Characterization of ScMAP4K1, a MAP kinase kinase kinase kinase involved in ovule, seed and fruit development in Solanum chacoense Bitt

In order to gain information about protein kinases acting during plant fertilization and embryogenesis, we used a reverse genetic approach to determine the role of protein kinases expressed in reproductive tissues in the wild potato species Solanum chacoense. Out of an EST library normalized for weakly expressed genes in fertilized ovaries, we isolated a cDNA clone named ScMAP4K1 that showed significant sequence similarities to members of the MAPKKKK family. ScMAP4K1 is a single copy gene predominantly expressed in stylar and ovary tissues. A phylogenetic analysis revealed that it belonged to the GCK-II subfamily of MAP4Ks. Unlike animal GCK-II, ScMAP4K1 has a centrally located kinase domain flanked by large N- and C-terminal putative regulatory domains. Yeast two-hybrid screens were conducted with all three domains separately and the N-terminal domain screen retrieved numerous interacting proteins including proteins involved in fruit ripening. Transgenic plant lines expressing an RNA interference construct for ScMAP4K1 showed abnormal fruit growth and lowered seed set ranging from ~6 to 60% when compared to WT plants. Analysis of ovules before fertilization showed that all transgenic plant lines had a higher than WT percentage of defective embryo sacs, but that did not correlate entirely with the number of seeds produced per line, suggesting both pre- and post-fertilization roles for the ScMAP4K1 protein kinase.