Plant Vacuole Biogenesis and Function

植物液泡的生物发生和功能

• 批准号: 9974429
• 负责人: John Rogers
• 金额: --
• 依托单位: Washington State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9974429&HistoricalAwards=false
• 关键词: Plant; Vacuole; Biogenesis; Function

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.

哺乳动物细胞或酵母细胞和植物细胞分别含有溶酶体或液泡，充当裂解室或消化室。然而，植物细胞还独特地在液泡中储存许多复杂的代谢产物和蛋白质。植物储存液泡在其膜的蛋白质组成及其腔内容物方面与裂解液泡不同。此外，植物细胞特有的囊泡途径从高尔基体运输到蛋白质储存液泡，而网格蛋白包被的囊泡（如在酵母和哺乳动物细胞中）从高尔基体运输到裂解液泡。 与酵母液泡相反，植物液泡膜含有大量（高达膜蛋白的 50%）液泡膜固有蛋白 (TIP)。 TIP 具有水通道蛋白活性，但其含量似乎远远超出了水运输所需的量。先前已经表明，特定的 TIP 亚型与特定的液泡功能相关：储存种子型储存蛋白的蛋白质储存液泡由 α-+ δ-TIP 标记；储存不同类型蛋白质、营养储存蛋白和储存色素的液泡单独用δ-TIP或用δ-加γ-TIP标记；通常存在且含有酸性 pH 值和活性蛋白酶的裂解液泡仅由 γ-TIP 标记；植物细胞饥饿时诱导的自噬液泡似乎仅用α-TIP 形式标记。据推测，TIP 亚型以某种方式决定液泡的内部环境，从而决定液泡功能。此外，植物细胞必须维持功能上不同的液泡以及各自的独立的囊泡途径这一事实表明，裂解液泡和储存液泡是不同的细胞器，而不仅仅是一个细胞器的两个极端。这种区别非常重要，因为单独的细胞器假说要求植物细胞维持膜蛋白流动的单独途径，以产生和维持不同的细胞器。此外，这意味着植物细胞具有独特的内膜系统其他细胞器成员的生物发生机制。由于三种 TIP 亚型中的每一种都可以在一个细胞内的液泡上单独发现，因此可能存在多达三种不同的植物液泡细胞器。目标是测试这些假设的各个部分。 TIP 同种型在确定液泡功能中的作用将通过仅敲除转基因矮牵牛和番茄植物中 delta-TIP 的表达来测试。如果液泡储存功能需要δ-TIP，那么这种基因敲除植物应该缺乏花瓣色素，并且番茄植物不应该能够在叶子和花瓣中积累和储存某些蛋白酶抑制剂，即充当防御分子的营养储存蛋白。此外，将鉴定并克隆与自噬液泡相关的 α-TIP 形式，以确定其与蛋白质储存液泡中的 α-TIP 的差异（如果有）。这也可能成为淘汰实验的目标。第二系列实验涉及从三个功能不同的液泡纯化的液泡膜的生化特征：蛋白质储存液泡、储存营养储存蛋白的液泡和裂解液泡。从酵母和哺乳动物的研究中，已经出现了这样的概念：分泌途径中细胞器的身份是通过每个细胞器膜上存在的独特突触蛋白来生物化学定义的。每种液泡类型上存在的突触融合蛋白将被纯化、克隆和表征，以测试功能不同的液泡作为独特的细胞器假设。 这些研究将提供有关植物细胞建立和维持功能独特的液泡过程的新信息。它们还可能为细胞器生物发生机制提供新的见解。