Signal Transduction in Root Gravitropism

重力是调节植物生长和形态的基本信号。尽管它对植物的成功很重要，但高等植物感知和响应重力信号的细胞和分子事件基本上是未知的。根为研究这些事件提供了几乎独特的优势，因为感知和反应发生在定义明确的空间上不同的区域。据认为，在根中，在根冠的柱细胞中感知重力。然后这些细胞产生转移到生长区的信号。然后，该信号通过可能涉及生长素、H+和Ca 2+的再分配的机制来激发不对称生长。在导致重力感知的小柱细胞中的事件仍然知之甚少。在一个被广泛接受的重力感知初始过程模型（淀粉平衡石假说）中，淀粉体在柱细胞中的沉降启动了导致重力感知的信号系统。然而，帽细胞中重力感知机制的分子组成仍然基本上是未知的。这个信号系统必须将平衡石的沉积转化为编码重力方向的细胞信号。拟南芥根的重力刺激后，细胞质pH值和细胞壁钙离子和pH值的变化迅速发生。这种离子通量的快速感应表明与初始重力感应事件密切相关的离子转运蛋白的激活。抑制Ca 2+或pH值的这些变化也抑制了根的gravesponse，这表明它们是根冠的向重力信号过程进行所需的。因此，本研究的目标是确定如何这些离子通量被激活的重力感应细胞的根冠拟南芥为模型系统。了解重力如何导致这些流量的离子转运蛋白的激活，应该提供洞察的一些初始分子的变化，编码的重力信号在柱细胞的根冠。这一问题的几个途径将被调查：（1）H+，Ca ~（2+）和K ~+流量将监测细胞质，细胞壁和周围的细胞质中的完整的根冠重力刺激根冠。这些离子的变化将在生活中，gravesponding根使用一系列新的，荧光，离子成像探针进行监测。(2)信号传导或离子转运活动的细胞质调节剂的活性，如第二信使和肌动蛋白和微管蛋白细胞骨架，将通过向柱细胞应用抑制剂和激活剂而改变。然后将评估这些因素对抑制或模拟H+和Ca 2+通量的重力调节的影响。(3)由于有大量的证据表明钙调素在离子转运蛋白调节以及重力反应中的作用，钙调素活性将被操纵，其对重力刺激离子通量的影响将被监测。此外，一种新的基于绿色荧光蛋白的钙调蛋白活性指示剂将被用来成像潜在的重力诱导的钙调蛋白激活结构域内的小柱细胞质。(4)为了检验重力感知的淀粉耳石模型，将使用激光镊子来置换非重力刺激的根中的柱细胞中的淀粉体。然后将评估对H+和Ca 2+通量的调节的类重力效应。诱导的重力样激活的柱细胞离子运输的淀粉体置换将强烈支持淀粉平衡石模型的重力感知在拟南芥根冠。这项研究的结果将扩大理解的重力感应机制所使用的植物。特别是，这些调查将有助于确定植物根重力感应系统的初始元素的分子候选人。

Gravity is a fundamental signal that regulates plant growth and form. Despite its importance to plant success, the cellular and molecular events whereby higher plants sense and respond to the gravity signal are essentially unknown. Roots offer an almost unique advantage for studying these events in that sensing and response occur in well defined, spatially distinct regions. It is thought that in the root gravity is perceived in the columella cells of the root cap. These cells then generate a signal that is translocated to the growth zone. This signal then elicits asymmetrical growth through a mechanism that may involve redistributions of auxin, H+ and Ca2+. The events in the columella cells that lead to gravity perception remain poorly understood. In a widely accepted model for the initial process of gravity sensing (the starch statolith hypothesis), the settling of amyloplasts in the columella cells initiates the signaling systems that lead to gravity perception. However, the molecular components of the gravity perception machinery in the cap cells remain essentially unknown. This signaling system must translate sedimentation of statoliths to a cellular signal encoding the direction of gravity. Changes in cytoplasmic pH and columella cell wall Ca2+ and pH have been shown to occur rapidly after gravistimulation of the root of Arabidopsis thaliana. This rapid induction of ion fluxes suggests activation of ion transporters that are closely associated with the initial gravity sensing events. Inhibition of these changes in Ca2+ or pH also inhibits the graviresponse of the root, suggesting they are required for the gravitropic signaling processes of the root cap to proceed. The goal of this research is therefore to define how these ion fluxes are activated in the gravity sensing cells of the root cap using Arabidopsis thaliana as a model system. Understanding how gravity leads to the activation of the ion transporters responsible for these fluxes should provide insight into some of the initial molecular changes that encode the gravity signal in the columella cells of the root cap.Several approaches to this problem will be investigated:(1) H+, Ca2+ and K+ fluxes will be monitored in the cytoplasm, cell walls and medium around columella cells in the intact gravistimulated root cap. These ionic changes will be monitored in living, graviresponding roots using a range of novel, fluorescent, ion imaging probes.(2) The activities of cytoplasmic regulators of signaling or ion transport activities, such as second messengers and the actin and tubulin cytoskeleton, will be altered by application of inhibitors and activators to the columella cells. The effect of these factors on inhibiting or mimicking the gravitational regulation of H+ and Ca2+ fluxes will then be assessed.(3) As there is extensive evidence for a role of calmodulin in ion transporter regulation as well as in the graviresponse, calmodulin activity will be manipulated and its effect on the gravistimulated ion fluxes monitored. In addition, a novel green fluorescent protein-based indicator of calmodulin activity will be used to image potential gravity-induced calmodulin activation domains within the columella cell cytoplasm.(4) In order to test the starch statolith model of gravity perception, laser tweezers will be used to displace amyloplasts in the columella cells in non-gravity stimulated roots. Gravity-like effects on the regulation of H+ and Ca2+ fluxes will then be assessed. Induction of a gravity-like activation of columella cell ion transport by amyloplast displacement would strongly support the starch statolith model for gravity perception in the Arabidopsis root cap.Results from this research will extend the understanding of the gravity sensing machinery used by plants. In particular, these investigations will help identify molecular candidates for the initial elements of the plant gravity sensing system of the root.