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

该建议描述了以谷物糊粉层细胞为模型系统，旨在理解植物液泡功能的实验。 大麦糊粉层细胞具有几种类型的液泡，这些细胞器的组织和功能在脱落酸（阿坝）和赤霉素（GA）处理的细胞中有显着的不同。 糊粉细胞在GA中孵育后变得高度空泡化;阿坝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溶解酶和接近中性的pH，而GA中孵育的细胞中的液泡具有升高的溶解酶水平和约5.5或更低的pH。 GA和阿坝也调节糊粉层细胞的程序性细胞死亡（PCD）。广泛的细胞重塑伴随着植物细胞的死亡，在糊粉层细胞中，裂解性液泡的形成可能是这一过程的关键特征。 细胞生物学的方法，侧重于溶解酶和他们居住的车厢是非常适合解开亚细胞的变化，伴随着死亡。 该项目将测试的假设，GA和阿坝改变亚细胞的关键溶解酶的位置，以及这些酶位于细胞器的pH值，从而影响细胞死亡（PCD）。 本项目主要研究糊粉细胞中半胱氨酸蛋白酶的作用。有待检验的假设是，蛋白酶的细胞内定位和翻译后加工在糊粉层细胞中调节PCD中起关键作用。 以前的实验表明，蛋白酶积累在GA处理的糊粉细胞的液泡，但在ABA处理的细胞中，只有这些蛋白质的非活性前体积累。在半胱氨酸蛋白酶糊粉蛋白酶的情况下，GA调节酶的翻译后加工和亚细胞定位。 在GA处理的细胞中，糊粉蛋白存在于蛋白质储存液泡中，但在ABA处理的细胞中，糊粉蛋白存在于不同的液泡样细胞器中。这一新的观察结果表明，GA既激活和改变降解酶的亚细胞定位，提供了一个有吸引力的模型糊粉细胞中的PCD的激素调节。 PI提出GA诱导的糊粉蛋白向蛋白质储存泡的递送是导致形成裂解环境和细胞死亡的一系列事件之一。 该细胞中至少有三个液泡区室：蛋白质储存液泡、次生液泡和含糊粉蛋白的液泡。 实验旨在了解这些细胞器的功能和生物起源。其他问题包括：空泡是否作为细胞成分降解的自噬细胞器发挥作用？ 如果自噬发生在糊粉层液泡中，那么细胞成分是如何传递到液泡中的？ 了解GA启动糊粉层从贮藏组织到分泌腺，最后到死亡组织的转化机制，将为植物生物学中的几个基本问题提供见解。

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.