Reactive oxygen species (ROS) act as signal molecules for a variety of processes in plants. However, many questions about the roles of ROS in plants remain to be clarified. Here, we report the role of ROS in gibberellin (GA) and abscisic acid (ABA) signaling in barley (Hordeum vulgare) aleurone cells. The production of hydrogen peroxide (H2O2), a type of ROS, was induced by GA in aleurone cells but suppressed by ABA. Furthermore, exogenous H2O2 appeared to promote the induction of α-amylases by GA. In contrast, antioxidants suppressed the induction of α-amylases. Therefore, H2O2 seems to function in GA and ABA signaling, and in regulation of α-amylase production, in aleurone cells. To identify the target of H2O2 in GA and ABA signaling, we analyzed the interrelationships between H2O2 and DELLA proteins Slender1 (SLN1), GA-regulated Myb transcription factor (GAmyb), and ABA-responsive protein kinase (PKABA) and their roles in GA and ABA signaling in aleurone cells. In the presence of GA, exogenous H2O2 had little effect on the degradation of SLN1, the primary transcriptional repressor mediating GA signaling, but it promoted the production of the mRNA encoding GAMyb, which acts downstream of SLN1 and involves induction of α-amylase mRNA. Additionally, H2O2 suppressed the production of PKABA mRNA, which is induced by ABA:PKABA represses the production of GAMyb mRNA. From these observations, we concluded that H2O2 released the repression of GAMyb mRNA by PKABA and consequently promoted the production of α-amylase mRNA, thus suggesting that the H2O2 generated by GA in aleurone cells is a signal molecule that antagonizes ABA signaling.

Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells.

Seed dormancy is one of the adaptive responses in the plant life cycle and an important agronomic trait. Reactive oxygen species (ROS) release seed dormancy and promote seed germination in several cereal crops; however, the key regulatory mechanism of ROS-mediated seed dormancy and germination remains controversial. Here, we focused on the relationship between hydrogen peroxide (a ROS) and abscisic acid (ABA) in dormant and non-dormant barley seeds. The hydrogen peroxide (H2O2) level produced in barley seed embryos after imbibition was higher in non-dormant seeds than in dormant seeds. H2O2 regulated the ABA content in the embryos through ABA-8′-hydroxylase, an ABA catabolic enzyme. Moreover, compared with non-dormant seeds, in dormant seeds the activity of NADPH oxidase, which produces ROS, was lower, whereas the activity of catalase, which is a H2O2 scavenging enzyme, was higher, as was the expression of HvCAT2. Furthermore, precocious germination of isolated immature embryos was suppressed by the transient introduction of HvCAT2 driven by the maize (Zea mays) ubiquitin promoter. HvCAT2 expression was regulated through an ABA-responsive transcription factor (HvABI5) induced by ABA. These results suggest that the changing of balance between ABA and ROS is active in barley seed embryos after imbibition and regulates barley seed dormancy and germination.

NADPH oxidase catalyzes the production of the superoxide anion (O 2 ¡ ), a reactive oxygen species (ROS), and regulates the germination of barley (Hordeum vulgare L.). Diphenyleneiodonium (DPI) chloride, an NADPH oxidase inhibitor, delayed barley germination, and exogenous H 2 O 2 (an ROS) partially rescued it. Six enzymes, ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA20-oxidase (GA20ox) and GA3-oxidase (GA3ox), catalyze the transformation of trans-geranylgeranyl diphosphate to active gibberellin, which promotes germination. Exogenous H 2 O 2 promoted the expressions of HvKAO1 and HvGA3ox1 in barley embryos. These results suggest that ROS produced by NADPH oxidase are involved in gibberellin biosynthesis through the regulation of HvKAO1 and HvGA3ox1.