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

这项建议描述了以谷物糊粉细胞为模型系统的旨在了解植物液泡功能的实验。大麦糊粉细胞有几种类型的液泡；在脱落酸(ABA)和赤霉酸(GA)处理的细胞中，这些细胞器的组织和功能有很大的不同。糊粉细胞在GA中孵育后变得高度空泡化；ABA处理的细胞不经历广泛的空泡化。ABA处理的糊粉细胞中的液泡具有相对较少的裂解酶和接近中性的pH，而在GA孵育的细胞中，液泡的裂解酶水平较高，pH为~5.5或更低。GA和ABA还调节糊粉细胞的程序性细胞死亡(PCD)。在植物中，广泛的细胞重塑伴随着细胞死亡；糊粉细胞中溶解空泡的形成可能是这一过程的一个关键特征。一种专注于裂解酶及其所在隔室的细胞生物学方法非常适合于解开伴随死亡而来的亚细胞变化。这个项目将检验这样的假设，即GA和ABA改变关键裂解酶的亚细胞位置以及这些酶所在细胞器的pH，从而影响细胞死亡(PCD)。本项目的重点是半胱氨酸蛋白酶在糊粉细胞中的作用。需要检验的假设是，在糊粉细胞中，蛋白水解酶的细胞内定位和翻译后处理在调节PCD方面起着关键作用。以前的实验表明，在GA处理的糊粉细胞中，蛋白水解酶会在液泡中积累，但在ABA处理的细胞中，只有这些蛋白的非活性前体会积累。在半胱氨酸蛋白酶Aleurain的情况下，GA调节该酶的翻译后加工和亚细胞定位。在GA处理的细胞中，Aleurain存在于蛋白质储存液泡中，但在ABA处理的细胞中，Aleurain存在于不同的液泡状细胞器中。这一新的观察表明，GA既激活并改变了降解酶的亚细胞定位，为糊粉细胞中PCD的激素调节提供了一个有吸引力的模型。PI认为，GA诱导的Aleurain输送到蛋白质储存液泡是导致裂解环境形成和细胞死亡的一系列事件之一。该细胞至少有三个液泡室：蛋白质储存液泡、次生液泡和含有糊蛋白的液泡。实验旨在了解这些细胞器的功能和生物发生。其他问题包括：液泡是否起自噬细胞器的作用，在那里细胞成分被降解？如果糊粉液泡中发生自噬，细胞成分是如何输送到液泡的？了解GA启动糊粉层从储藏组织到分泌腺，最后到死亡组织的转化机制，将有助于深入了解植物生物学中的几个基本问题。

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.