Plant Vacuole Biogenesis and Function

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Mammalian, or yeast and plant cells contain lysosomes or vacuoles, respectively, that function as lytic or digestive compartments. Plant cells uniquely, however, also store many complex metabolic products and proteins in vacuoles. Plant storage vacuoles differ from lytic vacuoles with respect to the protein composition of their membranes as well as their lumenal contents. Additionally, a vesicular pathway unique to plant cells traffics from Golgi to protein storage vacuoles, while clathrin coated vesicles, as in yeast and mammalian cells, traffic from Golgi to lytic vacuoles. In contrast to yeast vacuoles, plant vacuole membranes contain very large amounts (up to 50% of the membrane protein) of Tonoplast Intrisic Proteins (TIPs). TIPs have aquaporin activity, but the large amounts present appear to be in great excess over what would be needed for water transport. It has been shown previously that specific TIP isoforms are associated with specific vacuole functions: Protein storage vacuoles that store seed-type storage proteins are marked by alpha- plus delta-TIP; vacuoles that store different types of proteins, vegetative storage proteins, and vacuoles that store pigments are marked by delta-TIP alone or by delta- plus gamma-TIP; lytic vacuoles that are normally present and contain acidic pH and active proteases are marked by gamma-TIP alone; autophagic vacuoles that are induced when plant cells are starved appear to be marked with a form of alpha-TIP alone. It has been hypothesized that TIP isoforms in some way determine the interior environment of vacuoles and thereby determine vacuole functions. Additionally, the fact that plant cells must maintain functionally distinct vacuoles as well as separate vesicular pathways to each indicate that lytic and storage vacuoles are distinct organelles, not simply two extreme poles of one organelle. This distinction is of substantial importance, because the separate organelle hypothesis requires that plant cells maintain separate pathways for flow of membrane proteins to generate and maintain the different organelles. Additionally, it means that plant cells have unique mechanisms for biogenesis of additional organelle members of the endomembrane system. As each of the three TIP isoforms can be found separately on vacuoles within one cell, it is possible that as many as three distinct plant vacuole organelles may exist. The goal is to test various parts of these hypotheses. The role of TIP isoforms in determining vacuole function will be tested by knocking out expression of only delta-TIP in transgenic petunia and tomato plants. If delta-TIP is required for vacuole storage functions, such knockout plants should lack flower petal pigments, and tomato plants should not be able to accumulate and store certain protease inhibitors, vegetative storage proteins that function as defense molecules, in leaves and flower petals. Additionally, the form of alpha-TIP that is associated with autophagic vacuoles will be identified and cloned to determine its difference, if any, from alpha-TIP in protein storage vacuoles. This also will potentially be a target for knockout experiments. The second series of experiments deals with biochemical characterization of tonoplast membranes purified from three functionally distinct vacuoles: protein storage vacuoles, vacuoles storing vegetative storage proteins, and lytic vacuoles. From yeast and mammalian studies, the concept has emerged that identity of organelles within the secretory pathway is defined biochemically by the presence of a unique syntaxin protein on each organelle membrane. The syntaxins present on each vacuole type will be purified, cloned and characterized as a test of the functionally distinct vacuole as a unique organelle hypothesis. These studies will provide new information about processes by which plant cells establish and maintain functionally distinct vacuoles. They may also provide new insights into mechanisms for organelle biogenesis.