Extended Data Fig. 7: Subcellular localization of YFP fusion proteins of HPCA1 and HPCA1 mutants.

a, The fusion proteins HPCA1–YFP, mHPCA1-1–YFP (Q856*; hpca1-1 mutation in the kinase domain), and mHPCA1-3–YFP (G159D; hpca1-3 mutation in the LRR domain) were transiently expressed in Nicotiana benthamiana epidermal cells (CaMV 35S promoter-driven YFP constructs, p35S::HPCA1-YFP, p35S::mHPCA1-1-YFP and p35S::mHPCA1-3-YFP). The ectodomain mutant mHPCA1-3–YFP (G159D) showed lower fluorescence compared to wild-type HPCA1 and truncated mHPCA1-1-YFP (Q856*). These data suggest that the mutation in hpca1-1 may affect only the kinase activity but not the targeting of HPCA1, whereas the mutation in hpca1-3 may affect both the targeting and eH₂O₂ sensing. The experiments were repeated at least three times for each construct. Scale bars, 30 μm. b, Attenuated activation of MAPK by H₂O₂ in hpca1 and complementation by expressing wild-type HPCA1. Seedlings were treated with 4 mM H₂O₂, and phosphorylation of MAPK3 (p-MPK3) and MAPK6 (p-MPK6) was detected using pTEpY antibodies at indicated time points. α-tubulin was used as a loading control. Experiments were repeated independently three times. For gel source data, see Supplementary Fig. 1.