Extended Data Fig. 5: Conserved hydrogen peroxide domains between the last LRR and the transmembrane domain in the HPCA1 LRR-RK subfamily across species.

a, Schematic representation of conserved Cys residues in the region between the last LRR and the kinase domain. Analysis showed that the LRR domain is also flanked by domains with characteristic double-cysteine residue motifs (Cys pair) in these HPCAs. HPCA1 (or VIII-1 subfamily) proteins have six Cys residues that are conserved in this group. VIII-2 proteins have two conserved Cys residues. The others groups (except for V, IX, XIV and XV) have two conserved Cys residues that have been illustrated in a previous evolutionary study of LRR-RKs⁵³ (Methods). These conserved Cys residues in VIII-1, VIII-2 and the other groups appear as pale-blue lines, and their positions were referenced as HPCA1 (or AT5G49760) in VIII-1, AT1G29750 in VIII-2, and AT1G12460 in VIIa, and are shown on a relative scale. The HPCA subfamily contains a unique hydrogen peroxide (HP) domain. The last LRR, transmembrane domain (TM), and protein kinase domains are labelled in VIII-1, VIII-2 and the other LRR-RK groups. The transmembrane and protein kinase domains are conserved among all LRR-RK proteins. VIII-2 also contains a malectin domain. b, Multiple sequence alignment of the region containing the VIII-1-specific Cys residues (hydrogen peroxide domain) in LRR-RK proteins from seven land plant species. The classification of LRR-RKs was retrieved from the previous study⁵³ (Methods). Each protein is depicted as a protein ID, followed by the start and end position of the region. The seven species are the early land plant Physcomitrella patens (Potri), the early vascular plant Selaginella moellendorffii (Smo), two monocot genomes including Sorghum bicolor (Sb) as well as rice Oryza sativa Japonica (Os), and three dicot genomes including Arabidopsis thaliana (At), tomato Solanum lycopersicum (Soly) as well as black cottonwood Populus trichocarpa (Pp). Considering that these Cys residues are conserved in the hydrogen peroxide domain, we speculate that these HPCA1 homologues in Arabidopsis and other species could also function as H₂O₂ sensors. We also speculate that higher-order Arabidopsis mutants with HPCA1 homologues will show stronger impairment in eH₂O₂-induced responses. Given that all eight HPCA1 subfamily members in Arabidopsis contain these two Cys pairs—except HPCAL7, which misses Cys412 and Cys424—they could potentially act as eH₂O₂ sensors with similar kinetics and affinities, consistent with the reduction in amplitudes but not the K_d values of eH₂O₂-induced Ca²⁺ increases in hpca1 mutants (Fig. 1f). That is, the similar K_d values between wild-type and hpca1 seedlings (Fig. 1f) may indicate that the eH₂O₂-sensing systems that are both affected and unaffected by the hpca1 mutation share similar affinities to eH₂O₂.