Calcium-Independent Steps in Guard Cell Regulation by Abscisic Acid: The Kinase Connection

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The plant epidermis contains microscopic pores called stomata through which gas exchange with the environment occurs. Through the stomata, carbon dioxide is taken up for photosynthesis and water vapor and oxygen are lost. Stomatal apertures are regulated by pairs of guard cells which border and define the stomatal pores. Guard cells regulate stomatal apertures by osmotic swelling and shrinking, driven by uptake of ions and production of organic solutes (stomatal opening) or loss of ions and catabolism of organic solutes (stomatal closure). The plant hormone abscisic acid (ABA) inhibits stomatal opening and promotes stomatal closure when plants are droughted or otherwise stressed. A few years ago, the PI's laboratory used biochemical methods to identify in guard cells an ABA-activated, Ca2+-independent kinase (ABA-activated protein kinase; AAPK). This serine/threonine kinase is activated within one minute by physiological concentrations of ABA and is detected in guard cells but not in epidermal or mesophyll cells (Li and Assmann (1996) Plant Cell 8: 2359-2368). These characteristics suggested that AAPK could play an important role in triggering the rapid changes in guard cell solute content that drive stomatal closure upon ABA exposure.This project represents a request for additional funding for research related to that of PI's current NSF grant MCB 98-74438, initiated in March of 1999. Under the first year of funding of MCB 98-74438, the PI's laboratory succeeded in cloning the cDNA encoding AAPK, starting from AAPK peptide sequence obtained by mass spectrometric analysis of the purified guard cell protein. The PI's group has shown that biolistic transformation of guard cells with a dominant negative version of AAPK ("AAPK(K43A)") blocks ABA-induced stomatal closure. The PI's laboratory also has shown that AAPK(K43A) inhibits ABA-activation of a class of guard cell anion channels through which anion loss normally occurs during ABA-induced stomatal closure. This research has been published (Li et al., (2000) Science 287: 300-303). The research for which funding is sought under this request is the identification of proteins that interact with AAPK. The following approaches are proposed and prioritized: interaction cloning with labeled AAPK; yeast two-hybrid analysis; immunoprecipitation; and use of mass spectrometric analysis to identify the covalent modification of AAPK that occurs when ABA activates the kinase. Elucidation of the AAPK signal transduction pathway by these methods will increase understanding of hormonally-activated cellular signaling in plants and may provide an entry-point for biotechnological manipulation of stomatal responses to enhance ABA-induced stomatal closure when water is limiting, or to reduce ABA-induced stomatal closure and thus stomatal limitation of photosynthesis when water is abundantly available (e.g. during irrigation).