Extended Data Fig. 9: HPCA1 kinase activity is required for eH₂O₂ signalling.

a, Inhibition of H₂O₂-induced [Ca²⁺]_i increases in wild-type seedlings using the protein kinase inhibitor K252a, which has been shown to attenuate LRR-RK signalling pathways⁷⁷, such as those for flg22, pep1 and elf26. Seedlings were treated with 2.5 mM H₂O₂ in the absence (−K252a, blue) or presence (+K252a, brown) of 2 µM K252a, and [Ca²⁺]_i was analysed by aequorin luminometry spectroscopy. Data are mean ± s.e.m.; n = 12 seedlings. P < 0.001, two-way ANOVA. The quantified maximum [Ca²⁺]_i changes from the same experiments are shown in Fig. 3h. b, K252a inhibition of H₂O₂-induced Ca²⁺ currents in wild-type guard-cell protoplasts. Representative whole-cell currents in protoplasts treated with 5 mM H₂O₂ in the absence or presence of 2 µM K252a for 1.5 h are shown. The currents at −180 mV from similar experiments are shown in Fig. 3i. c, Inhibition of H₂O₂-induced stomatal closure by K252a. Detached leaves were treated with 2.5 mM H₂O₂ in the absence or the presence of 2 µM K252a, and stomatal apertures were analysed. Data are mean ± s.e.m.; n = 60 stomata. Stomatal apertures without treatment were arbitrarily set to 1 r.u. d, e, Phosphorylation of HPCA1–YFP induced by H₂O₂ and flg22 in planta. The hpca1-2 seedlings stably expressing the HPCA1::HPCA1-YFP construct were treated with 4 mM H₂O₂ (d) or 1 µM flg22 (e), and HPCA1–YFP proteins were prepared by immunoprecipitation. Phosphorylation levels were detected using pThr antibodies. YFP was used as a loading control. For gel source data, see Supplementary Fig. 1. f, HPCA1^CD was autophosphorylated. GST-tagged HPCA1^CD proteins—including wild-type HPCA1^CD and two kinase-inactive mutants (HPCA1(K859E)^CD and HPCA1(D773L)^CD) and kinase domain-truncated HPCA1 found in hpca1-1 (mHPCA1-1; HPCA1(Q856*)^CD)—were expressed in Escherichia coli (as in Fig. 3k). Purified proteins were incubated with or without λ-protein phosphatase (Lambda PPase; 30 °C) for 2 h, and separated by SDS–PAGE. Red and black arrows denote phosphorylated and unphosphorylated bands, respectively. Only the HPCA1^CD band was shifted owing to autophosphorylation, and the band shift was abolished by λ-protein phosphatase treatment. Experiments in d−f were repeated independently three times. g, The tandem mass spectrometry (MS/MS) spectra of a HPCA1 peptide (786-THVTTQVK-793; inset). Nine-day-old hpca1-2 plants stably expressing HPCA1–YFP were treated with water or 4 mM H₂O₂, and HPCA1–YFP was immunoprecipitated. The proteins were purified by PAGE, and in-gel digested with trypsin and GluC, and resulting peptides were extracted and analysed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Labelled peaks correspond to masses of y and b ions of the modified peptide. Analysis of y and b ion fragmentation patterns with MaxQuant showed that Thr789 and Thr790 were phosphorylated in vivo. These amino acid residues are illustrated in Fig. 3g. h, H₂O₂ treatment resulted in increased phosphorylation of Thr and Ser residues of HPCA1–YFP in vivo from experiments as in g. Data are mean ± s.d.; n = 3 independent experiments. P < 0.05, two-sided t-test. The functional relevance of these phosphosites could be determined further through complementation experiments of the hpca1-2-null mutant with the respective phosphosite mutants of HPCA1. These amino acid residues are illustrated in Fig. 3g. i, Highly conserved phosphorylated Thr residues in the catalytic core of the kinase domains of HPCA1, BAK1 and BRI1. Alignment of these three protein sequences in subdomain VIb to the invariant E in subdomain VIII. Bold residues are highly conserved in active kinases. Amino acid residues (orange) are confirmed phosphorylation sites in our study and previous reports^78,79,80. For the activation of LRR-RKs, it is thought that a conformational change mediated by ligand binding to the extracellular LRR domain activates the kinase by allowing trans-phosphorylation of the kinase domain within its homodimer^81,82. This, in turn, could allow further autophosphorylation of the kinase domain, providing docking sites for interacting proteins and funnelling the ligand signal to downstream events^27,81. The phosphorylation of these Thr residues has been shown to be required for kinase activation⁷⁸.

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