Hormone Receptors and Signal Transduction Pathways in Plants

小行星9303877 本研究的目的是定位大麦糊粉层中赤霉素（GAs）和脱落酸（阿坝）的受体，并鉴定和测试在该细胞中传递激素信号的分子的作用。 当单个分泌原生质体包埋在含有淀粉的琼脂糖基质中时，可以观察到α淀粉酶分泌。 这种分泌反应为GA和阿坝的作用提供了快速测定。 初步实验表明，用GA或阿坝显微注射的细胞对这些激素的反应不是改变它们的分泌速率，而是当GA或阿坝被呈递到细胞表面时，GA刺激α-淀粉酶的分泌，而阿坝抑制它。 糊粉原生质体还将用于检查从激素感知到细胞反应的事件。 糊粉层细胞中对GA的第一个可检测的反应之一是质子挤出。 由于质子挤出通过质膜质子泵与植物细胞的超极化，假设膜电位测量可以用作测定糊粉细胞对GA和阿坝的早期反应将被测试。 将绘制胞质溶胶的pH值，并对荧光染料和共聚焦显微镜进行定量，以确定pH值是否在信号转导中起作用。 质子和膜电位对质膜离子通道的门控作用的可能性将用膜片钳技术来探索。 钙调素参与糊粉细胞的信号转导。 本研究将研究钙调素与GA和阿坝的关系，并利用一种新的荧光探针定位糊粉层细胞中激活的钙调素的分布。 这些实验应确定是否钙调素基因，如分泌蛋白的基因，在糊粉细胞的转录水平上由GA和阿坝调节。 钙调素活性图谱的建立有助于阐明钙调素与受钙调素调控的膜转运蛋白之间的关系。 分子，如钙，钙调蛋白，和质子在信号转导中发挥的作用将被测试，询问是否从GA或阿坝的信号转导途径可以模仿糊粉细胞中的信号转导途径的成分，作为激素的激动剂或拮抗剂的引入。 本研究的目的是了解植物激素信号的感知和转导。 已经开发了一些技术来测试这样的假设，即一些激素在细胞膜上被感知，感知产生影响细胞内蛋白质活性的信号。分子，如钙，钙调蛋白和质子在信号转导中发挥的作用将使用各种生物化学和生物物理技术进行测试。这些结果将为研究GA和阿坝与糊粉细胞反应的受体性质和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. ***