Functional Characterization of the Arabidopsis thaliana ARG1 Gene Involved in Gravity Signal Transduction

植物器官使用重力矢量作为环境线索来指导它们的生长。向重力性允许根向下生长到土壤中，在那里它们吸收植物生长和发育所需的水和矿物质离子。它还迫使嫩芽朝着光线向上生长，从而使它们进行光合作用。这一重要的植物生长对环境的反应也使植物在被风和雨的作用后恢复向上生长，并使根在克服生长途径中的障碍后恢复向下生长。在重力场中，特定植物器官方向的变化主要通过在被称为状态细胞的特殊细胞的细胞质中沉积致密的淀粉体来感知。淀粉体的沉积激活了一条信号转导途径，导致生理信号的产生，据信是生长素和/或质外体钙离子梯度穿过重力刺激的器官。然后，该信号被传输到反应部位，在那里它促进了负责曲率发育的不同细胞生长。在根中，重力感应被认为发生在根冠的柱状细胞中，而差异生长反应发生在远端和中央伸长区。关于植物器官将淀粉体沉积产生的物理信息转化为生理信号的分子机制，人们知之甚少。利用拟南芥的分子遗传学方法现在可以进行研究，这应该有助于更好地理解这一过程。ARG1基因的突变导致根和下胚轴向重力性的特殊缺陷。突变器官表现出野生型的生长速度、对植物激素的生长敏感性和野生型的光致反应动力学，强烈表明重力信号转导的早期阶段存在特定的缺陷。ARG1编码一种新的dna J样蛋白，含有一个假定的卷曲结构域，与在许多已知与细胞骨架相互作用的蛋白质中发现的卷曲结构域具有氨基酸相似性。有趣的是，细胞骨架被认为与向重力性的感知阶段有关，但与曲率反应阶段无关。基于这些数据，假设ARG1蛋白与状态细胞中的细胞骨架相互作用，促进淀粉体沉积产生的物理力转化为生理信号。为了验证这一模型，将分析ARG1在拟南芥幼苗中的表达模式，将定义在转基因Arg1植物中完全恢复向重力性所需的最低空间ARG1表达要求，并将研究其向重力性功能所需的ARG1区域。利用原位免疫荧光技术定位ARG1蛋白在根冠小柱细胞和下胚轴细胞中的分布，并将其定位与统计细胞中微管和微丝的分布进行比较。与ARG1不同结构域相互作用的蛋白质将被识别，编码它们的基因将被克隆和鉴定。最后，将确定ARG1类似物并对其进行功能表征。这种遗传学、生理学和免疫细胞学的结合研究将有助于确定arg1和arg1的分子功能(S)。有趣的是，在Conowhabditis elegans中发现了高度保守的ARG1同源基因。这一观察表明，相应的蛋白质在植物和动物的生长、发育和/或对环境的反应中发挥着重要的作用。因此，研究将为更好地理解这种新型的dna J样蛋白在高等真核生物中所发挥的功能提供基础。

Plant organs use the gravity vector as an environmental cue to direct their growth. Gravitropism allows roots to grow downward into the soil where they take up the water and mineral ions required for plant growth and development. It also forces shoots to grow upward toward light, allowing them to photosynthesize. This important plant growth response to its environment also allows plant shoots to resume upward growth after having been prostrated by the action of wind and rain, and roots to resume downward growth after having overcome an obstacle placed in their growth path.A change in the orientation of a specific plant organ within the gravity field is perceived mainly by the sedimentation of dense amyloplasts in the cytoplasm of specialized cells named statocytes. Amyloplast sedimentation activates a signal transduction pathway that result in the production of a physiological signal, believed to be an auxin and/or apoplastic Ca2+ gradient across the gravistimulated organ. That signal is then transmitted to the site of response where it promotes a differential cellular growth responsible for the development of curvature. In roots, gravity sensing is thought to occur in the columella cells of the root cap, while the differential growth response occurs in the distal and central elongation zones.Very little is known about the molecular mechanisms allowing plant organs to convert the physical information derived from amyloplast sedimentation into a physiological signal. A molecular genetic approach using Arabidopsis thaliana now allows investigations that should help to better understand that process. Mutations in the ARG1 gene result in a specific defect in root and hypocotyl gravitropism. Mutant organs show wild-type growth rates, growth sensitivities to phytohormones and develop wild-type kinetics of phototropic response, strongly suggesting a specific defect in the early phases of gravity signal transduction. ARG1 encodes a novel dnaJ-like protein containing a putative coiled coil domain with amino acid similarity with coiled coils found in a number of proteins known to interact with the cytoskeleton. Interestingly, the cytoskeleton has been proposed to be implicated in the sensing phase, but not in the curvature response phase, of gravitropism. Based on these data, it is hypothesized that the ARG1 protein interacts with the cytoskeleton in the statocytes, facilitating the transduction of physical forces derived from amyloplast sedimentation into a physiological signal. To test this model the pattern of ARG1 expression will be analyzed in Arabidopsis thaliana seedlings, minimal spatial ARG1 expression requirements will be defined for complete restoration of gravitropism in transgenic arg1 plants, and the regions of ARG1 that are required for its function in gravitropism will be investigated. In situ immunofluorescence strategies will be used to localize the putative ARG1 protein within the columella cells of the root cap and in hypocotyl cells, and to compare its localization with the distribution of microtubules and microfilaments in statocytes. Proteins that interact with the various domains of ARG1 will be identified, and the genes encoding them will be cloned and characterized. Finally, ARG1 paralogs will be identified and functionally characterized. This combination of genetic, physiological and immunocytological studies will help define the molecular function(s) of ARG1 and ARG1 paralogs. Interestingly, a highly conserved ARG1 ortholog was found in Coenorhabditis elegans. This observation suggests an important role for the corresponding protein in the regulation of plant and animal growth, development and/or responses to the environment. Thus, it is expected that research will provide the foundation for a better understanding of the functions played by this novel type of dnaJ-like protein in higher eukaryotes.