Functional Analysis Of Two E3 Ligases Genes In Rice(Oryza Sativa L.) Tolerant To Abiotic Stresses

Unfavourable environmental conditions,such as salinity,drought,and cold extensively affect agricultural productivity and quality of agricultural products worldwide.Molecular dissection of the complex plant responses to abiotic stresses has obviously more important.Ubiquitination serves as a versatile post-translational modification in all eukaryotic species.It functions in plant growth and development,and responses to biotic and abiotic stresses during the plant growth.In this study,by combining plant molecular biology,biochemistry and genetics methods,we characterized the biological and biochemical functions of the rice RING E3 ubiquitin ligases ZFIP1?ZFP182 Interacting Protein 1?and ZFRG1?ZFP252 Regulated Gene 1?,and elucidated their molecular function in response to abiotic stresses.ZFIP1 was expressed mainly in root,spikelet and node.Consistent with our qRT-PCR results,we detected strong GUS signals in root,panicle and node of the pZFIP1:GUS transgenic plants.Expression of ZFIP1 was up-regulated by salt,dehydration,ABA,cold,oxidative stresses,while down-regulated by heat treatment.In vitro ubiquitination assays showed that ZFIP1 possessed E3 ubiquitin ligase activity,and that the RING finger conserved region was required for the activity.Rice protoplast transfection indicated that ZFIP1 was localized predominantly in the nucleus,as well as some localized in the cytoplasm.Transactivation activity assay showed that ZFIP1 had a strong transcription activation activity in yeast cells.Deletion analysis showed that the RING finger domain was essential for its transactivation activity.Transient expression of ZFIP1 fused with GAL4 BD domain can increase the luciferase activity 4-fold relative to the control with an effector plasmid containing only the GAL4 DNA binding domain in rice leaf protoplasts.These results support a role of ZFIP1 in transcriptional regulation of gene expression in the nucleus.Both ZFIP1 overexpression and RNAi transgenic lines were produced through Agrobacterium-mediated method.Stress treatments showed that overexpression of ZFIP1 reduced ABA sensitivity and cold,oxidative tolerance,and increased salt sensitivity in transgenic rice plants,while RNAi silencing of ZFIP1 enhanced stress tolerance.The ZFIP1-OE plants suppressed the expression of the gene encoding proline synthase under cold stress.The ZFIP1-RNAi plants accumulated greater amounts of proline and less amounts of H₂O₂,malondialdehyde.The enhanced activities of antioxidant enzymes,including peroxidase,superoxide dismutase,and catalase,may underlie the lower H₂O₂ and O^2- contents in ZFIP1-RNAi plants.Microarray analysis showed that many stress responsive genes were differentially expressed in the ZFIP1 overexpression plants under normal and cold conditions.These findings suggest that ZFIP1 encodes a stress-responsive transcription factor that plays a negative role through transcriptional regulation of diverse stress-related genes in regulation of rice to salt,cold,and oxidative stress.ZFRG1 transcripts were detected in various rice tissues examined,but its expression was most abundant in root and different periods of spikelet.Expression of ZFRG1 was up-regulated by salt,dehydration,ABA,cold,oxidative stress and heat.In vitro ubiquitination assays showed that ZFRG1 possessed E3 ubiquitin ligase activity,and that the RING finger conserved domain was required for the activity.Rice protoplast transfection and tabacco transient expression assays indicated that ZFRG1 was localized predominantly in the cytomembrane.Both ZFRG1 overexpression and RNAi transgenic lines were produced through Agrobacterium-mediated method.Stress treatments showed that overexpression of ZFRG1 reduced salt and drought tolerance in transgenic rice plants,while RNAi silencing of ZFRG1 enhanced stress tolerance.Yeast two-hybrid?Y2H?screening showed that ZFRG1 interacted with two of NAC2 family members,NAC2 and NAC2-like.The interaction was confirmed by in vivo yeast assay and in vitro GST Pull-Down assay.Remarkably,yeasts grow well on SD/-Trp/-Leu/-His/-Ade/X-a-Gal plate transformed with either pGADT7-NAC2 and pGBKT7-ZFRG1,or pGADT7-NAC2 and pGBKT7-ZFRG1?H171Y?,pGADT7-NAC2-like was not able to coexist with pGBKT7-ZFRG1 while the mutant pGBKT7-ZFRG1?H171Y?did.Together,these results demonstrate that ZFRG1 plays a negative role in drought and salt stress responses through post-translational regulation in rice.