-d-old cotyledons of wild-type (wt), tmm, and T2 generation seedlings expressing functional TMM (two independent lines) or TMM with E379R substitution in the tmm background (three independent lines). The stomatal index was analyzed on the mature stem (bottom) of wild-type, tmm, and T2 generation plants expressing functional TMM or TMM with E379R substitution in the tmm background. Values are means ± SE. N = 18–30. Values significantly different from the tmm mutant (P < 0.0001) are indicated by asterisks.

Figure 3.

ERL1–TMM complexes function as receptors of EPF1 and EPF2 in vitro. (A) ERfs alone fail to interact with EPF1 or EPF2 in pull-down assays. The 6xHis-tagged ER^LRR, ERL1^LRR, and ERL2^LRR were incubated individually with EPF1 or EPF2, and the mixtures were then flowed through Ni-NTA beads. After extensive washing, the bound proteins were eluted and analyzed with SDS-PAGE followed by Coomassie blue staining. (B) EPF1 directly binds to the ERL1^LRR–TMM^LRR complex. (Left panel) Size exclusion chromatography analysis of the interaction between EPF1 and ERL1^LRR–TMM^LRR; the elution volumes and the molecular weight markers are indicated at the top. (Right panel) SDS-PAGE analysis of peak fractions from the left panel. (C) Quantification of the binding affinity of EPF1 (left) or EPF2 (right) to ERL1^LRR or ERL1^LRR–TMM^LRR by ITC. (nd) No detectable binding. EPF1 or EPF2 was titrated into ERL1^LRR or ERL1^LRR–TMM^LRR protein in the ITC cell. Integrated heat measurements from ITC are shown. The calculated stoichiometry (N) and the dissociation constant (K_d) are indicated.

Figure 4.

Recognition mechanism of EPF1 by ERL1^LRR–TMM^LRR. (A) Overall structure of the EPF1–ERL1^LRR–TMM^LRR complex. (N) N terminus; (C) C terminus. Color codes are indicated. (B) Detailed interactions of the N-terminal side of EPF1 with ERL1^LRR–TMM^LRR. (C) Detailed interactions of the central region of EPF1 with ERL1^LRR–TMM^LRR. (D) Detailed interactions of EPF1 with ERL1^LRR–TMM^LRR. (E) Mutagenesis analysis of EPF1-6xHis's interaction with ERL1^LRR–TMM^LRR in vitro. The pull-down assays were performed as described in Figure 3A. (F) Effects of the mutations of EPF1 on the stomatal density of Arabidopsis cotyledons; the concentration of peptide was 10 µM. The error bars show SD. n > 8. (*) P < 0.05; (**) P < 0.01, versus the controls by Student's t-test.

Figure 5.

EPFL9 competes with EPF1 and EPF2 for binding to the ERf^LRR–TMM^LRR complexes. (A, left panel) Size exclusion chromatography analysis of the interaction between EPFL9 and ERL1^LRR–TMM^LRR. (Right panel) SDS-PAGE analysis of peak fractions from the left panel. The elution volumes and molecular weight markers are indicated at the top. (B) TMM promotes EPFL9 binding to ERL1^LRR. Quantification of the binding affinity of EPF9 with ERL1^LRR or ERL1^LRR–TMM^LRR by ITC. EPFL9 was titrated into ERL1^LRR or ERL1^LRR–TMM^LRR protein in the ITC cell. Raw data (top panel) and integrated heat measurements (bottom panel) from ITC are shown. The calculated stoichiometry (N) and the dissociation constant (K_d) are indicated. (C) EPFL9 competes with EPF1,2 for binding to ERf^LRR–TMM^LRR. ER^LRR–TMM^LRR or ERL1^LRR–TMM^LRR was incubated with 10 µM EPF2-6xHis or EPF1-6xHis with increasing concentrations of EPFL9 and subjected to Ni⁺ resin. The bound proteins were eluted and further analyzed with SDS-PAGE followed by Coomassie blue staining.

Figure 6.

EPFL4 directly binds to ERfs independent of TMM. (A) ERfs alone are sufficient for interaction with EPFL4 or EPFL6. The 6xHis-tagged ER^LRR, ERL1^LRR, or ERL2^LRR was incubated individually with EPFL4 or EPFL6 and subjected to Ni⁺ resin. Next, the bound proteins were eluted from Ni-NTA and further analyzed by SDS-PAGE with Coomassie blue staining. (B, left panel) Size exclusion chromatography analysis of the interaction between EPFL4 and ERf^LRR. (Right panel) SDS-PAGE analysis of peak fractions from the left panel. (C) TMM dampens EPFL4 binding to ERL1^LRR in vitro. Quantification of the binding affinity of EPFL4 with the ERL1^LRR or ERL1^LRR–TMM^LRR by ITC. EPFL4 was titrated into ERL1^LRR or ERL1^LRR–TMM^LRR protein in the ITC cell. Raw data (top panel) and integrated heat measurements (bottom panel) from ITC are shown. The calculated stoichiometry (N) and the dissociation constants (K_d) are indicated.

Figure 7.

Recognition mechanism of EPFL4 by ERL2^LRR. (A) Overall structure of EPFL4–ERL2^LRR. (N) N terminus; (C) C terminus. (B) Structural alignment of ERL2^LRR–EPFL4 and EPF1–ERL1^LRR–TMM^LRR complexes. The structure of ERL1^LRR was used as the template for the alignment. (C) Detailed interactions of the N-terminal side of EPFL4 with ERL2^LRR. (D) Detailed interactions of the central region of EPFL4 with ERL2^LRR.