| Early researches in our laboratory have shown that ZFRG1(ZFP252 Regulated Gene 1)is typical RING type E3 ubiquitin ligase.The RNAi silencing of ZFRG1 transgenic plants can significantly improve the resistance of rice,also suggests that ZFRG1 play the negative role in the process of rice response to abiotic stress.In order to further clarify the tolerance mechanisms of ZFRG1 response to drought and high salt stress in rice,Yeast two-hybrid(Y21H)screening showed that ZFRG1 interacted with two NAM conservative structure domain proteins ZRIP1(ZFRG1 Interacting Protein 1)and ZRIP2(ZFRG1 Interacting Protein 2),and the interaction was confirmed by GST pull-down.In this study,by combining plant molecular biology,biochemistry and genetics methods,we characterized the biological and biochemical functions of the RING E3 ubiquitin ligases ZFRG1 and two NAM conservative structure domain proteins ZRIP1 and ZRIP2.Analysis using pZFRGl:GUS transgenic plants showed that higher expression level in young panicle and floretin rice.Nicotiana benthamiana transient expression assays and rice protoplast transfection indicated that ZFRG1 was localized predominantly in the cell membrane and nucleus.The ABA sensitivity assay showed that RNAi silencing of ZFRGI transgenic plant significantly increased ABA sensitivity.ZFRGl interacted with ZRIP1 was confirmed by co-immunoprecipitation,Bimolecular fluorescence complementation assays.The Nicotiana benthamiana transient expression results showed the ZRIP1 localization in the cell membrane and cytoplasm.Co-sublocalization of ZFRG1 and ZRIP1 showed that they were able to coexist in the cell membrane.In vitro ubiquitination assays showed that ZRIP1 can be ubiquitinated by ZFRG1.ZRIP1 was highly expressed in the leaf and stem and induced by salt stress(100 mm NaC1)and ABA(50?M).By the agrobacterium-mediated transformation,we obtained the ZRIP1 overexpression of transgenic lines and the biological function analysis showed that overexpression of ZRIP1 had slightly lower ABA sensitivity in the late germination,salt sensitivity analysis,the RNAi silencing of ZFRG1 transgenic plants and the overexpression of ZRIP1 had slightly lower salt sensitivity,while the overexpression of ZRIP1 increased the salt,and PEG sensitivity of transgenic rice plants.ZFRG1 interacted with ZRIP2 was confirmed by co-immunoprecipitation,Bimolecular fluorescence complementation assays.The Nicotiana benthamiana transient expression results showed the ZRIP2 localization in the cell membrane and cytoplasm.Co-sublocalization of ZFRG1 and ZRIP2 showed that they were able to coexist in the cell membrane.ZRIP2 was highly expressed in the leaf and stem and induced by ABA(50?M).By the agrobacterium-mediated transformation,we obtained the ZRIP2 overexpression of transgenic lines and the biological function analysis showed that overexpression of ZRIP2 higher ABA sensitivity and reduced salt sensitivity in the late germination.Abiotic Stresses seriously affect agronomic traits and constrain the global agricultural production,which has become the major environmental challenges for sustainable development of agriculture.Rice is the main food crops in the world.Therefore,it is important to improve the crop resistance to abiotic stresses.Ubiquitin-proteasome pathway(UPS)is important for protein modification in plant growth and development.This pathway majorly functions in plant morphogenesis,metabolism regulation,hormone signal transduction and DNA damage repair and other processes.The increasing evidences have suggested that ubiquitin proteasome pathway is involved in plant response to abiotic stress.In this study,we functionally characterized a novel RING E3 ubiquitin ligase ZFRG1 and two NAM conservative structure domain proteins.Above results demonstrate that ZFRG1 may plays a negative role in drought stress responses through post-translational regulation of ZRIPl,at the same time ZFRG1 may interact with the positive regulator ZRIP2 to develop resistance mechanism of rice response to abiotic stress. |
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