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

质子在谷物发芽过程中整合胚乳和胚胎活性的作用

• 批准号: 9306355
• 负责人: Russell Jones
• 金额: $ 33万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9306355&HistoricalAwards=false
• 关键词: Role; Protons; Integrating; Activities; Endosperm

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. ***

9306355琼斯这项建议的目标是了解糊粉、淀粉胚乳和萌发谷物的盾片的活动是如何整合的。将研究谷类淀粉胚乳的动员，这种组织完全由死亡的储存细胞组成，并将测试一个简单的模型，在该模型中，pH在胚乳动员的调节中起关键作用。这个模型是建立在以下观察基础上的：低pH对于糊粉和盾片水解酶的合成和分泌、胚乳中这些酶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。为了验证胚乳酸化是由糊粉和盾片细胞质膜(PM)上的质子泵引起的这一假说，提出了一些实验。糊粉和盾片酸化外部介质的能力将通过将这些组织培养在含有赤霉酸(GA)和脱落酸(ABA)的无缓冲和缓冲介质中来评估。将使用pH电极和滴定来监测培养介质的酸化情况。我们将研究糊粉和盾片酸化胚乳的机制。由于大麦糊粉的质膜不具有可检测到的P型ATPase水平，因此将测试PM中V型ATPase引起酸化的可能性。V型ATPase的特异性抑制剂将被用来研究体内的酸化作用，从糊粉和盾片中分离的质膜小泡的H+转运将被用来研究体外的酸化过程。分离的糊粉层在加入GA后迅速发生酸化，这一观察表明H+转运体在该组织中有结构性表达。我们将用特异的基因和抗体探针来检测糊粉和盾片中质膜H+-ATPase的RNA和L蛋白的水平，并将进行实验以确定GA和脱落酸是否调节这些转运蛋白的基因表达。由于盾片含有内源GAs，GA在调节该组织酸化中的作用将在大麦GA缺失突变体中进行检测。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。在糊粉和盾片细胞中，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将被用孵育介质的高效液相色谱来探索。由于这一建议的目的之一是了解H+在协调发芽谷物活动中所起的作用，因此将使用31P核磁共振波谱来监测完整发芽谷物胚乳的pH变化。胚乳pH在调节胚乳运动中起关键作用的假说将被用核磁共振来检验，以监测暴露在已知抑制萌发或幼苗生长的胁迫或暴露于施加的ABA和GA的谷物中的pH。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，将设计一种简单的测试来测量这一参数。这项研究的成功完成将有助于了解植物生理过程的基本知识，并为谷类作物的种植者提供具有实用性质的信息。***