Calcium signalling mediates self-incompatibility response in the Brassicaceae.

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 Ca(2+) 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 Ca(2+) ([Ca(2+)]cyt) in papilla cells. Direct application of SP11/SCR to the papilla cell protoplasts induced Ca(2+) increase, which was inhibited by D-(-)-2-amino-5-phosphonopentanoic acid (AP-5), a glutamate receptor channel blocker. An artificial increase in [Ca(2+)]cyt in papilla cells arrested wild-type (WT) pollen hydration. Treatment of papilla cells with AP-5 interfered with self-incompatibility, and Ca(2+) increase on the self-incompatibility response was reduced in the glutamate receptor-like channel (GLR) gene mutants. These results suggest that Ca(2+) influx mediated by GLR is the essential self-incompatibility response leading to self-pollen rejection.

Actin dynamics in papilla cells of Brassica rapa during self- and cross-pollination.

The self-incompatibility system of the plant species Brassica is controlled by the S-locus, which contains S-RECEPTOR KINASE (SRK) and S-LOCUS PROTEIN11 (SP11). SP11 binding to SRK induces SRK autophosphorylation and initiates a signaling cascade leading to the rejection of self pollen. However, the mechanism controlling hydration and germination arrest during self-pollination is unclear. In this study, we examined the role of actin, a key cytoskeletal component regulating the transport system for hydration and germination in the papilla cell during pollination. Using rhodamine-phalloidin staining, we showed that cross-pollination induced actin polymerization, whereas self-pollination induced actin reorganization and likely depolymerization. By monitoring transiently expressed green fluorescent protein fused to the actin-binding domain of mouse talin, we observed the concentration of actin bundles at the cross-pollen attachment site and actin reorganization and likely depolymerization at the self-pollen attachment site; the results correspond to those obtained by rhodamine-phalloidin staining. We further showed that the coat of self pollen is sufficient to mediate this response. The actin-depolymerizing drug cytochalasin D significantly inhibited pollen hydration and germination during cross-pollination, further emphasizing a role for actin in these processes. Additionally, three-dimensional electron microscopic tomography revealed the close association of the actin cytoskeleton with an apical vacuole network. Self-pollination disrupted the vacuole network, whereas cross-pollination led to vacuolar rearrangements toward the site of pollen attachment. Taken together, our data suggest that self- and cross-pollination differentially affect the dynamics of the actin cytoskeleton, leading to changes in vacuolar structure associated with hydration and germination.

Characterization of the SP11/SCR high-affinity binding site involved in self/nonself recognition in brassica self-incompatibility.

In Brassica self-incompatibility, the recognition of self/nonself pollen grains, is controlled by the S-locus, which encodes three highly polymorphic proteins: S-locus receptor kinase (SRK), S-locus protein 11 (SP11; also designated S-locus Cys-rich protein), and S-locus glycoprotein (SLG). SP11, located in the pollen coat, determines pollen S-haplotype specificity, whereas SRK, located on the plasma membrane of stigmatic papilla cells, determines stigmatic S-haplotype specificity. SLG shares significant sequence similarity with the extracellular domain of SRK and is abundant in the stigmatic cell wall, but its function is controversial. We previously showed that SP11 binds directly to its cognate SRK with high affinity (K(d) = 0.7 nM) and induces its autophosphorylation. We also found that an SLG-like, 60-kD protein on the stigmatic membrane forms a high-affinity binding site for SP11. Here, we show that the 60-kD stigmatic membrane protein is a truncated form of SRK containing the extracellular domain, transmembrane domain, and part of the juxtamembrane domain. A transiently expressed, membrane-anchored form of SRK exhibits high-affinity binding to SP11, whereas the soluble SRK (eSRK) lacking the transmembrane domain exhibits no high-affinity binding, as is the case with SLG. The different binding affinities of the membrane-anchored SRK and soluble eSRK or SLG will be significant for the specific perception of SP11 by SRK.

Immunohistochemical studies on translocation of pollen S-haplotype determinant in self-incompatibility of Brassica rapa.

The self-incompatibility system in Brassica is controlled by the S-locus, which contains S-receptor kinase (SRK) and S-locus protein 11 (SP11). SRK and SP11 control stigma and pollen S-haplotype specificity, respectively. SP11 binding to SRK induces the autophosphorylation of SRK, which triggers the signaling cascade that results in the rejection of self-pollen. The localization of SP11 protein during pollen development and pollination, however, have never been demonstrated. In this study, we examined the localization of S(8)-SP11 protein in the anther or pollinated stigma by immuno-electron microscopy. The immunostaining suggested that S(8)-SP11 was secreted from the tapetal cell into the anther locule as a cluster and translocated to the pollen surface at the early developmental stage of the anther. During the pollination process, SP11 was translocated from the pollen surface to the papilla cell, and then penetrated the cuticle layer of the papilla cell to diffuse across the pectin cellulose layer. Furthermore, SP11 protein could only penetrate the cuticle layer of the papilla cell in the presence of pollen grains, and could not penetrate on its own. This suggests that another factor from the pollen grain is needed for SP11 protein to penetrate the papilla cell wall.

Isolation of a cDNA for a nucleoside diphosphate kinase capable of phosphorylating the kinase domain of the self-incompatibility factor SRK of Brassica campestris.

SRK is a plant receptor kinase involved in the self-incompatibility system of Brassica species. During a cDNA screening for the phosphoproteins from a stigma expression library, a clone encoding the nucleoside diphosphate kinase III (Bc-NDPK III) was obtained. After in vitro phosphorylation assays with recombinant proteins, Bc-NDPK III contained mostly phosphoserine. By contrast, the kinase domain of SRK contained phosphoserine and phosphothreonine, both of which were significantly increased by the addition of Bc-NDPK III in the presence of an SRK inhibitor KN-62. The result suggested the possible involvement of Bc-NDPK III in the signal transduction pathway through SRK.

The dominance of alleles controlling self-incompatibility in Brassica pollen is regulated at the RNA level.

Self-incompatibility (SI) in Brassica is controlled sporophytically by the multiallelic S-locus. The SI phenotype of pollen in an S-heterozygote is determined by the relationship between the two S-haplotypes it carries, and dominant/recessive relationships often are observed between the two S-haplotypes. The S-locus protein 11 (SP11, also known as the S-locus cysteine-rich protein) gene has been cloned from many pollen-dominant S-haplotypes (class I) and shown to encode the pollen S-determinant. However, SP11 from pollen-recessive S-haplotypes (class II) has never been identified by homology-based cloning strategies, and how the dominant/recessive interactions between the two classes occur was not known. We report here the identification and molecular characterization of SP11s from six class II S-haplotypes of B. rapa and B. oleracea. Phylogenetic analysis revealed that the class II SP11s form a distinct group separated from class I SP11s. The promoter sequences and expression patterns of SP11s also were different between the two classes. The mRNA of class II SP11, which was detected predominantly in the anther tapetum in homozygotes, was not detected in the heterozygotes of class I and class II S-haplotypes, suggesting that the dominant/recessive relationships of pollen are regulated at the mRNA level of SP11s.