Extended Data Fig. 6: HPCA1 genomic structure, mutations in three hpca1 mutants, HPCA1 subcellular localization and expression patterns.

a, Schematic illustration of the exon–intron structure of the HPCA1 gene, with boxes representing exons. The mutations in hpca1-1, hpca1-2 and hpca1-3 are illustrated. Primers for genotyping a splicing error in hpca1-2 are shown. FP, HPCA1 forward primer in the thirteenth exon; RP, HPCA1 reverse primer in the fourteenth exon. Genomic DNA sequence analysis confirmed that the hpca1-1 mutant contains a mutation in the kinase domain (Q856*, stop codon) that leads to a protein with a truncated kinase domain; hpca1-2 contains a mutation that leads to intron mis-splicing and a premature stop codon in the extracellular domain and a truncated HPCA1 protein without the transmembrane and kinase domains (a knockout line); and hpca1-3 contains a mutation in the LRR domain (G159D). The possible loss-of-function mutations are illustrated in the schematic in the kinase domain (C4425T) and LRR domain (C1663T) in hpca1-1 and hpca1-3, respectively, consistent with the importance of these two domains in LRR-RKs. b, The splicing mutation in hpca1-2. The hpca1-2 mutant has a single base mutation (G-to-A in the DNA sense strand) that corresponds to a change from 5′-AG2627 to 5′-AA2627 in the splice acceptor site of the thirteenth intron. This makes the site unrecognizable by the splicing enzymes, and leads to the predicted 76-bp addition in HPCA1 mRNA and a premature stop codon. PCR analyses of cDNA showed that hpca1-2 cDNA was about 76 bp longer than wild-type cDNA, which was confirmed by sequencing the HPCA1-2 cDNA clone of hpca1-2. Experiments were repeated independently three times. c, Expression patterns of HPCA1–YFP in Arabidopsis seedlings stably expressing the pHPCA1::HPCA1-YFP construct, which were also used in Fig. 3f. YFP fluorescence was analysed using a Zeiss stereo microscope, and images were merged to generate the whole-seedling image. Enlargements of upper epidermis and a root tip are shown at the top and bottom right, respectively, illustrating the plasma membrane localization of HPCA1–YFP and the high expression in epidermal cells and guard cells but the low expression in root tips. More than 15 homozygous single-insertion transgenic lines were generated, and similar results were observed from these lines. Scale bars, 3 mm (seedling) or 100 μm (leaf section and root tip). d, YFP fluorescence of Arabidopsis seedlings stably overexpressing YFP (CaMV 35S promoter-driven YFP construct, p35S::YFP) was analysed using confocal microscopy as a control, as in Fig. 3f. YFP fluorescence was observed in the cytosol and nucleus of the turgid cells (left) and plasmolysed cells (right). Scale bar, 20 μm. Data in c and d are representative of more than ten independent lines examined.