NASA/NSF Collaborative Research: Mechanosensing in Arabidopsis: A Genetic Analysis Based on Mechano-Inducible Luminescence

小行星9416025 该建议描述了一种对拟南芥中的机械传感的分子分析的遗传方法。 机械力对植物对环境的反应的重要性历来被大大低估，在基础植物研究中对这种环境刺激的关注相对较少。 然而，许多观察指出，机械刺激在植物中对于模式形成和对环境的适应性反应都具有根本的重要性。 虽然已经在许多物种中进行了这些效应的生理学研究，但尚未描述所涉及的机械感觉通路的分子和遗传表征。 这样的分析将不仅有助于理解机械感觉信号，而且还有助于理解该途径如何与其他环境刺激的转导途径交叉和整合。 这个实验室以前已经发现了一种快速的分子反应--TCH（“触摸”）基因的诱导- -以及拟南芥中机械刺激的形态发生效应。 最近，它已被证明与转基因含aqueorin的幼苗，像tabbella，拟南芥也响应机械刺激与胞质Ca 2+的立即和短暂的elevatimn。 Ca ~（2+）可能作为第二信使将机械刺激转导到核基因激活事件中，这一点得到以下观察结果的支持：外源加入的Ca ~（2+）可以诱导培养细胞中TCH基因的表达，并且五个鉴定的TCH基因中有三个编码具有预测的Ca ~（2+）结合结构域的蛋白质，其中一个是钙调素。 也有可能这些分子机械反应（Ca 2+升高和TCH基因激活）中的一个或两个可以调节随后的发育事件，例如抑制伸长和延迟开花。 已经建立了两类转基因拟南芥植物，每一种都将这些早期机械响应之一转化为光发射响应。 一类，含有TCH 4：荧光素酶（Luc）基因融合，监测TCH 4基因激活;另一类，含有组成型表达的CaMV 35 S：水蛋白基因融合，监测胞浆CA 2+升高。 低光成像系统已经建立，并确定实验条件，以允许识别机械传感突变体异常发光表型。 这样的突变体将允许在机械感知中的早期信号事件的特异性靶向，而基于对机械刺激的改变的形态发生反应鉴定的突变体通常在执行许多信号通路共有的基本发育反应（例如细胞伸长）方面有缺陷。 在这些筛选中鉴定的机械感觉突变体将进行遗传和生理分析，以开始建立细胞内信号传导回路的图谱，同时努力绘制和克隆基因，以启动机械感觉途径的分子表征。 %%% 这项研究旨在扩大我们对生物体如何在称为“机械感知”的过程中感知和响应物理（也称为“机械”）刺激的理解。 虽然机械感知的性质通常与听觉、平衡、本体感觉和触觉中涉及的专门感觉细胞有关，但实际上，它是细胞在被触摸、摩擦、弯曲、拉伸、压缩或振动时表现出的一般性质。 这种刺激既可以是外部的，如风雨使植物和树木弯曲，也可以是内部的，如血液有节奏地脉动血管系统的壁，并且可以深刻地影响植物和动物的生长、发育和功能。 在人类和其他哺乳动物中，机械感知关键地涉及由重力对骨骼的压缩力引起的骨形成过程;在正常和病理性骨形成过程中，机械感知是骨形成过程的关键。（例如动脉粥样硬化）血管衬里对流动血液产生的剪切力的反应;通过拉伸肺细胞产生表面活性剂，这减轻了肺扩张的工作;改变骨骼肌的结构和特性以响应拉伸，或响应血压升高而使过度劳累的心脏增大（心脏肥大）;以及松弛扩张的膀胱内壁的平滑肌。 至于植物，园艺家和农民早就认识到，风引起的弯曲会降低作物的大小和产量，而机械刺激通常会减缓植物的发育。 已经进一步表明，机械扰动影响植物对许多其他环境刺激（例如重力、光、冷和干旱胁迫）的响应。 甚至一个植物分化成它的特征形式也可以通过对发育中的植物施加外部物理限制来改变。 这项研究计划利用模式遗传生物，拟南芥（一种小型芥菜植物），解剖机械感觉过程。 它已经被改造成具有一种新的表型，由此它响应机械刺激治疗而发光。 这种容易测定的反应应该能够快速鉴定在机械感觉途径的组分中具有突变的植物，并且将允许对负责这种细胞性质的细胞机器进行分子表征。 到目前为止，在植物和动物细胞的mechanosensory过程中发现的明显相似之处表明，从这项调查中获得的mechanosensing的更充分的知识可能具有生物医学和农业效益。 ***

9416025 Davis This proposal describes a genetic approach towards the molecular analysis of mechanosensing in Arabidopsis. The importance of mechanical forces on plant responses to the environment have historically been greatly under-appreciated and relatively little attention is given to this environmental stimulus in basic plant research. However, numerous observations point to the fundamental importance of mechano-stimulation in plants both for pattern formation and for adaptive responses to the environment. Although physiological studies of these effects have been carried out in a number of species, molecular and genetic characterization of the mechanosensory pathway(s) involved have not been described. Such an analysis would develop an understanding of not only mechanosensory signaling, but also of how this pathway intersects with and integrates with transduction pathways from other environmental stimuli. This laboratory has previously discovered both a rapid molecular response -- induction of the TCH ("touch") genes - - and morphogenetic effects of mechano-stimulation in Arabidopsis. More recently, it has been shown with transgenic aqueorin-containing seedlings that, like tabacco, Arabidopsis also responds to mechano-stimulation with an immediate and brief elevatimn of cytosolic Ca2+ . That Ca2+ may act as 2nd messenger in transducing mechanical stimuli into nuclear gene activation events is supported by the observations that exogenously added Ca2+ can induce TCH gene expression in cultured cells and that three of the five identified TCH genes encode proteins with predicted CA2+ - binding domains, one being calmodulin. It is also possible that either or both of these molecular mechano-responses (Ca2+ elevation and TCH gene activation) may regulate subsequent developmental events, e.g. inhibition of elongation and delay of flowering. Two classes of transgenic Arabidopsis plants have been created, each of which translates one of these early m echano-responses into a light emission response. One class, containing a TCH4:luciferase (Luc) gene fusion, monitors TCH4 gene activation; the other, containing a constitutively expressed CaMV 35S:aqueorin gene fusion, monitors cytosolic CA2+ elevation. A low-light imaging system has been set up, and experimental conditions determined, to permit identification of mechanosensory mutants with aberrant luminescent phenotypes. Such mutants will allow the specific targeting of the early signaling events in mechanoperception, whereas mutants identified on the basis of altered morphogenetic responses to mechano-stimulation may often be defective in executing basic developmental responses (e.g. cell elongation) common to many signaling pathways. Mechanosensory mutants identified in these screens will be subjected to genetic and physiological analysis to begin establishing maps of signaling circuitry within the cell, while efforts to map and clone the genes are undertaken to initiate a molecular characterization of the mechanosensory pathway. %%% This research aims to expand our understanding of how organisms sense and respond to physical (also referred to as "mechanical) stimulation in a process known as "mechanosensing". Although the property of mechanosensing is usually associated with the specialized sensory cells involved in hearing, balance, proprioception and touch, it is in fact a general property exhibited by cells when they are touched, rubbed, flexed, stretched, compressed or vibrated. Such stimulation can arise both externally, as in the flexing of plants and trees by wind and rain, or internally, as in the rhythmic pulsing of blood against the walls of the vascular system, and can profoundly influence the growth, development and functioning of plants and animals alike. In humans and other mammals, mechanosensing is critically involved in the bone-building processes elicited by the compressive force of gravity on the skeleton; in the normal and pathological (e.g. atherosclerotic) responses of the vascular lining to the sheer forces generated by flowing blood; in the production of surfactant by stretching lung cells, which eases the work of lung expansion; in modifying the structure and properties of skeletal muscle in response to stretching, or the enlargement of an over-worked heart (cardiac hypertrophy) in response to elevated blood pressure; and in the relaxation of smooth muscle lining a distending bladder. As for plants, horticulturalists and farmers have long appreciated that wind-induced flexing decreases both the size and yield of crop plants, and that mechanostimulation in general slows the development of plants. It has been shown further that mechanical perturbation affects a plant's responses to many other environmental stimuli, such as gravity, light, cold and drought stress. Even a plant's differentiation into its characteristic form can be altered by imposing external physical constraints on the developing plant. This research program utilizes the model genetic organism, Arabidopsis ( a small mustard plant), to dissect the mechanosensory process. It has been engineered so as to have a novel phenotype whereby it emits light in response to mechanostimulatory treatments. This easily assayable response should enable the rapid identification of plants which have mutations in components of the mechanosensory pathway, and will permit the molecular characterization of the cell machinery responsible for this cellular property. Apparent similarities so far uncovered in the mechanosensory processes of plant and animal cells suggest that the fuller knowledge of mechanosensing gained from this investigation may well have biomedical as well as agricultural benefits. ***