Calcium-dependent protein kinases (CDPKs) constitute a specific protein kinase family containing both a kinase domain and a Ca2+ binding domain. There are 34 CDPKs encoded in Arabidopsis genome, but no phenotypes have been published for any loss-of-function cdpk mutants. The preliminary results of the sponsor's lab suggest that double mutants in two guard cell expressed CDPKs (CDPK-GCs) exhibit defects in ABA-induced stomatal closure. The phenotype of this cdpk-gc double mutant in ABA signaling provides a genetic tool to analyze the role of CDPKs in Ca2+ sensing and ABA signaling. [Ca2+]cyt oscillations are essential for long-term stomatal closure. The relative location of CDPK-GCs to [Ca2+]cyt oscillations in ABA signaling pathway will be determined by calcium imaging and stomatal movement analyses. The subcellular localization of CDPK-GCs will be determined by confocal fluorescent microscopy and immunogold electron microscopy. Substrates and interacting proteins of CDPK-GCs will be isolated by yeast two-hybrid assay and tandem affinity purification/mass spectrometry. The long-term goal is to use tagged CDPK substrates to test the hypothesis that CDPKs function as decoders of [Ca2+]cyt oscillations. Understanding the decoding mechanism of [Ca2+]cyt oscillation will contribute to the broad field of Ca2+ signaling including memory study in human.