The Expression Patterns And Function Of One RING Zinc Finger Protein Gene Regulated By Phytochrome In Rice

Zinc finger proteins play a critical role in many cellular functions, includingtranscriptional regulation, RNA binding, regulation of apoptosis, and protein-proteininteractions. The RING (really interesting new gene) finger motif was defined as a novelzinc-finger domain. The RING zinc-fingermotif, a small Cys/His-rich motif (C3HC/HC3),is represented in two distinct variants, RING-HC and RING-H2, depending on whichamino acid (Cys or His) occupies the fifth position of the motif. The RING-H2finger is asubgroup of the RING finger motif specifically found in eukaryotic cells, which can bedescribed as Cys-X2-Cys-X9-39-Cys-X1-3-His-X2-3-Cys/His-X2-Cys-X4-48-Cys-X2-Cys(X: arbitrary amino acid residue) with the Cys and His representing zinc-coordinatingresidues. The RING finger is subcategorized into RING-HC and RING-H2depending onthe fifth coordination site, Cys or His, respectively. Moreover, there are twotrans-membrane domains in the N-terminal and the RING domains in the C-teminal.In this work, we annalsized the expression and the function of an RING finger proteinregulated by phytochrome. The the expression of OsASRP was analysised by real-timePCR, and we constructed the OsASRP-overexpression vector, then generated transgenicrice plant overexpressing OsASR. We studied the function of OsASRP in Physiology. Themain results were as follows.1OsASRP was detected in the cytoplasmTo examine the subcellular localization of OsASRP, we fused OsASRP to theN-terminal region of a green fluorescent protein (GFP) reporter gene under the control ofthe CaMV35S promoter. This construct was transfected into rice protoplasts andtransiently expressed. the OsASRP-GFP signal was detected in the cytoplasm, whereasGFP alone was distributed throughout the cell. 2phyB-mediated R signaling repressed OsASRP gene expression, whereasphyA-mediated FR signaling also repressed OsASRP gene.To comprehensively determine the molecular characteristics of phytochromesfunctioning in regulating OsASRP gene expression, we compared the transcripts levels ofOsASRP gene in WT and, phyB, phyAphyB mutants grown under continuous R and FR.These results suggest that phyB-mediated R signaling repressed OsASRP gene expression,whereas phyA-mediated FR signaling induced OsASRP gene.3OsASRP mRNA levels accumulated to quite higher levels in leavesTo better understand the function of OsASRP, we examined the transcript levels ofOsASRP in different organs.OsASRP was detected in all organs. OsASRP mRNA levelsaccumulated to quite higher levels in leaves compared to other organs, while OsASRPmRNA levels were relatively low in the spikes.4ABA、PEG、NaCl increased the OsASRP mRNA expressionABA, PEG, NaCl treated four-weeks old plants, the leaves for further study. Theresults showed than exogenous ABA treatment also rapidly induced OsRNS4mRNA geneexpression.The same results as PEG and NaCl.5Transgenic plants overexpressing OsASRP show hyposensitivity to R and FR duringseedling growth and involved in photomorphogenesisTo analyze the role of OsASRP in phytochrome-mediated responses, we compared thelength of the first leaves in WT and OsASRP-OE lines grown under continuous R or FR for8days, since phytochrome-mediated R and FR signalings inhibit the growth of first leavesin rice. OsASRP-OE transgenic lines exhibited longer first leaves and the second leafsheathes than WT plants under R and FR. Thus, overexpression of OsASRP hashyposensitivity to continuous R and FR. We further compared the relative length ofcoleoptiles in WT and OsASRP-OE seedlings treated with R-or FR-pulse. The coleoptileswere obviously inhibited by R-pulse treatment in OsASRP-OE lines and WT. However, the inhibitory effects were more obvious in WT plants compared with OsASRP-OE lines.Moreover, the difference in coleoptile growth was more obvious at high rather than lowfluence intensity. Similarly, FR-pulse treatment caused the stronger inhibition of coleoptilegrowth in WT than in OsASRP-OEseedlings. Taken together, these results suggest thatOsASRP-OE lines have hyposensitivity to R and FR during seedling growth.6Transgenic plants overexpressing OsASRP show hypersensitivity to exogenous ABAand enhanced tolerance to abiotic stress in riceSince ABA is an important hormone involved in abiotic stress responses, we thusinvestigated the possible function of OsASRP in ABA responses by measuring the effect ofexogenous ABA on seedling growth. The results suggest that OsASRP-OE lines havehypersensitivity to exogenous ABA. We further analyzed the roles of OsASRP in regulatingtolerance to drought stress and high salinity in rice. We found that overexpression ofOsASRP confers improved tolerance to high salinity and drought.7OsASRP involved in ABA metabolismSelected NCED1, NCED3, NCED5in the ABA biosynthetic pathway and ABAOX1,ABAOX3in ABA decomposition pathway for further research. Results show that:exogenous ABA treated1hour, the expression of NCED1and NCED3was significantlyhigher in the over-expression plants expression than that in WT, NCED5was no significantchanged; however, the expression of ABAOX1and ABAOX3was significantly lower inthe over-expression plants expression than that in WT. These results show that when thereis presence of exogenous ABA, OsASRP gene can inhibit the decomposition of ABApromote the synthesision of the ABA, involved in ABA metabolism.