Cellular Mechanisms of Mechanotransduction in Arabidopsis

科学目标：尽管植物对机械力的反应在塑造它们的进化和形式中起着根本性的作用，但植物生物学家仍然对植物中触摸感知发生的分子机制有一个非常不完整的想法。虽然植物机械传感器的身份是未知的，它似乎很清楚，这样的传感器的机械刺激触发细胞质Ca 2+的快速和短暂的增加。 然后，Ca 2+的这种变化被认为会引发各种反应，从仅限于受刺激细胞的防御反应，到整个器官（如根或芽）的生长变化，甚至影响整个植物的生长习性的变化。 然而，将这些触摸相关的Ca 2+变化与随后的反应联系起来的精确事件还有待确定。来自Gilroy实验室的初步数据表明，这些Ca 2+变化引起信号分子水平的快速和局部变化，如pH和活性氧，这些信号分子反过来在调节随后对机械刺激的发育反应中发挥作用。因此，这项研究计划旨在提供一个更清晰的细胞和分子框架，定义Ca 2+信号在植物机械传感响应中的作用。为了实现这一目标，Gilroy实验室将：（1）确定细胞内机械诱导的Ca 2+变化的空间和时间“指纹"（2）确定机械感知/响应中依赖于Ca 2+的ROS产生的作用（3）确定Ca 2+，pH和生长之间的关系。这些目标将通过产生表达蛋白质的植物来实现，这些蛋白质被设计成可使用光学显微镜检测到的Ca 2+和pH传感器。然后，这些传感器将被用于可视化触摸相关的Ca 2+信号所表现出的空间和时间上的“指纹”。然后通过使用这些Ca 2+响应组分的两种突变体破坏植物中可能的Ca 2+响应元件并评估用破坏这些Ca 2+依赖元件活性的药物处理的植物中的触摸反应来评估这些Ca 2+变化的作用。特别是，在吉尔罗伊实验室这些反应的初步表征意味着，Ca 2+依赖性ROS的生产是这些反应的关键组成部分。因此，Ca 2+作用的机制将在突变体植物中进行探索，其中负责ROS产生的蛋白质已被破坏。 因此，这项研究计划将有助于确定负责触发和协调植物机械感知和反应的细胞和分子信号传导元件。更广泛的影响：除了对植物的机械感觉系统提供基本的了解外，了解这种触摸感测如何操作具有重要的实际意义。作物的耐旱性通常与根系渗透土壤的效率和深度有关。因此，了解根系如何感知和响应土壤障碍和阻抗有可能帮助指导育种和工程策略，以产生更抗旱的农业。该项目还将以研究和教育的强有力整合为特色。博士后研究人员、研究生和本科生将接受现代分子和细胞生物学技术的培训。PI和CoPI致力于增加妇女和代表性不足的少数群体在各级科学中的代表性。这一重点将通过广泛的多样性为导向的方案，如威斯康星州少数民族参与联盟，中西部联盟在科学，技术，工程和数学，三角洲计划和多样性研究所在NSF赞助的教学研究和学习的整合中心提供便利。PI实验室也正在进行翻新，专门为残疾学生设计了完全可用的实验室空间，以促进通过这些方案招募的残疾学生充分参与实验室内的研究。

Scientific goals: Despite the fundamental role of plant responses to mechanical forces in shaping their evolution and form, plant biologists still have a remarkably incomplete idea of the molecular mechanism whereby touch perception occurs in plants. Although the identity of the plant mechanical sensor is unknown, it seems clear that mechanical stimulation of such a sensor triggers rapid and transient increases in cytoplasmic Ca2+. This change in Ca2+ is then thought to trigger reactions ranging from defense responses limited to the stimulated cell, to alterations in growth that extend across entire organs, such as the root or shoot, and even changes in growth habit that affect the whole plant. However, the precise events linking these touch-related Ca2+ changes to subsequent responses also remain to be determined. Preliminary data from the Gilroy Lab suggests that these Ca2+ changes elicit rapid and localized alterations in the levels of signaling molecules such as pH and reactive oxygen species that in turn play roles in regulating the subsequent developmental response to mechanical stimulation. This research program therefore seeks to provide a clearer cell and molecular framework defining the roles of Ca2+ signals in the mechanical sensing response of plants. To achieve this aim, the Gilroy lab will:(1) Define the spatial and temporal ''fingerprints'' of mechanically-induced Ca2+ changes within the cell(2) Define the role of Ca2+-dependent ROS production in mechanical perception/response(3) Define the relationship between Ca2+, pH and growth.These goals will be accomplished through the generation of plants expressing proteins engineered to act as Ca2+ and pH sensors detectable using light microscopy. These sensors will then be used to visualize the ''fingerprints'' in space and time exhibited by the touch-related Ca2+ signals. The role of these Ca2+ changes will then be assessed by disrupting likely Ca2+-responsive elements in the plant through use of both mutants of these Ca2+ responsive components and assessing touch responses in plants treated with drugs that disrupt the activity of these Ca2+-dependent elements. In particular, initial characterization of these responses in the Gilroy Lab implies that Ca2+-dependent ROS production is a key component of these responses. Therefore, the mechanism of Ca2+ action will be probed in mutant plants in which the proteins responsible for ROS production have been disrupted. This research program will therefore help to define the cell and molecular signaling elements responsible for triggering and coordinating plant mechanical perception and response. Broader impact: In addition to providing a fundamental insight into the mechanosensory systems of the plant, understanding how this touch sensing operates has important practical implications. Drought tolerance in crops is often linked to how efficiently and deeply the root system can penetrate the soil. Thus, understanding how roots sense and respond to soil obstacles and impedance has the potential to help direct breeding and engineering strategies to generate more drought resistant agriculture.This project will also feature a strong integration of research and education. Postdoctoral researchers, graduate students and undergraduates will be trained in modern molecular and cell biological techniques. The PI and CoPI are committed to increasing the representation of women and underrepresented minorities at all levels of science. This emphasis will be facilitated through the extensive diversity-oriented programs available at UW Madison such as the Wisconsin Alliance for Minority Participation, the Midwest Alliance in Science, Technology, Engineering and Mathematics, the DELTA program and the Diversity Institute within the NSF sponsored Center for the Integration of Teaching Research and Learning. The PIs laboratory is also being renovated with customized laboratory space designed to be fully useable by disabled students to facilitate full inclusion of the students with disabilities recruited through these programs into research within the laboratory.