Role of mitochondria and chloroplasts during stomatal closure: Subcellular location of superoxide and H2O2 production in guard cells of Arabidopsis thaliana

Stomatal guard cells are unique in that they have more mitochondria than chloroplasts. Several reports emphasized the importance of mitochondria as the major energy source during stomatal opening. We re-examined their role during stomatal closure. The marked sensitivity of stomata to both menadione (MD) and methyl viologen (MV) demonstrated that both mitochondria and chloroplasts helped to promote stomatal closure in Arabidopsis. As in the case of abscisic acid (ABA), a plant stress hormone, MD and MV induced stomatal closure at micromolar concentration. All three compounds generated superoxide and H₂O₂, as indicated by fluorescence probes, BES-So-AM and CM-H₂DCFDA, respectively. Results from tiron (a superoxide scavenger) and catalase (an H₂O₂ scavenger) confirmed that both the superoxide and H₂O₂ were requisites for stomatal closure. Co-localization of the superoxide and H₂O₂ in mitochondria and chloroplasts using fluorescent probes revealed that exposure to MV initially triggered higher superoxide and H₂O₂ generation in mitochondria. In contrast, MD elevated superoxide/H₂O₂ levels in chloroplasts. However, with prolonged exposure, MD and MV induced ROS production in other organelles. We conclude that ROS production in mitochondria and chloroplasts leads to stomatal closure. We propose that stomatal guard cells can be good models for examining inter-organellar interactions.