Ion Channel Regulation in Higher Plants

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Stomatal pores in the epidermis of leaves regulate the diffusion of CO2 into leaves for photosynthetic carbon fixation and control water loss of plants via transpiration to the atmosphere. A network of signal transduction mechanisms in guard cells integrate environmental stimuli to regulate stomatal apertures for optimization of plant growth under diverse conditions. Control of stomatal movements in response to environmental stress conditions is an important factor in determining crop productivity and plant water loss during drought. The long-term goal of this project is to achieve a detailed quantitative understanding of the network of signal transduction events that regulate stomatal movements using the easily manipulated plant, Arabidopsis thaliana. Guard cells provide a powerful system for quantitative and time-resolved dissection of the complex molecular machinery mediating specificity in early plant signaling cascades. During the preceding funding period a signal transduction model has been developed, suggesting that several types of both positively- and negatively-regulating protein kinases and PP1- or PP2A-type protein phosphatases play central roles in mediating stomatal closing by the phytohormone, abscisic acid (ABA). However, the genes encoding these protein kinases and PP1/2A-type phosphatases, as well as their locations and functions within cascades and their downstream targets remain largely unknown. From an Arabidopsis guard cell cDNA libraries, the cDNAs that encoded for the receptor-like kinases (RLKs), the Ca2+-dependent protein kinases (CDPKs), and the catalytic PP2A subunit have been isolated.. The large gene families and redundancies of these signal transducers in plants have limited direct genetic characterization of individual kinases and PP2As. Direct functional analyses of guard cell-expressed members will now allow determination of their redundancies and of their precise functions in signal transduction. Preliminary studies show that an insertional mutation in a regulatory PP2A subunit causes recessive ABA insensitivity in guard cells. Furthermore, disruption of a receptor kinase abolishes light-induced stomatal opening, illustrating the importance and feasibility of the proposed research. The following hypotheses will be tested in this project: That both positively- and negatively-regulating protein kinases and PP2As function in ABA-induced stomatal closing. That CDPKs control ABA-induced stomatal closing. That specific PP2As may function either as positive or negative regulators of ABA signaling. To test these, the following specific aims will be done: Insertional disruption mutant alleles will be identified and transgenic repression lines will be generated in the isolated guard cell RLK, CDPK and catalytic PP2A genes. Stomatal movement analyses will be pursued to characterize phenotypic responses to ABA and light stimuli in disruption mutants or double/multi mutants of redundant homologous CDPKs and PP2As. Effects of two to three kinase and PP2A mutations with the strongest stomatal phenotypes will be characterized on stimulus-induced regulation of guard cell anion or K+ channels and signal-induced cytosolic Ca2+ changes. Epistasis analyses with known guard cell signaling mutants will be pursued to determine the relative sequence of events in the guard cell signaling network. Selected CDPKs/PP2As will be analyzed for modulation of cloned guard cell ion channels. Furthermore screens for guard cell-expressed interacting proteins will be pursued for one of the regulators. The knowledge gained from these studies will reveal novel fundamental mechanisms by which a network of kinases and PP2As control signal transduction and mediate signaling specificity in a plant cell. Furthermore, these studies will lead to elucidation of key signal transduction mechanisms by which plants balance CO2 influx into leaves and transpirational water loss and may contribute to future strategies for manipulating gas exchange in plants.