Regulations On Target Genes By Grape Bud Dormancy Release Related ERF Transcription Factors

Grape?Vitis?is second largest cultivated fruit trees in China with high economic value.In recent years,due to the introduction of shelter cultivation techniques and screening of new cultivars of grape in southern China region,grape industry is developing rapidly,and becomes one of important wine-producing regions in China.Due to lack of chilling in warm winter climate,grape can not break dormancy by itself,hydrogen cyanamide?HC?is usually used to break grape bud dorancys.Studies have shown that HC treatment activates ethylene biosynthesis,and exogenous ethylene can break dormancy of grape bud.ERF transcription factors as downstream components in the ethylene signal transduction pathways,regulate dormancy breaking,growth,maturity,aging stress response related gene expression in plants.To understand the grape bud dormancy mechanism in southern China region,the grape bud dormancy curves and the effect of HC on bud break of‘Wink'?V.vinifera cv.Wink?and‘Summer Black'?V.vinifera L.×Vitis labrusca L.cv.Summer Black?grown in Hunan province were studied in this study.To study the regulation of ERF transcription factors on downstream target genes in ethylene transduction pathways during grape dormancy release,VvERF57,VvERF58,VvERF59 that were known to response to HC in our previous study were seleted.Here we use Wink bud to analyze the expression pattern of candidate genes of VvERFs.Using yeast-one hybrid and EMSA assay to study the interaction of target genes and the ERFs.Furthermore,we use the Dual-Luciferase detection system to investigate ERFs influences on the promoter activity of target genes.Results are as follows:?1?Comparing the dormancy curve of‘Summer Black'and‘Wink',we found that‘Summer Black'entered endodormancy in November 10^th,while‘Wink'was ten days later.In addition,‘Wink'had a longer period of endodormancy than‘Summer Black'.?2?After treating with HC?hydrogen cyanamide?,dormancy of Wink grape bud was broken to a certain extent,but not obviously.?3?Quantitive real-time PCR assay showed the expression of 12 candidate target genes of ERFs after treatment of HC.The results showed that,VvAMY expressed high in12 h after HC treatments,VvCYP,VvGST,VvPFK,VvWRKY75 expressed high in 24 h after HC treatments,VvWRKY31,VvSDH,VvACS1,VvSTK,VvBG expressed high in 48 h after HC treatments,VvPGIG,VvNIT4B-like expressed high in 96 h after HC treatments.?4?The results of yeast-one hybrid showed VvERF57 interacted with VvSTK,VvNIT4B-like,VvWRKY31.VvERF58 interacted with VvACS1,VvSTK,VvNIT4B-like,VvWRKY31,VvERF59 interacted with VvACS1,VvWRKY31.?5?Using EMSA assay to analysis the interactions of 12 promoter fragment containing GCC-boxeswith these ERFs.The results showed that VvERF57 could interact with all 12 promoter fragment;VvERF58 could interact with promoter fragment of VvAMY,VvCYP,VvPFK;VvERF59 could interact with promoter fragment of VvAMY,VvCYP,VvACS1,VvBG.?6?The results of the dual-luciferase detection system revealled that VvERF57increased the promoter activity of VvBG,VvWRKY31 by 3 flods,but decreased the promoter activity of VvSTK to 3 flods.The promoter activity of VvBG increased after treated with ethylene.Taken together,we finally choose VvERF57,VvERF59 as the possible transcription factors in the pathway of grape bud dormancy release and consider VvERF57 may activate the expressions of VvBG,VvWRK31,VvSTK;VvERF59 may activate the expressions of VvACS,and then regulate bud dormancy release.