Hormones, Programmed Cell Death and DNA Breakdown in Aleurone Cells

小行星9722956 本项目的目标是表征大麦糊粉层细胞中伴随程序性细胞死亡（PCD）的细胞事件。糊粉层细胞的PCD受赤霉酸（GA）和脱落酸（阿坝）的调控。GA诱导大麦糊粉层PCD，阿坝阻止细胞死亡。实验被提出来表征DNA分解，询问DNA是如何降解的，并确定参与这一过程的酶。 动物细胞的细胞凋亡伴随着核DNA的核内切割，导致特征性DNA梯的形成。以前发表的工作，其他人提出，DNA梯状是一个功能的PCD在大麦糊粉层细胞。 来自主要研究者实验室的初步数据显示，虽然PCD伴随着GA处理的糊粉细胞中的DNA断裂，但DNA梯状条带的形成不会发生。 本研究以大麦糊粉层和原生质体为材料，通过对细胞程序性死亡的激素处理，研究了大麦糊粉层和原生质体的DNA降解过程，以探讨大麦糊粉层和原生质体的DNA降解机制。 糊粉层细胞中的核酸酶将被表征以确定这些酶是否仅具有外切核酸酶活性，这将解释糊粉层细胞中不存在DNA梯状化。还将进行实验以确定糊粉细胞中何时发生DNA断裂。主要研究者已经确定，DNA断裂发生在GA处理后，而不是阿坝处理后。 DNA降解是在活细胞中发生还是仅在PCD后发生？ 在阿坝处理的细胞中没有DNA降解是由于这些细胞中核酸酶活性水平低，还是DNA被修饰为在阿坝处理的细胞中降解不太快？ 荧光染料染色的DNA在活的糊粉细胞将被用来监测DNA降解和相关的变化与PCD的DNA。 如果糊粉层细胞死亡后发生DNA降解，将从原生质体中纯化死细胞以监测DNA如何降解。 在大麦种子中，有一个富含淀粉的组织，称为胚乳，它是种子内发育中的植物胚胎的营养库。 糊粉层是胚乳周围的细胞组织，其产生并分泌水解酶，所述水解酶将淀粉质胚乳消化成可被发育中的胚直接用作食物的单糖。 糊粉层因此可以被认为是萌发种子内发育胚的消化器官。 当糊粉层完成了酶的生产，它就没有进一步的用途，组织细胞经历程序性细胞死亡。 糊粉层细胞的发育调控的生死受两种主要植物激素赤霉酸和脱落酸的控制。这项工作的结果将提供有价值的见解的过程中发育调节PCD的植物和激素在调节这一过程中发挥的作用。 ***

9722956 Jones The goals of this project are to characterize the cellular events that accompany programmed cell death (PCD) in the barley aleurone cell. PCD in aleurone cells is controlled by the hormones gibberellic acid (GA) and abscisic acid (ABA). GA induces PCD in barley aleurone and ABA prevents cell death. Experiments are proposed to characterize DNA breakdown, to ask how DNA is degraded, and to identify the enzymes that are involved in this process. Apoptosis in animal cells is accompanied by intranucleosomal cleavage of nuclear DNA resulting in the formation of characteristic DNA ladders. Previously published work by others has suggested that DNA laddering is a feature of PCD in the barley aleurone cell. Preliminary data from the Principal Investigator's laboratory show that whereas PCD is accompanied by DNA breakdown in GA-treated aleurone cells, formation of DNA ladders does not occur. A detailed study is planned of DNA degradation from barley aleurone layers and protoplasts treated with hormones that regulate PCD to characterize the mechanism of DNA breakdown. The nucleases in aleurone cells will be characterized to determine whether these enzymes only possess exonuclease activity, which would account for the absence of DNA laddering in the aleurone cell. Experiments will also be carried out to determine when DNA breakdown occurs in aleurone cells. The Principal Investigator has established that DNA breakdown occurs after GA but not following ABA treatment. Is DNA degraded in living cells or does DNA degradation only occur after PCD? Is the absence of DNA degradation in ABA treated cells a result of the low levels of nuclease activity in these cells, or is DNA modified to be less rapidly degraded in ABA treated cells? Fluorescent dyes that stain DNA in living aleurone cells will be used to monitor DNA degradation and to correlate changes in DNA with PCD. If DNA breakdown occurs after aleurone cells die, dead cells will be purified from prot oplasts to monitor how the DNA is degraded. In the barley seed, there is a starch-rich tissue called the endosperm, which serves as a reservoir for nourishment of the developing plant embryo inside the seed. The aleurone layer is a cellular tissue surrounding the endosperm, which produces and secretes hydrolytic enzymes that digest the starchy endosperm into monosaccharides that are can be used directly as food by the developing embryo. The aleurone can thus be thought of as the digestive organ of the developing embryo within the germinating seed. When the aleurone has finished producing the enzymes, it has no further purpose and the cells of the tissue undergo a programmed cell death. The developmentally regulated life and death of the cells of the aleurone are under the control of two major plant hormones, gibberellic acid and abscisic acid. The results of this work will provide valuable insights into the process of developmentally regulated PCD in plants and the roles that hormones play in regulating this process. ***