The Role of Protons in Integrating the Activities of Endosperm and Embryo in Germinating Cereal Grains

[306355 Jones]本提案的目标是了解萌发谷物的糊粉、淀粉胚乳和胚乳的活性是如何整合的。谷类淀粉胚乳是一种完全由死亡储存细胞组成的组织，其动员将被研究，并将测试一个简单的模型，其中pH值在胚乳动员的调节中起关键作用。该模型是基于以下观察：低pH值对于糊粉和黄花瓣的水解酶的合成和分泌、这些酶在胚乳中的活性以及由分泌的水解酶活性产生的糖和氨基酸的吸收是必不可少的。提出了一些实验，旨在验证位于糊粉质膜和胚乳细胞质膜（PM）的质子泵引起胚乳酸化的假设。通过将这些组织在赤霉素酸（GA）和脱落酸（ABA）存在的无缓冲和缓冲培养基中孵育，来评估糊粉和鳞片对外部培养基的酸化能力。将使用pH电极和滴定法监测培养培养基的酸化情况。探讨了糊粉粉和鳞片对胚乳酸化的机理。由于大麦糊粉的PM不具有可检测的p型atp酶水平，因此将测试由PM中的v型atp酶引起酸化的可能性。将使用v型atp酶的特异性抑制剂来检测体内酸化，并使用相分配法从淀粉粉和黄芪中分离的PM囊泡进行H+转运来研究体外酸化过程。添加GA后，被分离的糊粉层迅速酸化，这表明H+转运蛋白在该组织中组成性表达。我们将使用特异性cDNA和抗体探针检测糊粉和刀柄中PM H+ATPase RNA和蛋白l的水平，并通过实验确定GA和ABA是否调控这些转运体基因的表达。由于稻壳中含有内源性气体，因此我们将在缺乏GA的大麦突变体中研究GA在调节该组织酸化中的作用。由于糊粉细胞分泌大量的H+，它们必须具有维持电荷平衡的机制。利用高效液相色谱的培养介质，探索糊粉和鳞状细胞中通过有机和无机阴离子的合成和输出来实现电荷平衡的可能性。由于本课题的目标之一是了解H+在协调萌发籽粒活动中的作用，因此将利用31P核磁共振波谱技术监测完整萌发籽粒胚乳的pH变化。胚乳pH值在调节胚乳动员中起关键作用的假设将通过核磁共振来检测暴露于已知抑制发芽或幼苗生长的胁迫或暴露于ABA和GA的谷物中的pH值进行验证。如果胚乳pH值被证明是谷物对胁迫反应的可靠和早期指标，将设计一种测量该参数的简单试验。这项研究的成功完成将有助于了解植物生理过程的基础知识，并为谷物作物种植者提供实用的信息。＊＊＊

9306355 Jones The goals of this proposal are to understand how the activities of the aleurone, starchy endosperm and scutellum of germinating cereal grains are integrated. The mobilization of the starchy endosperm of cereal grains, a tissue that consists entirely of dead storage cells, will be studied, and a simple model in which pH plays a key role in the regulation of endosperm mobilization will be tested. This model is based on the observation that low pH is essential for the synthesis and secretion of hydrolytic enzymes by the aleurone and scutellum, for the activity of these enzymes in the endosperm, and for the uptake of sugars and amino acids produced by the activity of secreted hydrolases. Experiments are proposed that have been designed to test the hypothesis that acidification of the endosperm is brought about by proton pumps located at the plasma membrane (PM) of aleurone and scutellum cells. The capacity of the aleurone and scutellum to acidify the external medium will be evaluated by incubating these tissues in unbuffered and buffered media in the presence of gibberellic acid (GA) and abscisic acid (ABA). Acidification of the incubation medium will be monitored using pH electrodes and by titration. The mechanism of acidification of the endosperm by aleurone and scutellum will be examined. Since the PM of barley aleurone does not possess detectable levels of a P-type ATPase, the possibility that acidification is brought about by a V-type ATPase at the PM will be tested. Specific inhibitor of V-type ATPases will be used to examine acidification in vivo, and H+ transport by PM vesicles isolated from aleurone and scutellum by phase partitioning will be used to study the process in vitro. Acidification by isolated aleurone layers occurs rapidly after addition of GA, an observation that indicates that H+ transporters are constitutively expressed in this tissue. Specific cDNA and antibody probes will be used to measure PM H+ATPase RNA and protein l evels in aleurone and scutellum, and experiments will be carried out to establish whether GA and ABA regulate the expression of genes for these transporters. Because the scutellum contains endogenous GAs, the role of GA in regulating acidification in this tissue will be examined in GA deficient mutants of barley. Since aleurone cells secrete large amounts of H+, they must possess a mechanism for maintaining charge balance. The possibility that charge balance in aleurone and scutellum cells is achieved by the synthesis and export of organic and inorganic anions will be explored using high performance liquid chromatography of incubation media. Since one of the goals of this proposal is to understand the role played by H+ in coordinating the activities of germinating grain, 31P NMR spectroscopy will be used to monitor pH changes in the endosperm of intact germinating grain. The hypothesis that endosperm pH plays a key role in regulating endosperm mobilization will be tested using NMR to monitor pH in grain exposed to stresses known to inhibit germination or seedling growth or exposed to applied ABA and GA. If endosperm pH proves to be a reliable and early indicator of the response of the cereal grain to stress, a simple test for measuring this parameter will be devised. The successful completion of this research will contribute to fundamental knowledge about physiological processes in plants as well as well as provide information of a practical nature to growers of cereal crops. ***