a, Schematics of the covalent modification of HPCA1 Cys residues by the membrane impermeable alkyne reagents IA-biotin (top) and MTSEA-biotin (bottom) (as in Fig. 4a–d). IA-biotin-mediated irreversible modification of extracellular Cys residues prevents disulfide bond formation between Cys residues. The Cys residues are specifically alkanethiolated by MTSEA-biotin (a cysteine-disulfde-bond-forming reagent) under mild conditions, and then form a covalent disulfide bond. This disulfide bond is readily reduced by neighbour Cys residues to form a disulfide bond between Cys residues (asterisk). b, hpca1 complementation using HPCA1 with Cys mutations could not complement the hpca1-2 phenotype. Wild-type and hpca1-2 mutant seedlings, and hpca1-2 seedlings expressing HPCA1 or HPCA1 with Cys mutations (used in Fig. 4e) were treated with 0.9 M CaCl 2 and 10% (v/v) ethanol (aequorin discharge), and the remaining aequorin luminescence was imaged. Similar luminescence intensities were detected for all genotypes. The relative [Ca2+] i is scaled by a pseudo-colour bar. The experiment was repeated independently 20 time with similar results. c, Plasma membrane localization of HPCA1 and Cys-pair-mutated HPCA1 used in Fig. 4e. The hpca1-2 seedlings stably expressing the pHPCA1::HPCA1-YFP construct or mutated Cys-pair HPCA1 were used, and YFP fluorescence was analysed by confocal microscopy. YFP fluorescence of HPCA1 and mutated Cys-pair HPCA1 was observed in the periphery of turgid and plasmolysed cells, which suggests that these Cys mutations did not alter HPCA1 subcellular localization. Data are representative of more than ten independent lines examined. Scale bar, 20 μm. d, Schematics of the covalent modification of HPCA1 Cys residues by iodoacetamide and NEM, for experiments in Fig. 4h–j. HPCA1–YFP proteins from transgenic plants were labelled first with iodoacetamide to form Cys-CAM and represent the reduced form, and then treated with dithiothreitol (DTT) to reduce disulfide bonds, which were then labelled with NEM to represent the oxidized form. Modifications of proteins were analysed by LC–MS/MS. The CAM- and NEM-labelled peptides were analysed and their relative amounts were calculated. e−g, Representative MS/MS spectra of the STLPTNC421SPC424EPGME peptide in the hydrogen peroxide domain from HPCA1–YFP proteins prepared using hpca1-2 plants stably expressing the pHPCA1::HPCA1-YFP construct with or without 4 mM H 2 O 2 treatment, as in Fig. 4h–j. HPCA1–YFP proteins were labelled with iodoacetamide and NEM as in d, and digested using GluC and chymotrypsin for mass spectrometry. Cys421 and Cys424 labelled by CAM (e), Cys421 by CAM and Cys424 by NEM (f), and Cys421 by NEM and Cys424 by CAM (g) were all detected from the HPCA1–YFP proteins prepared with or without H 2 O 2 treatment. Note that H 2 O 2 treatment increased the relative amount of NEM-labelled Cys residues (Fig. 4j). The amounts of Cys red and Cys ox residues were represented by CAM- and NEM-labelled peptides, respectively, and normalized to those of CAM-labelled peptides (Fig. 4j). The relative percentages of Cys red and Cys ox from the total amounts of Cys residues were determined (Fig. 4j). Note that the relative amount of Cys red + the relative amount of Cys ox = 100% Cys residues. Data are from four independent experiments. We have demonstrated that both Cys red and Cys ox residues in the hydrogen peroxide domain exist, and that these Cys red residues could be oxidized by eH 2 O 2 to Cys ox residues in planta, which transduces the eH 2 O 2 signal to downstream events. To fulfil the receptor function, the HPCA1 receptor should be regenerated. Given that plants grow almost constantly to form new cells, these new cells should carry the HPCA1 receptors with Cys red residues. For the eH 2 O 2 -oxidized HPCA1 receptors with Cys ox residues, it is possible that these oxidized receptors are either recycled via the endocytic process, or reduced by unknown mechanisms, which need to be determined in the future.