9506191 Schroeder Stomatal pores in leaves close to reduce transpirational water loss of plants in response to environmental stress conditions such as drought. Control of stomatal movements by guard cells in response to environmental stimuli and stress conditions is a primary factor in determining water use efficiency and productivity of crop plants. Guard cells provide an ideal system to elucidate early events in higher plant signal transduction. Several key guard cell ion channels have been identified which have been proposed to function as important signal transducers and mediators of stomatal closing. Recent data suggest that both Ca2+- dependent and Ca2+-independent transduction cascades may control stomatal closing. However, the intracellular signaling cascades which link signal reception to ion channel modulation during stomatal closing remain largely unknown. Furthermore, the molecular mechanisms have remained unknown, by which guard cell vacuoles release ions, which is essential for stomata closing. The long term goal of this research is to characterize the chain of events of the signaling cascade which integrates physiological stimuli, such as abscisic acid, intracellular coupling proteins, second messengers, and ion channels to produce stomatal closing. To elucidate signaling mechanism in guard cells, we propose studies using a combination of cell biological, patch clamp, biochemical and genetic analyses. Recent research has led to the model that slow anion channels and outward-recitifying K+ channels in the plasma membrane and newly identified vacuolar K+ (VK) and Ca2+- activated Ca2+-permeable (SV) channels in the tonoplast of guard cells are central transducers and mediators of stimulus-dependent stomatal closing. The regulation and roles of the newly identified VK channels and the proposed Ca2+-induced Ca2+ release by SV channels in guard cell vacuoles will be investigated in detail to test the hypothesis that these vacuolar ion chan nels are crucial for signal transduction and vacuolar ion release during stomatal closure. Furthermore, studies of these vacuolar ion channels together with analyses of rate-limiting slow anion channels in guard cell plasma membrane will be pursued to determine intermediate Ca2+-dependent and putative Ca2+- independent signaling steps which produce the integrated response of stomatal closing. Regulation of guard cell vacuole channels and phosphorylation-dependent plasma membrane slow anion channels will further be studied by using isoforms of the Ca2+-dependent protein kinase (CDPK). In addition, cell biological characterization of guard cell signaling mutants in Arabidopsis will be pursued to elucidate the function of genetic loci within the cascade of events which produced stomatal closing. Stomatal regulation ion abscisic acid-insensitive Arabidopsis mutants (abi1 and abi2) and effects of the purified recombinant ABI1 protein phsophatase on guard cell ion channels will be pursued for enhanced ABA sensitivity and for phenotypic stomatal responses in existing Arabidopsis mutants to identify and characterize additional genetic loci involved in stomatal and ABA signaling. The proposed research will contribute significantly to the understanding of the molecular mechanisms of the signal transduction cascade which produces the integrated response of stomatal closing. These studies may further provide important information for the design of strategies for future engineering of improved water use efficiency in crop plants. %%% Plants "breathe" through tiny openings or "mouths" called stomata which are often found on the underside of leaves. However, a fine balance must be achieved between respiration and loss of water vapor through the stomata. To achieve this balance, the plant is sensitive to a wide variety of environmental stimuli which induce stomatal closure, such as elevated carbon dioxide levels, darkness and the phytohormone abscisic acid (ABA). It is known that stomatal closing is, in part, brought about by ion efflux through ion channels in the outer cell membrane and the membrane which surrounds the vacuole, the tonoplast of the cells which comprise the stomate, the guard cells. This project is concerned with the way the environment stimuli bring about changes in the amount and rate of ion transport through these channels. The channels have been identified. Potassium is released from the vacuole through the tonoplast, into the cytoplasm where it is released into the cell wall via an outward-rectified potassium channel. A rise in intracellular calcium and pH stimulates this release. The regulation and role of the newly-identified vacuolar potassium transporter is explored, as is the coordination of these channels with others at the plasma membrane to produce an integrated response to intracellular calcium signals. The integration of the signaling pathways is also explored using molecular genetics. Mutants which have the phenotype of an altered guard cell response to the phytohormone, ABA, are analyzed in order to identify more genes involved in stomatal opening and ABA signaling. The role of one of these signaling genes, one which encodes a protein phosphatase, is being explored by purifying the protein and determining its direct effect on channel physiology. This project has biotechnical application through the design of crop plants which have improved water use efficiency. ***
