| Apple is one of the most important fruits in the world,and its peel color is mainly determined by anthocyanin content.Ethylene and jasmonic acid can promote fruit ripening,which is also accompanied by the rapid accumulation of anthocyanins.Although there have been many reports on the mechanism of the two hormones promoting the synthesis of anthocyanins,in the complex process of fruit ripening,two kinds of hormones are involved in the rapid accumulation of anthocyanins.Whether there is an interaction between plant hormones in promoting the accumulation of anthocyanins in fruit is still unclear,and the related mechanism needs to be further explored.In this study,we found that the ethylene signaling pathway gene Md ERF1 B was strongly induced by both ethylene and jasmonic acid.As a positive regulator of anthocyanin synthesis,Md ERF1 B can directly bind to the Md MYC2 promoter and upregulate its expression.In addition,Md ERF1 B can interact with the jasmonic acid signaling pathway inhibitors Md JAZ5 and Md JAZ10,The Md ERF1B-Md JAZ5/10 protein complex reduces the activation activity of Md ERF1 B on the Md MYC2 promoter.In addition,the key protein of ethylene signal transduction,Md EIL1,can directly bind to the Md ERF1 B promoter and upregulate its expression.The key research findings are as follows:1.Ethephon,methyl jasmonate（Me JA）,1-MCP and Me JA+1-MCP treatments were performed on apple fruit of ’Zaojie?,and it was found that ethephon and Me JA could significantly promote the accumulation of anthocyanins in apple peel.The expression patterns of genes related to jasmonic acid and ethylene signaling pathways were detected by RT-q PCR,showing that the expression levels of Md MYC2 and Md ERF1 B were significantly induced by jasmonic acid and ethylene.2.Overexpression or interference of Md MYC2 and Md ERF1 B in ’Orin’ apple callus,it was found that overexpression of Md MYC2 and Md ERF1 B significantly promoted anthocyanin accumulation and the expression of anthocyanin synthesis pathway-related genes,However,the callus with Md MYC2 and Md ERF1 B interfering expression did not change in color,but decreased the expression levels of genes related to anthocyanin synthesis pathway.The same results were also obtained by transient overexpression or silencing of Md MYC2 and Md ERF1 B in apple fruit.In addition,the expression levels of Md MYC2 and Md ERF1 B showed a consistent trend in Md ERF1 B transgenic calli and injected fruits.3.Yeast one-hybrid（Y1H）and Electrophoretic Mobility Shift Assay（EMSA）verified that Md ERF1 B can specifically bind to the promoter of Md MYC2,and Ch IP-q PCR assay also verified that Md ERF1 B can bind to the promoter of Md MYC2 in vivo.Further LUC experiments demonstrate that Md ERF1 B enhances Md MYC2 transcriptional activity.4.Interfering with the expression of Md MYC2 in apple callus of Md ERF1B-OE,it was found that the anthocyanin content and the expression levels of anthocyanin synthesis-related genes in the co-transformed calli were higher than those in the control,but significantly lower than those in the transgenic calli overexpressing Md ERF1 B alone,indicating that Md MYC2 plays an important role in the regulation of anthocyanin synthesis by Md ERF1 B.5.The transgenic callus of Md EIL1-OE was obtained by introducing 35S::Md EIL1 into’Orin’ apple callus.It was found that overexpression of Md EIL1 significantly promoted the accumulation of anthocyanin in the callus,and the expression level of Md ERF1 B was also increased accordingly.The Yeast one Hybrid（Y1H）,EMSA,and Ch IP-q PCR experiments verified that Md EIL1 specifically binds to the promoter of Md ERF1 B in vivo and in vitro.Further LUC activity experiments showed that Md EIL1 activates the expression of Md ERF1 B by binding to the promoter of Md ERF1 B.6.Using Yeast two-hybrid（Y2H）,Pull down and bimolecular fluorescent complementation（Bi FC）experiments,it was verified that Md ERF1 B interacts with JAZ5 and JAZ10 in vitro and in vivo.In addition,using the LUC reporter gene experiment,it was proved that JAZ5 and JAZ10 weakened the transcriptional activation ability of Md ERF1 B on the promoter of the target gene Md MYC2.In summary,our results indicate that Md ERF1 B,as a core factor,mediates the interaction between jasmonic acid and ethylene signaling,and regulates the synthesis of apple anthocyanins under the joint participation of Md MYC2,Md JAZ5,Md JAZ10,and Md EIL1. |
