Hormone Receptors and Signal Transduction Pathways in Plants

9303877琼斯，这项研究的目标是定位大麦糊粉层中赤霉素(GAS)和脱落酸(ABA)的受体，并识别和测试在该细胞中传递激素信号的分子的作用。当单个分泌的原生质体被嵌入含有淀粉的琼脂糖基质中时，可以看到α-淀粉酶的分泌。这种分泌反应提供了一种快速检测GA和ABA效应的方法。初步实验表明，微量注射GA或ABA的细胞不会通过改变它们的分泌速度来响应这些激素，但当GA或ABA出现在细胞表面时，GA刺激α-淀粉酶的分泌，而ABA抑制它的分泌。这些激素受体的细胞位置将被确定。糊粉原生质体也将被用来研究从激素感知到细胞反应的事件。糊粉细胞对GA的第一个可检测反应之一是质子挤出。由于质膜质子泵的质子排出与植物细胞的超极化有关，膜电位测量可作为糊粉细胞对GA和ABA的早期反应的假设将被检验。用荧光染料和共聚焦显微镜对胞浆的pH值进行测绘和定量，以确定pH值是否在信号转导中起作用。利用膜片钳研究质子和膜电位作用于质膜门控离子通道的可能性。钙调蛋白参与糊粉细胞的信号转导。将研究钙调蛋白与GA和ABA的关系，并将使用一种新的荧光探针绘制激活的钙调蛋白在糊粉细胞中的分布。这些实验应该确定，在糊粉细胞中，钙调蛋白基因是否像分泌蛋白基因一样，在转录水平上受到GA和ABA的调控。钙调蛋白活性的映射应阐明钙调蛋白与膜转运蛋白之间的关系，膜转运蛋白的活性受钙调蛋白的调节。钙、钙调蛋白和质子等分子在信号转导中的作用将通过以下问题来测试：在糊粉细胞中，是否可以通过引入信号通路的组件作为激素的激动剂或拮抗剂来模拟GA或ABA的信号转导途径。这项研究的目标是了解植物对激素信号的感知和转导。已经开发出一些技术来检验这一假设，即某些激素是在细胞膜上感知的，而感知产生的信号会影响细胞内蛋白质的活性。钙、钙调蛋白和质子等分子在信号转导中所起的作用将使用各种生化和生物物理技术进行测试。这些结果将提供关于受体的性质和将GA和ABA与谷类糊粉细胞的反应联系起来的信号转导途径的基本新知识。***

9303877 Jones The objectives of this research are to localize the receptors for gibberellins (GAs) and abscisic acid (ABA) in the aleurone layer of barley and to identify and test the roles of the molecules that transduce hormonal signals in this cell. Alpha amylase secretion can be visualized when single, secreting protoplasts are embedded in an agarose matrix containing starch. This secretory response provides a rapid assay for the effects of GA and ABA. Preliminary experiments show that cells microinjected with GA or ABA do not respond to these hormones by changing their rate of secretion, but when GA or ABA are presented to the cell surface, GA stimulates alpha-amylase secretion and ABA inhibits it. The cellular location of the receptors for these hormones will be determined. Aleurone protoplasts will also be used to examine the events that lead from hormone perception to cellular responses. One of the first detectable responses to GA in the aleurone cells is proton extrusion. Since proton extrusion via the plasma membrane proton pump is associated with hyperpolarization of plant cells, the hypothesis that membrane potential measurements can be used as an assay for the early response of aleurone cells to GA and ABA will be tested. The pH of the cytosol will be mapped and quantitated fluorescent dyes and confocal microscopy to determine whether pH plays a role in signal transduction. The possibility that protons and membrane potential act to gate ion channels in the plasma membrane will be explored using patch clamp. Calmodulin has been implicated in signal transduction in the aleurone cell. The relationship between calmodulin and GA and ABA will be studied, and the distribution of activated calmodulin in aleurone cells will be mapped using a new fluorescent probe. These experiments should establish whether the calmodulin gene, like the genes for secretory proteins, are regulated by GA and ABA at the transcriptional level in aleurone cells. The m apping of calmodulin activity should clarify the relationship between calmodulin and membrane transporters whose activities are regulated by calmodulin. The roles that molecules such as calcium, calmodulin, and protons play in signal transduction will be tested by asking whether the signal transduction pathway from GA or ABA can be mimicked in aleurone cells by the introduction of components of the signaling pathway that act as agonists or antagonists of the hormones. %%% The goals of this research are to understand the perception and transduction of hormonal signals in plants. Techniques have been developed to test the hypothesis that some hormones are perceived at the cell membrane, and perception generates signals that affect the activity of proteins within the cell. The roles that molecules such as calcium, calmodulin, and protons play in signal transduction will be tested using a variety of biochemical and biophysical techniques. The results will provide fundamental new knowledge about the nature of the receptors and signal transduction pathways linking GA and ABA to the response of the cereal aleurone cell. ***