Molecular Mechanisms for Polar Growth in Pollen Tubes

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The mechanisms by which cell polarity arises are fundamental to development and morphogenesis of multicellular organisms, particularly of plants because their cells are non-motile. The goal of this project is to use tip-growing pollen tubes as a model system to elucidate how polarity arises during plant growth and development. Work from prior NSF funding has demonstrated that a plant-specific Rho family GTPase, Rop, acts as a central switch to control polar growth in pollen tubes. This switch is controlled by a positive feedback loop of Rop activation and recruitment at the plasma membrane that is initiated locally and amplified laterally by unknown mechanisms. This feedback loop is inhibited globally by Rop GTPase activating proteins and guanine nucleotide dissociation inhibitors to generate a tip-focused gradient of active Rop at the plasma membrane. Recent findings indicate that the active Rop specifies the apical plasma membrane domain for tip growth and then activates growth via the regulation of both dynamic tip F-actin and tip-focused cytosolic Ca2+ gradients. Two active Rop interacting proteins (ARIPs) have been identified that may link Rop to tip actin and Ca2+, respectively. These advances have relied heavily on biochemical and cellular Rop signaling assays such as a fluorescence-based Rop activity assay, actin imaging, and complementary Arabidopsis and tobacco pollen tube systems that are suited for a multifaceted functional approach that includes genetics, transient expression of proteins, and microinjection. This research will continue to use a similar approach but also develop new assays and methods to address several significant outstanding questions regarding Rop-dependent mechanisms for polar growth in pollen tubes: 1) What is the functional interplay between Rop, Ca2+ and actin? 2) How does Rop regulate Ca2+ and actin? 3) What is the molecular basis underlying the Rop positive feedback loop? Specific experiments will include identification of Rop effectors and potential factors that may activate and recruit Rop at the plasma membrane using interactive cloning and mutant isolation, alteration of specific components in Rop signaling using genetic and chemical methods, and analysis of changes in Rop recruitment and activation and in signaling targets such as tip actin and Ca2+. This work should uncover key components and mechanisms in Rop-dependent signaling to pollen tube growth and may lead to new insights into how cells control development, polar growth and morphogenesis.