Hormone Receptors and Signal Transduction Pathways in Plants

9303877 Jones本研究的目的是定位大麦淀粉粉层中赤霉素（GAs）和脱落酸（ABA）受体，并鉴定和测试这些分子在该细胞中转导激素信号的作用。α -淀粉酶的分泌可以被观察到，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。这种分泌反应为GA和ABA的作用提供了一种快速测定方法。初步实验表明，微注射GA或ABA的细胞对这些激素的分泌速度没有反应，但当GA或ABA呈现在细胞表面时，GA刺激α -淀粉酶分泌，而ABA抑制α -淀粉酶分泌。这些激素受体的细胞位置将被确定。糊粉原生质体也将用于检查从激素感知到细胞反应的事件。在糊粉细胞中对GA的第一个可检测的反应是质子挤压。由于质子通过质膜质子泵的挤压与植物细胞的超极化有关，因此膜电位测量可用于测定糊粉细胞对GA和ABA的早期反应的假设将得到验证。细胞质溶胶的pH将被绘制和定量荧光染料和共聚焦显微镜来确定pH是否在信号转导中起作用。利用膜片钳探讨质子和膜电位作用于质膜中离子通道的可能性。钙调素参与糊粉细胞的信号转导。研究钙调蛋白与GA和ABA之间的关系，并利用新型荧光探针绘制活化钙调蛋白在糊粉细胞中的分布。这些实验将确定钙调蛋白基因是否像分泌蛋白基因一样，在糊粉细胞的转录水平上受到GA和ABA的调控。钙调蛋白活性的测定有助于阐明钙调蛋白与受钙调蛋白调控的膜转运蛋白之间的关系。钙、钙调素和质子等分子在信号转导中所起的作用将通过询问GA或ABA的信号转导途径是否可以通过引入作为激素激动剂或拮抗剂的信号通路成分在淀粉粉细胞中模拟来测试。本研究的目的是了解植物激素信号的感知和转导。一些激素在细胞膜上被感知，并且感知产生影响细胞内蛋白质活性的信号。钙、钙调素和质子等分子在信号转导中的作用将使用各种生化和生物物理技术进行测试。这一结果将为了解受体的性质和连接GA和ABA与谷物糊粉细胞反应的信号转导途径提供新的基础知识。* * *

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. ***