Stomatal movement is a dynamic process that is regulated by environmental and hormonal cues. In response to drought stress, stomatal closure is mediated by the hormone abscisic acid (ABA). Upon binding to its receptor ABA rapidly induces accumulation of reactive oxygen species (ROS), as detected with fluorescent dyes and ROS biosensors in Arabidopsis guard cells, which precedes changes in stomatal aperture. Moreover, these sensors and reporters show that mutants with decreased synthesis of flavonol antioxidants have enhanced rate of ABA-dependent guard cell closure and increased ROS. Flavonol mutants also exhibit increased quantities of unidentified punctate structures, which display the most striking ROS increase of any organelle. Arabidopsis mutants with defects in plasma membrane respiratory burst oxidase homolog (RBOH) enzymes RBOHD and RBOHF have impaired guard cell closure and reduced ABA-induced ROS accumulation. However, we detect ABA-induced ROS production in the cytoplasm, chloroplasts, peroxisomes, nucleus, and endomembrane puncta, some of which do not coincide with plasma membrane localized RBOHs. We are asking whether ROS is produced by RBOH enzymes in sites other than the plasma membrane or by alternate mechanisms. Monitoring the colocalization of RBOHD-GFP with the plasma membrane dye FM4-64 raises the possibility that these puncta contain internalized RBOH enzymes. We also test the identity of these punctate structures and how RBOHs and flavonol levels regulate them using mutant and transgenic lines containing organelle-specific reporters. Together, these results allow us to determine how guard cell ROS signals are temporally and spatially communicated during drought stress.
Coauthors: Xiaotian Jiao – Wake Forest University;Gloria Muday – Wake Forest University