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.

哺乳动物或酵母和植物细胞分别包含溶酶体或液泡，它们起着裂解或消化室的作用。但是，植物细胞独特地在液泡中存储了许多复杂的代谢产物和蛋白质。相对于其膜的蛋白质组成以及其腔内含量，植物储存液泡与裂解液泡不同。此外，从高尔基体到蛋白质储存液的植物细胞运输特有的囊泡途径，而网状蛋白涂层的囊泡，如酵母和哺乳动物细胞中，从高尔基体到裂解液泡。 与酵母菌液泡相反，植物液泡膜含有大量（膜蛋白的50％）的块状骨膜散发蛋白（TIPS）。尖端具有水通道活动，但存在的大量似乎比水运输所需的大量过多。先前已经显示，特定的尖端同工型与特定的液泡功能相关：储存种子型储存蛋白的蛋白储存液泡由α-加上delta-tip标记；储存不同类型的蛋白质，营养储存蛋白和液泡的液泡，单独或以Delta-plus gamma-tip为标志；通常存在并包含酸性pH和活性蛋白酶的溶液液泡仅由γ-TIP标记。当植物细胞饥饿时诱导的自噬液泡似乎单独用α-TIP形式标记。已经假设，尖端同工型在某种程度上决定了液泡的内部环境，从而确定了液泡功能。此外，植物细胞必须保持功能上不同的液泡以及到每种囊泡途径的独立囊泡途径这一事实表明，裂解和存储液泡是不同的细胞器，而不仅仅是一个细胞器的两个极端杆。这种区别具有重要的重要性，因为单独的细胞器假设要求植物细胞维持单独的膜蛋白流动途径，以生成和维持不同的细胞器。此外，这意味着植物细胞具有内膜系统其他细胞器成员生物发生的独特机制。由于三个尖端同工型中的每一个都可以在一个细胞内的液泡上分别找到，因此可能存在多达三个不同的植物液泡细胞器。目的是检验这些假设的各个部分。尖端同工型在确定液泡功能方面的作用将通过在转基因矮牵牛和番茄植物中仅敲出Delta-Tip的表达来测试。如果液泡存储功能需要三角尖，则这种基因敲除植物应缺乏花瓣色素，番茄植物不应能够积累和存储某些蛋白酶抑制剂，在叶子和花瓣中充当防御分子的营养储存蛋白。此外，将确定与自噬液泡相关的α-TIP的形式，并克隆以确定蛋白质储存液泡中α-TIP的差异（如果有的话）。这也可能是敲除实验的目标。第二系列实验涉及从三个功能上不同的液泡中纯化的块状膜的生化表征：蛋白质储存液泡，储存营养储存蛋白和裂解液泡的蛋白质储存液泡。从酵母和哺乳动物的研究中，该概念表明，分泌途径内的细胞器的身份是通过在每个细胞器膜上存在独特的语法蛋白在生化上定义的。每种液泡类型上存在的语法素将被纯化，克隆和表征为功能上不同的液泡作为独特的细胞器假设的测试。 这些研究将提供有关植物细胞建立和维持功能不同液泡的过程的新信息。他们还可以提供有关细胞器生物发生机制的新见解。