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，因为感知和响应发生在定义明确的、空间上不同的区域。据认为，在根部，重力是在根冠的柱状细胞中感受到的。然后，这些细胞产生一个信号，该信号被转移到生长区。这一信号通过一种可能涉及生长素、H+和钙离子重新分配的机制引起不对称生长。小柱细胞中导致重力感知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(淀粉状态石假说)中，小柱细胞中淀粉体的沉淀启动了导致重力感知的信号系统。然而，帽子细胞中重力感知机制的分子组成基本上仍然未知。这个信号系统必须将雕像的沉积转化为编码重力方向的细胞信号。拟南芥根在重力刺激后，细胞质pH和柱状细胞壁的Ca~(2+)和pH迅速发生变化。这种离子通量的快速诱导表明，与最初的重力探测事件密切相关的离子转运体被激活。抑制这些钙离子或pH的变化也会抑制根部的重力反应，这表明它们是根冠向重力信号过程进行所必需的。因此，这项研究的目标是以拟南芥为模型系统，确定这些离子通量是如何在根冠的重力感应细胞中被激活的。了解重力如何导致负责这些通量的离子转运体的激活，将有助于深入了解根冠柱状细胞中编码重力信号的一些初始分子变化。解决这个问题的几种方法将被研究：(1)在完整的重力刺激的根冠中，H+、Ca~(2+)和K+通量将在柱状细胞周围的细胞质、细胞壁和介质中进行监测。这些离子变化将使用一系列新型的荧光离子成像探测器在活生生的妊娠反应根中进行监测。(2)对柱状细胞应用抑制剂和激活剂将改变信号或离子传输活动的细胞质调节器的活动，如第二信使以及肌动蛋白和微管蛋白细胞骨架。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)由于有广泛的证据表明钙调蛋白在离子转运体调节和重力反应中发挥作用，因此将操纵钙调蛋白的活性并监测其对重力刺激离子通量的影响。此外，一种新型的基于绿色荧光蛋白的钙调蛋白活性指示剂将被用于成像重力诱导的柱状细胞胞浆内潜在的钙调蛋白激活结构域。(4)为了检验重力感知的淀粉定位石模型，将使用激光镊子来置换非重力刺激的根中柱状细胞中的淀粉体。然后将评估重力对H+和Ca~(2+)通量调节的影响。通过淀粉体置换诱导柱状细胞离子转运的重力激活将有力地支持拟南芥根帽中重力感知的淀粉状态石模型。本研究结果将扩大对植物使用的重力传感机制的理解。特别是，这些研究将有助于确定植物根部重力传感系统的初始元件的分子候选者。

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.