Identification of pollen components regulating pollination-specific responses in the stigmatic papillae of Brassica oleracea

When components of the mature pollen grain of Brassica oleracea are applied to the cuticularised surfaces of the stigmatic papilla, a number of changes can take place in the architecture of the subjacent cell wall. Treatment of the papillar surface with isolated pollen coating evokes a rapid and extensive expansion of the outer of the two stigmatic wall layers. This response occurs within 4 h but is restricted to those regions where no callose is formed and where the coating exhibits a characteristic increase in electron opacity. This study establishes that the coating alone is responsible for initiating the expansion of the outer wall, which is considered to be an essential step preceding penetration of the stigma surface by the pollen tube. Vesicle-like inclusions, some staining intensely, occasionally occur in regions of the wall expanded by isolated coating, sometimes fusing to form a subcuticular 'boundary layer'. These structures are not observed under compatible grains in vivo and their presence is regarded as artefaetual. However, close examination of the plasma membrane at sites beneath areas of expanded cell wall reveals membrane-bound structures resembling the vesicles being generated by the cytoplasm and moving into the apoplast. These data indicate that signals from the coating initiate the loosening of the wall matrix by stimulating an unusual form of localized secretion which appears to be an essential prerequisite for stigmatic penetration. Isolated pollen coating contaminated with fragments of the grain itself engender a very different response, including the formation of densely-staining vesicles accompanied by extensive accumulations of callose; wall expansion never occurs under these circumstances. Self-pollinations in B. oleracea are frequently accompanied by the synthesis of stigmatic callose, the presence of which has, in the past, been interpreted as forming a structural barrier to incompatible pollen tubes. However, callose elicited both by self-pollinations and coating supplemented by killed grains is independent of protein synthesis and occurs in the presence of the phosphatase inhibitor, okadaic acid. Since both these inhibitors also overcome self-incompatibility (SI) in B. oleracea, these data strongly suggest that callose synthesis is unrelated to the operation of SI, and it is proposed that callose, when formed, is elicited by molecules released from the necrotic pollen protoplast. Interestingly, isolated pollen coating of self- and cross-genotypes does not readily elicit callose. The significance of these changes in the stigmatic wall is discussed in the perspective of current views on pollination and self-incompatibility in Brassica.