Functional Analysis Of C2H2-Type Zinc Finger Protein ZFP182and ZFP36in ABA-Induced Antioxidant Defense In Rice

The adaptability of plants especially crops to abiotic stress such as drought, cold and high salinity has a very significant impact for the production and quality of crops. Drought is the most common form of plant stresses and is critical bottleneck to the development of agriculture in many countries. The accumulation of the plant hormone abscisic acid (ABA) plays important roles in the responses to water stress. ABA induced by water stress can cause an enhancement in the generation of ROS and antioxidant defences to enhance plant tolerance to water stress. In this signal network, a number of transcription factors such as MYB, WRKY, heat shock factor and multiple types of zinc finger protein content increased, they participate in maintaining stable levels of ROS. So that, these transcription factors play important regulation in plant responses to abiotic stresses. However, it is not clear whether these members of C2H2-type ZFPs are involved in the ABA-induced antioxidant defense and if so, what the relationship between the C2H2-type ZFPs, H2O2, MAPK and NADPH oxidase in the ABA signaling is.In this study, two ABA-and H2O2-responsive C2H2-type ZFP genes, ZFP36and ZFP182were identified in rice. By protoplast transient expression system and transgenic technology, we made the targen gene over-expression or silence. The enzyme activity and gene expression of antioxidant enzymes, NADPH oxidase and MAPK were investigated in transgenic rice and wild type rice to explore the mechanism of ZFP36and ZFP182in rice stress tolerance. It has an important theoretical significance to improve rice resistance. The results as follows:In this study, two zinc finger protein genes ZFP182and ZFP36was identified from rice (Oryza sativa Japonica) by bioinformatics method and RT-PCR approach which are clustered with Zat12and Zat10respectively. Sequence analysis showed that ZFP182and ZFP36both contained two typical C2H2zinc finger domains. ZFP182encodes a18.215kDa polypeptide with170amino acids and ZFP36encodes a22.804kDa polypeptide with 220amino acids. It was found by subcellular localization, the two zinc finger protein genes are located in the nucleus. Real-time quantitative RT-PCR analysis showed that:Treatments with ABA and H2O2, a biphasic response in the expression of ZFP182and ZFP36was observed. Pretreatment with reactive oxygen species scavengers (DMTU) substantially arrested ABA-induced gene expression. NO can also up-regulate the expression of ZFP182and ZFP36. These results suggest that ABA, H2O2and NO can increase ZFP182and ZFP36gene expression and H2O2is required for the ABA-induced up-regulation expressions of ZFP182and ZFP36in rice.For further study of the function of ZFP182and ZFP36, overexpression (sense) vector, antisense inhibition (anti sense) expression vector, and RNA interference (sense/antisense, dsRNA) expression vector of ZFP182and ZFP36gene were constructed, and successfully transferred to rice callus, using agrobacterium-mediated transformation approach. We have get the RNA interference plants of ZFP182; overexpression and antisense suppression and RNA interference plants of ZFP36. We used F1generation of transgenic rice and the wild type rice. Respectively as materials, exogenous ABA could increase intracellular SOD and APX activity. Compare with the wild type, SOD and APX activity of the transgenic plants which were RNA interfered or antisense inhibited decreased significantly. By protoplast transient expression system, we found that overexpression of ZFP182and ZFP36gene could increase the SOD and APX activity, while interfering the two zinc finger protein, SOD and APX activity decrease significantly. After ABA treatment, SOD and APX activity were increased slightly. These results suggest that ZFP182and ZFP36not only participate but also play an important role in ABA-induced antioxidant protection. It is significant to enhance plant antioxidant protection and increase stress tolerance.MAPK cascade in the eukaryotic system play a pivotal role in the downstream processes that exogenous stimuli into intracellular responses receptors/sensors. MAPK cascade also involve ABA signaling pathway in plants. In this study, we use pharmacological approaches and quantitative RT-PCR technique to discuss the relationship of MAPK and ZFP182/ZFP36gene transcription between the ABA signaling pathway in leaves of rice seedlings. MAPKK inhibitor (PD98059and U0126) pretreatment can effectively inhibit the ABA-induced gene transcription of ZFP182and ZFP36. In rice protoplasts, ABA treatment induced the expression of ZFP36and ZFP182, and the ABA-induced up-regulation in the expression of ZFP36and ZFP182was almost fully blocked in the protoplasts transfected with dsRNAs against OsMPK1and OsMPK5, which show the highest similarity with AtMPK6and AtMPK3respectively. By contrast, the reduction in the expression of ZFP36and ZFP182in the protoplasts silencing transiently ZFP36and ZFP182and in the RNAi mutants of ZFP36and ZFP182did not affect the ABA-induced up-regulation in the expression OsMPK1and OsMPK5. These data indicate that there are a linear relationship between OsMPK1/OsMPK5and ZFP36/ZFP182in ABA signaling, and OsMPKl and OsMPK5function upstream of ZFP182and ZFP36in the ABA signaling.Previous studies showed that ABA and H2O2induced the expression of OsDMI3, which suggested that OsDIM3may be involved in drought and oxidative stress signal transduction. However, the downstream components involved in the responses of this kinase to stress are unclear. The relationship between OsDIM3and ZFPs is not reported. Our studies showed pretreatment with Calcium channel blockers (EGTA and LaC13), CaM inhibitor (W7and TFP) or CaMK inhibitor KN-93substantially arrested ABA-induced OsZF182and OsZF36gene expression. Using rice protoplast transient expression and dsRNA technology to have OsDMI3gene silencing, ABA-induced ZFP182and ZFP36gene expression was significantly decreased, it indicates that OsDMI3gene locate upstream of the two zinc finger proteins gene ZFP182and ZFP36and control the expression of transcription factors in the ABA signaling system. Interestingly, while interference ZFP182and ZFP36genes respectively, ABA-induced OsDMI3gene expression and enzyme activity were significantly decreased, it indicate that ZFP182and ZFP36genes regulate OsDMI3gene expression. These results clearly suggest that there is a cross talk between OsDMJ3and OsZF182IOsZF36in the ABA signaling:OsDMJ3as a upstream factor regulates the expression of ZFP182and ZFP36, while ZFP182and ZFP36as transcription factors maybe act directly on the promoter of OsDM3, or regulate OsDIM3indirectly through acting on other kinases or factors. Specific mechanism needs further study.Previous studies showed that there is a positive feedback loop involving NADPH oxidase, H2O2, and MAPK in ABA signaling in maize leaves. ABA-induced H2O2production activates MAPKs, which can amplify H2O2signal by regulating the activity of NADPH oxidase in ABA signaling. However, it is unknown whether ZFP36and ZFP182are involved in the positive feedback loop in the ABA signaling. Our studies showed pretreatment with NADPH inhibitor DPI can effectively inhibit the ABA-induced gene transcription of ZFP182and ZFP36. it indicates that Osrbohs locate upstream of the two zinc finger proteins gene ZFP182and ZFP36in the ABA signaling system. The RNAi silencing of ZFP36and ZFP182in rice protoplasts did not affect the ABA-induced up-regulation in the expression of the NADPH oxidase genes, OsrbohB and Osrbohl. The results of DAB staining showed ABA treatment caused the similar increases in the production of H2O2in the leaves of the wild-type plants and the RNAi mutants of ZFP36and ZFP182during the4h of treatment. The above points suggest that ZFP36and ZFP182maybe not mediate the feedback regulation of H2O2production in the ABA signaling. However, we cannot exclude the possible involvement of other C2H2-type ZFPs in the positive feedback loop in ABA signaling.