Cellular Mechanisms of Mechanotransduction in Arabidopsis

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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.