Regulation of Calcium Transport in Barley Aleurone Cells

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The goals of the proposed research are to achieve an understanding of calcium homeostasis in the barley aleurone cell. Prior work from this laboratory on the synthesis of the calcium- containing metalloenzyme, alpha-amylase, has established that the cytoplasmic calcium concentration is maintained within narrow limits while a constant flux of calcum occurs through the cytoplasm. Experiments are proposed to determine the source of the calcium that assures cytoplasmic calcium homeostasis. Patch clamp techniques will be used to identify the channels in the plasm membrane and tonoplast of the aleurone cell that regulate the flux of ions into the cytoplasm. The experimental approach relies on the ability to isolate functional protoplasts from the aleurone layer and to use these protoplasts as a source of intact vacuoles. The activties of channels in the plasma membrane of aleurone protoplasts and in vacuoles isolated from protoplasts have already been measured in this laboratory; thus, this approach should succeed. During seed germination metabolites stored in the vacuoles of storage organs such as aleurone layers are mobilized for use by the growing plant embryo. Although these stored reserves are crucial, little is known about how these materials are mobilized. It is thought that giberellic acid is involved, but the mechanism by which it acts is not known. This laboratory has shown that mobilization of calcium is of central importance to the giberellic acid-induced synthesis of alpha-amylase, an enzyme important in mobilizing storage products in the aleurone cell. This project is directed toward understanding how calcium is regulated in barley aleurone cells. The results should elucidate an important step in the regulation of metabolite mobilization during seed germination.