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Self-incompatibility in the Brassicaceae is controlled by multiple haplotypes encoding the pollen ligand (S-locus protein 11, SP11, also known as S-locus cysteine-rich protein, SCR) and its stigmatic receptor (S-receptor kinase, SRK). A haplotype-specific interaction between SP11/SCR and SRK triggers the self-incompatibility response that leads to self-pollen rejection, but the signalling pathway remains largely unknown. Here we show that Ca2+ influx into stigma papilla cells mediates self-incompatibility signalling. Using self-incompatible Arabidopsis thaliana expressing SP11/SCR and SRK, we found that self-pollination specifically induced an increase in cytoplasmic Ca2+ ([Ca2+]cyt) in papilla cells. Direct application of SP11/SCR to the papilla cell protoplasts induced Ca2+ increase, which was inhibited by D-(?)-2-amino-5-phosphonopentanoic acid (AP-5), a glutamate receptor channel blocker. An artificial increase in [Ca2+]cyt in papilla cells arrested wild-type (WT) pollen hydration. Treatment of papilla cells with AP-5 interfered with self-incompatibility, and Ca2+ increase on the self-incompatibility response was reduced in the glutamate receptor-like channel (GLR) gene mutants. These results suggest that Ca2+ influx mediated by GLR is the essential self-incompatibility response leading to self-pollen rejection.Flowering plants have developed self-incompatibility as a genetic system to prevent inbreeding and thus promote outcrossing. In many species, self-incompatibility is controlled by an S locus with multiple haplotypes1. Each S-haplotype encodes both male- and female-specificity determinants (S-determinants), and self/non-self-discrimination is accomplished by the S-haplotype-specific interaction between these S-determinants., In the Brassicaceae, the male and female S-determinants have been identified as SP11/SCR and SRK, respectively1. SP11/SCR is a polymorphic small peptide secreted from the anther tapetum that localizes to the pollen surface, whereas SRK is a polymorphic Ser/Thr receptor kinase that localizes to the plasma membrane of stigma papilla cells. SP11/SCR and SRK from each S-haplotype function respectively as a ligand and its cognate receptor. Upon self-pollination, the S-haplotype-specific interaction between SP11/SCR and SRK induces autophosphorylation of SRK, which is thought to trigger a signalling cascade in the papilla cells, resulting in the rejection of self-pollen2. Although self-pollination is known to evoke multiple physiological changes in the papilla cells, including disruption of actin bundles, fragmentation of vacuolar structure and modification of microtubules3,4, the signalling pathway downstream of SRK that leads to these processes remains largely unknown.Thus far, two candidate molecules, M-locus protein kinase (MLPK) and Arm-repeat containing 1 (ARC1), have been identified as the direct downstream effectors of SRK. MLPK was identified as a gene responsible for a self-compatibility mutation in Brassica rapa5, and encodes a membrane-anchored cytoplasmic protein kinase that interacts with SRK on the papilla cell membrane6. Recent studies have suggested that MLPK is also involved in intraspecies unilateral incompatibility of B. rapa7, but it remains unclear whether MLPK is required for self-incompatibility throughout the Brassicaceae., ARC1 is known to interact with, and is phosphorylated by, the kinase domain of SRK in Brassica napus9,10. ARC1 is a U-box protein with E3 ubiquitin ligase activity11, and interacts with Exo70A112, a putative component of the exocyst complex, which generally functions in polarized secretion13. These results suggested a model in which activated SRK phosphorylates ARC1, leading to the preclusion of as-yet unknown ‘compatibility factors’ secretion to the stigmatic surface and inhibiting pollen entrance14. However, the suppression of ARC1 expression results in incomplete breakdown of self-incompatibility in both B. napus and Arabidopsis lyrata10,14, and self-compatible Arabidopsis thaliana that lacks ARC1 acquires the self-incompatibility phenotype by introducing SRK and SP11/SCR genes15. Therefore, the extent ARC1 to which is involved in the signalling pathway downstream of SRK remains unclear8,16,17.In this study, we focused on investigating the cytoplasmic Ca2+ dynamics in stigma papilla cells during the self-incompatibility response. A previous study injected dyes to monitor Ca2+ dynamics in the self-incompatibility response18. By combining the in vivo imaging using genetically encoded [Ca2+]cyt probes and pharmacological approaches, we found that cytoplasmic Ca2+ drastically increases in the papilla cells after self-pollination, which can be efficiently blocked by the inhibitors of glutamate receptor channels that mediate the influx of extracellular Ca2+. Pretreatment of papilla cells with glutamate receptor channel inhibitor compromised the self-incompatibility response in vivo, whereas an artificial increase in [Ca2+]cyt in papilla cells induced arrest of pollen hydration to compatible pollen. The [Ca2+]cyt increase in papilla cells during the self-incompatibility response of GLR mutants was significantly reduced. Our results overall strongly suggest that the Ca2+ influx in papilla cells mediated by GLR is the key self-incompatibility response that leads to self-pollen rejection.