Functional Analysis Of Zinc Finger Proteins ZFP150 And ZFP214 In Responses To Abiotic Stress In Rice (Oryza Sativa L.)

Rice is one of the most important crop plants through the world,and also an important model plant in molecular biology research of monocotyledon plants.Abiotic stresses,such as drought,salinity and cold seriously affect the plants normal growth,development and crop production.Plant TFI?A type zinc finger proteins play important roles in plant response to abiotic stresses.In this research,two rice TF?A type zinc finger proteins ZFP150 and ZFP214 were functionally studied and the mechanisms of them in regulating stress responses were analyzed.1.Expression of rice zinc finger protein gene ZFP150 were observed in various tissues of adult rice plants,and regulated by different abiotic stress treatments including temperature stress(4 ? and 42 ?),H2O2,NaCl,PEG6000 and ABA.Subcellular localization assay revealed that ZFP150 was a nuclear protein.Transgenic rice plants were generated via Agrobacterium-mediated method with Zhonghual 1 background.GUS histochemical staining suggests that ZFP150 was mainly distributed in elongation intemodes and immature spike internode.Phenotype of transgenic rice plants showed that the plant height was related to the expression level of ZFp150,and the cell length of ZFP150-OE transgenic rice seedlings was significantly shorter than WT.Exogenous GA3 treatment could recover this phenotype.Meanwhile overexpressing of ZFP150 could increase rice seedlings tolerance to drought,salinity and chilling stress,and also increase the sensitivity of transgenic rice to exogenous ABA.ZFP150 function as a transcription repressor in rice protoplast.Interaction proteins were identified from yeast two hybrid screening and totally obtained 14 positive clones.Among them,E47 encoded a LEA family protein.Subcellular localization of E47::GFP fusion protein revealed that it distributed all through the cells.Expression level of E47 could be induced by drought,PEG6000 and ABA.BiFC assay suggested that ZFP150 and E47 could interact in nuclear of tobacco epidermal cells,where ZFP150 could form homodimer.2.ZFP214 was expressed in all rice tissues at adult stage,and could be induced by NaCl,PEG6000 and ABA treatment.ZFP214::GFP fusion protein was localized in the whole cell,including nuclear,cytoplasm and plasma membrane.By transgenic technology,overexpression and RNAi transgenic rice plants with Zhonghuall background were generated.Overexpressing of ZFP214 increased drought tolerance of rice seedlings.Under drought condition,ZFP214-OE seedlings accumulated more free proline,less H2O2 and MDA content than WT,and elevated higher ability to scavenge ROS.The expression of drought related genes were induced in ZFP214-OE seedlings,including SODA1,POD,bZIP46,DREB1A,P5CS1,LEA3-1 and RAB21.DLN-box/EAR motif,at C-terminal of ZFP214 amino acid sequence,usually functions as a repress domain according to previous research.In our study,yeast one hybrid assay suggested that ZFP214?C had trans-activation activity in yeast cells and ZFP214FL and ZFP214AN did not.In rice protoplasts,ZFP214FL,ZFP214?N and ZFP214?C all had no transcriptional activities,suggesting ZFP214 might function as a trans-repressor.ZFP214FL was used as the bait for yeast two hybrid by screening yeast expression libraries.Totally 7 ZFP214-interacting proteins were identified and among them,OsSAMS 1 was shown to interact with both ZFP214FL and ZFP214?C in yeast cells.3.Rice membrane protein gene SALP1(Stress Associated Little Protein 1)was mainly expressed in root,leaf blade and immature panicle.SALP1 could be induced by various abiotic stress treatments.The fusion protein of SALP1::GFP was distributed in cell membrane of rice protoplast.Under NaCl condition,SALP1 overexpression transgenic seedlings had a higher survival rate and accumulated more free proline than WT,P5CS was also induced in OE seedlings.SALP1 transgenic rice showed reduced sensitivity to ABA treatment and expression of SALP1 is not related to ABI5-like 1 protein.Conclusively,SALP1,a previously unknown membrane protein,is involved in salt tolerance through an ABA-independent signaling pathway in rice.