Minisymposium 3: Hormones I

Abs # 13001: Dissection of guard cell ABA signal transduction mechanisms using combined single cell-type functional genomics and cell biological approaches

Presenter:	Kwak, June M, jkwak@biomail.ucsd.edu
Authors	Kwak, June M (A)   Leonhardt, Nathalie  (A)   Mori, Izumi  (A)   Bloom, Rachel  (A)   Torres, Miguel A (B)   Dangl, Jeff  (B)   Jones, Jonathan  (C)   Pei, Zhen-Ming  (D)   Robert, Nadia  (A)   Waner, David  (A)   Schroeder, Julian I (A)
Affiliations:	(A): Cell and Developmental Biology Section, Division of Biological Sciences, UC, San Diego, La Jolla, CA 92093-0116 (B): Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280 (C): The Sainsbury Laboratory, John Innes Center, Norwich NR4 7UH, UK (D): Biology Department, Duke University, Durham, NC 27708

Guard cells have been developed for dissecting early signal transduction mechanisms. Relatively few signal transduction components have been identified from recessive ABA insensitive disruption mutants known to function during early ABA signal transduction upstream of transcription. The limited number of genetically identified positive ABA transducers is most likely due to redundancy in genes encoding ABA signaling components. To overcome this limitation and to dissect redundant signal transduction proteins, we have developed an alternative "single cell-type genomics" approach. Comprehensive analyses of microarray experiments with Arabidopsis guard cell and mesophyll cell RNA will be presented, which would provide a basis for cell-type specific genomic scale analyses of gene function. From the microarray results, we identified guard cell-expressed protein phosphatase 2C and protein phosphatase 2A (RCN1) genes. A T-DNA disruption mutation in the protein phosphatase 2C gene confers ABA-hypersensitive regulation of stomatal closing and seed germination. Reactive oxygen species (ROS) have been proposed to function as 2nd messengers in ABA signaling in guard cells (Pet et al., 2000, Nature, 406: 731-734). However, the question whether ROS production is indeed required for ABA signal transduction in vivo has not yet been addressed and the molecular mechanisms mediating ROS production during ABA signaling remain unknown. Two partially redundant Arabidopsis guard cell-expressed NADPH oxidase catalytic subunit genes, AtrbohD and AtrbohF, were identified in which gene disruption impairs ABA signaling. atrbohD/F double mutations impair ABA-induced stomatal closing, ABA promotion of ROS production, ABA-induced cytosolic Ca²⁺ increases and ABA-activation of plasma membrane Ca²⁺-permeable channels in guard cells. Exogenous H₂O₂ rescues both Ca²⁺ channel activation and stomatal closing in atrbohD/F. These data provide direct molecular genetic and cell biological evidence that ROS are rate-limiting second messengers in ABA signaling and that the AtrbohD and AtrbohF NADPH oxidases function in guard cell ABA signal transduction.