Structural and Functional analysis of Lytic Organelles and Lytic Enzymes in Aleurone Cells

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This proposal describes experiments aimed at an understanding of vacuolar function in plants using cereal aleurone cells as a model system. Barley aleurone cells have several types of vacuoles; the organization and function of these organelles is dramatically different in abscisic acid (ABA) and gibberellic acid (GA) treated cells. Aleurone cells become highly vacuolate following incubation in GA; ABA treated cells do not undergo extensive vacuolation. Vacuoles in ABA-treated aleurone cells have relatively few lytic enzymes and a pH near neutrality, whereas vacuoles in cells incubated in GA have elevated levels of lytic enzyme and a pH of ~ 5.5 or below. GA and ABA also regulate programmed cell death (PCD) in aleurone cells. Extensive cellular remodeling accompanies cell death in plants; the formation of lytic vacuoles may be a key feature of this process in aleurone cells. A cell biological approach which focuses on lytic enzymes and the compartments in which they reside is highly suited for unraveling the subcellular changes that accompany death. This project will test the hypothesis that GA and ABA alter the subcellular location of key lytic enzymes as well as the pH of the organelles in which these enzymes are located, thereby affecting cell death (PCD). This project focuses on the roles of cysteine proteases in aleurone cells. The hypothesis to be tested is that intracellular location and posttranslational processing of proteases play a key role in regulating PCD in the aleurone cell. Previous experiments show that proteases accumulate in the vacuoles of GA-treated aleurone cells, but in ABA-treated cells only inactive precursors of these proteins accumulate. In the case of the cysteine protease aleurain, GA regulates the posttranslational processing and subcellular location of the enzyme. Aleurain is found in the protein storage vacuole in GA-treated cells, but in a different vacuole-like organelle in ABA-treated cells. This novel observation indicates that GA both activates and changes the subcellular localization of degradative enzymes, providing an attractive model for the hormonal regulation of PCD in aleurone cells. The PI proposes that GA-induced delivery of aleurain to the protein storage vacuole is one of a series of events that leads to the formation of a lytic environment and cell death. There are at least three vacuolar compartments in this cell: the protein storage vacuole, the secondary vacuole and the aleurain containing vacuole. Experiments are aimed at understanding the function and biogenesis of these organelles. Other questions include: Do vacuoles function as autophagic organelles where cellular components are degraded? If autophagy occurs in aleurone vacuoles, how are cellular components delivered to the vacuole? An understanding of the mechanisms by which GA initiates the transformation of the aleurone layer from a storage tissue to secretory gland and finally to a dying tissue will provide insights into several fundamental questions in plant biology.