OsHK3 Acts As A Key Regulator In ABA-induced Antioxidant Defense And Mediates The Production Of H₂O₂ In ABA Signaling

Drought and soil salinity are among the most damaging abiotic stresses affecting today's agriculture. Under water stress conditions, plants accumulate increased amounts of ABA, which stimulate stomatal closure, changes in gene expression, the accumulation of osmo-compatible solutes, and the up-regulation of antioxidant defense systems, thus increasing the plant's capacity to cope with stress conditions. Reactive oxygen species (ROS) is a universal second messenger and acts as a mediator of stimulus-response coupling in the regulation of ABA-induced antioxidant defence. In plants, the two-component system (TCS) has been implicated in regulating various cellular processes responsive to extracellular stimuli, in which Phosphorylation is mediated by two-component system (TCS) or histidine-to -aspartate (His-Asp) phosphorelays. A recent study shown that ATHK1 participated in the process of ABA-induced stomatal closure and ATHK1 functions downstream of H2O2 to regulates calcium channel and adjustes Ca2+ concentration in guard cells. However, it is unknown whether HK is involved in ABA-induced antioxidant defense and, if so, what the relationship between HK and other components in ABA signaling is. Work from our lab shown that OsHK3 which responsed significantly in the expressions induced by ABA, was an ABA-regulated OsHK of the OsHK gene family (OsHK1-6), and H2O2 is required for ABA-induced increase in the expression of OsHK3 under water stress.In view of the above-mentioned facts, in this study, to investigate whether OsHK3 regulates the expression of several important genes involved in ABA-induced antioxidant defense, the transient gene expression analysis and the transient RNAi test in rice protoplasts were used. Our results indicated that transient expression-of OsHK3 in protoplasts resulted in significant increases in the expression of MAPK (OsMPK1, OsMPKS),OsDMI3, and TF (transcription factor) genes such as ZFP36 OsHsfA2, and transient silencing of OsHK3 markedly reduced the expression of these genes, when compared with the controls. Alternatively, OsDMI3 or OsMPK1 knockout did not affect the expression of OsHK3 under the control condition, also did not affect the ABA-induced increase in the expression of OsHK3. Moreover, a similar result was also observed in the protoplasts transiently expressing OsDMB, OsMPK1 and OsMPK5. These results suggested that OsMPKl, OsMPK5 and OsDMI3 do not mediate the ABA-induced up-regulation in the expression of OsHK3 in rice.To determine whether OsHK3 functions upstream of OsDMI3 and OsMPK1 in the regulation of antioxidant enzyme activity, the protoplasts of OsDMI3 and OsMPK1 mutants were isolated and a transient gene expression analysis in the protoplasts was conducted. in the protoplasts of OsDMI3 mutant or OsMPK1 mutant, the transient expression of OsHK3 could hardly induce the increases in the activities of SOD and APX, compared with the controls. ABA-induced increases in the activities of SOD and APX were also inhibited in the protoplasts of OsDMI3 and OsMPK1 mutants and in the protoplasts of OsDMI3 and OsMPKl mutants in combination with the transiently expressed OsHK3. These results suggest that in ABA-induced antioxidant defence, OsHK3-mediated increases in the activities of SOD and APX were blocked by the loss-of-function of OsDMI3 and OsMPKl; obviously, OsHK3 acts upstream of OsDMI3 and OsMPK1 to regulate the activities of antioxidant enzymes in ABA signaling.Early studies from our lab shown that ABA-induced H2O2 production activates CCaMK and MAPKs, which can amplify H2O2 signal by regulating the activity of NADPH oxidase in ABA signaling. To investigate whether OsHK3 is involved in ABA-induced H2O2 production, the protoplasts transfected with dsRNA against OsHK3 were used and H2O2 production in the protoplasts was monitored using the fluorescent probe 2',7'-dichlorofluorescein diacetate (H2DCF-DA). The RNAi silencing of OsHK3 in the protoplasts not only reduced the H2O2-mediated fluorescence under the control condition, but also blocked the ABA-induced increase in the fluorescence. Moreover, the expression of OsrbohB and OsrbohE was analyzed in protoplasts transiently expressing OsHK3 or silencing OsHK3 resulted in that the expression of OsrbohB and OsrbohE was higher in the protoplasts with transiently expressed OsHK3, and the expression of these genes was lower in the protoplasts with transiently silenced OsHK3 than that in the protoplasts transfected with the empty vector. These results suggested OsHK3 regulated the expression of the NADPH oxidase genes, OsrbohB and OsrbohE, and the production of H2O2 in ABA signaling.