Dissection Of Zinc Finger Protein PhZFP1 Regulating Cold Stress Tolerance In Petunia Hybrida

Low temperature,as a major environmental stress,has a negative effect on plant growth,development,and plant distribution.Petunia hybrida is a kind of important landscape ornamental plant,known as the king of bedding flowers.However,the plant is sensitive to low temperature and hard to overwinter in many parts of the country,which hinders its promotion and application.Therefore,improving basal freezing tolerance of petunia will extend the planting season and increase the planting area,which is of great significance to improve its garden application value.In previous work,one gene encoding a C2H2 zinc finger protein,PhZFP1 is regarded as a particularly promising candidate based on its unique stress-induced expression profile compared with other cold-responsive genes.However,the function of this gene remains unclear.Based on these studies,the PhZFP1 gene was isolated,and its role in cold tolerance was evaluated.Additionally,a lot physiological and molecular experiments to explore its mechanism in cold tolerance of petunia.The main results are as follows:1.PhZFP1 contained two dispersed C2H2 zinc finger domains,is a typical C2H2zinc finger protein,and each zinc finger domain contains the conserved structure of QALGGH.PhZFP1 was predominantly localized in the nucleus,PhZFP1 exhibited high expression level mainly in the roots,stems,leaves and other nutritional tissues of Petunia hybrida.Overexpression of PhZFP1 conferred enhanced cold tolerance of transgenic plants including Petunia hybrida,Solanum lycopersicum,and Arabidopsis thaliana whereas RNAi mediated suppression of PhZFP1 led to increased cold susceptibility in Petunia hybrida.2.The levels of hydrogen（H₂O₂）and superoxide radicals（O₂.^-）were determined in petunia leaves by ROS histochemical staining.The results showed that PhZFP1-OE transgenic lines had lighter staining degree and smaller staining area than WT after cold treatment.Consistent with this result,PhZFP1-OE transgenic lines had higher SOD activity and lower H₂O₂ content than those in WT.However,both histochemical staining and quantitative measurement indicated that PhZFP1-RNAi lines accumulated more ROS than the WT plants.Moreover,the expression levels of ROS scavenging-related gene PhMn-and PhPrx4 were significantly increased in the PhZFP1-OE transgenic petunia compared with the WT plants after the cold treatment.3.Overexpression of PhZFP1 promoted the expression levels of raffinose synthesis-related genes PhGol S1-1,PhRS.Moreover,PhZFP1-OE lines accumulated more galactinol and raffinose than WT and RNAi lines.Furthermore,Yeat one-hybrid（Y1H）,EMSA,Ch IP-q PCR and dual luciferase reporter（DLR）assay proved that PhZFP1 could bind to the promoter of PhGol S1-1 and activate its expression.In addition,silencing of PhGol S1-1 mediated by VIGS compromised the cold tolerance of petunia.4.Y1H and DLR assay proved that PhDREB1F/1I could bind to DRE cores in PhZFP1 promoter and activate its expression.In addition,PhDREB1F and PhDREB1I expressions were rapidly and strongly induced by cold treatment,and they culminated at 2 h and 6 h,respectively.Petunia plants with silencing of PhDREB1F/1I mediated by VIGS displayed hypersensitivity to freezing stress.Furthermore,the expression levels of PhZFP1 and PhGol S1-1 were inhibited in PhDREB1F/1I-VIGS plants.5.A RED family protein PhRED was screened as interaction protein of PhZFP1by yeast two-hybrid（Y2H）screening system,and the interactions were further confirmed by bimolecular fluorescence complementation（Bi FC）,and luciferase complementation imaging（LCI）assay.Further studies showed that the interaction between PhZFP1 and PhRED promotes the transcription of PhZFP1 on PhGol S1-1.Additionally,the q RT-PCR results showed that PhRED was found to be highly expressed in roots and stems during seedling stage of petunia,which was in line with the expression pattern of PhZFP1.In summary,this study analyzed the molecular mechanism and physiological mechanism of PhZFP1 regulating cold tolerance in Petunia.On one hand,cold rapidly induced the transcription level of PhDREB1F and PhDREB1I,and their proteins activated PhZFP1 gene expression.Then,PhZFP1 protein activated the expression of PhGol S1-1,in turn accumulating more galactinol and raffinose,eventually enhancing plant cold tolerance.Moreover,PhRED physically interacts with PhZFP1 protein to further enhance the transcriptional activation activity of PhZFP1 on PhGol S1-1.On the other hand,overexpressing PhZFP1 promoted the increase of SOD activity and reduced the accumulation of ROS（H₂O₂and O₂.^-）by increasing the transcription levels of antioxidant enzyme genes PhMn-SOD and PhPrx4 under low temperature stress,thereby improving the cold tolerance of petunia plants.