Abscisic acid-induced apoplastic H2O2 accumulation up-regulates the activities of chloroplastic and cytosolic antioxidant enzymes in maize leaves

The histochemical and cytochemical localization of abscisic acid (ABA)-induced H2O2 production in leaves of maize (Zea mays L.) plants were examined, using 3,3-diaminobenzidine (DAB) and CeCl3 staining, respectively, and the relationship between ABA-induced H2O2 production and ABA-induced subcellular activities of antioxidant enzymes was studied. H2O2 generated in response to ABA treatment was detected within 0.5 h in major veins of the leaves and maximized at about 2-4 h. In mesophyll and bundle sheath cells, ABA-induced H2O2 accumulation was observed only in apoplast, and the greatest accumulation occurred in the walls of mesophyll cells facing large intercellular spaces. Meanwhile, ABA treatment led to a significant increase in the activities of the leaf chloroplastic and cytosolic antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), the O-2 scavenger Tiron and the H2O2 scavenger dimethylthiourea (DMTU) almost completely arrested the increase in the activities of these antioxidant enzymes. Our results indicate that the accumulation of apoplastic H2O2 is involved in the induction of the chloroplastic and cytosolic antioxidant enzymes. Moreover, an oxidative stress induced by paraquat (PQ), which generates O-2 and then H2O2 in chloroplasts, also up-regulated the activities of the chloroplastic and cytosolic antioxidant enzymes, and the up-regulation was blocked by the pretreatment with Tiron and DMTU. These data suggest that H2O2 produced at a specific cellular site could coordinate the activities of antioxidant enzymes in different subcellular compartments.