The Role of Protons in Integrating the Activities of Endosperm and Embryo in Germinating Cereal Grains

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9306355 Jones The goals of this proposal are to understand how the activities of the aleurone, starchy endosperm and scutellum of germinating cereal grains are integrated. The mobilization of the starchy endosperm of cereal grains, a tissue that consists entirely of dead storage cells, will be studied, and a simple model in which pH plays a key role in the regulation of endosperm mobilization will be tested. This model is based on the observation that low pH is essential for the synthesis and secretion of hydrolytic enzymes by the aleurone and scutellum, for the activity of these enzymes in the endosperm, and for the uptake of sugars and amino acids produced by the activity of secreted hydrolases. Experiments are proposed that have been designed to test the hypothesis that acidification of the endosperm is brought about by proton pumps located at the plasma membrane (PM) of aleurone and scutellum cells. The capacity of the aleurone and scutellum to acidify the external medium will be evaluated by incubating these tissues in unbuffered and buffered media in the presence of gibberellic acid (GA) and abscisic acid (ABA). Acidification of the incubation medium will be monitored using pH electrodes and by titration. The mechanism of acidification of the endosperm by aleurone and scutellum will be examined. Since the PM of barley aleurone does not possess detectable levels of a P-type ATPase, the possibility that acidification is brought about by a V-type ATPase at the PM will be tested. Specific inhibitor of V-type ATPases will be used to examine acidification in vivo, and H+ transport by PM vesicles isolated from aleurone and scutellum by phase partitioning will be used to study the process in vitro. Acidification by isolated aleurone layers occurs rapidly after addition of GA, an observation that indicates that H+ transporters are constitutively expressed in this tissue. Specific cDNA and antibody probes will be used to measure PM H+ATPase RNA and protein l evels in aleurone and scutellum, and experiments will be carried out to establish whether GA and ABA regulate the expression of genes for these transporters. Because the scutellum contains endogenous GAs, the role of GA in regulating acidification in this tissue will be examined in GA deficient mutants of barley. Since aleurone cells secrete large amounts of H+, they must possess a mechanism for maintaining charge balance. The possibility that charge balance in aleurone and scutellum cells is achieved by the synthesis and export of organic and inorganic anions will be explored using high performance liquid chromatography of incubation media. Since one of the goals of this proposal is to understand the role played by H+ in coordinating the activities of germinating grain, 31P NMR spectroscopy will be used to monitor pH changes in the endosperm of intact germinating grain. The hypothesis that endosperm pH plays a key role in regulating endosperm mobilization will be tested using NMR to monitor pH in grain exposed to stresses known to inhibit germination or seedling growth or exposed to applied ABA and GA. If endosperm pH proves to be a reliable and early indicator of the response of the cereal grain to stress, a simple test for measuring this parameter will be devised. The successful completion of this research will contribute to fundamental knowledge about physiological processes in plants as well as well as provide information of a practical nature to growers of cereal crops. ***