Effects of Inhibitors of Protein Serine/Threonine Phosphatases on Pollination in Brassica

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Abstract
We have examined the effect of the protein phosphatase inhibitors okadaic acid and microcystin on pollen-pistil interactions in Brassica. Inhibitor-treated flowers or floral buds were pollinated with untreated pollen and examined for pollen tube growth by fluorescence microscopy. Our results show that type 1 or type 2A serine/threonine phosphatases play a crucial role in the pollination responses of Brassica. We observed two distinct effects of protein phosphatase inhibitors on pollination: (a) the inhibition of pollen tube growth during cross-pollination in flowers, and (b) the break-down of self-incompatibility or promotion of pollen tube growth during self-pollination in flower buds just prior to anthesis. Thus, treatment of flower pistils with protein phosphatase inhibitors resulted in the inhibition of pollen tube growth at the surface of the papillar cells of the stigma in crosses between different self-incompatible Brassica oleracea strains, in an interspecific cross between B. oleracea and Brassica campestris, and in self-pollinations of a self-fertile Brassica napus cultivar. With four different self-incompatibility genotypes, treatment of mature flowers with protein phosphatase inhibitors had no effect on self-pollination response. In contrast, treatment of flower buds just prior to the anthesis stage allowed self-pollen tube invasion of papillar cells. However, the magnitude of this effect was genotype dependent, being most pronounced in the S22 genotype. The data support the conclusion that pollinations in Brassica are controlled in part by the presence of phosphorylated proteins in the papillar cells of the stigma, and that the quantity of these proteins or their levels of phosphorylation changes during stigma development.