The Crabapple McMYB10 Modulates The Expression Of A Ubiquitin Ligase,McCOP1 To Regulate Its Own Expression

Crabapple is an important resourse of ornamental value and economical value in the Malus Mill of Rosaceae.The transcription factor MYB10 plays a vital role in the colour development of higher plants.In the dark,MYB10 can be specifically degraded by E3 ubiquitin ligase COP1.To explore the correlation between MYB transcription factor and COP1 protein in transcription level,in this study,to understand the regulation mechanism of anthocyanin by McMYB10 and McCOP1,two crabapple cultivars,�Royalty' and �Flame' were used as research materials.Here,we first analyzed the content of flavonoids at 5 different development stages in the ever-red leaf �Royalty' and ever-green leaf �Flame' leaf.The results showed that the leaves of �Royalty' are rich in cyanidin-3-O-glucoside at 5 development stage.In contrast,the cyanidin-3-O-glucoside are not detected at all development stages in the leaf of �Flame'.Moreover,the content of flavonols and aicularin decreased gradually with the development of the leaves in the �Royalty',and the content was less in the �Flame' than that in leaves of �Royalty'.The expression of McMYB10 and its specific E3 ubiquitin ligase,McCOP1 at 5 development stage in two different crabapple cultivars were determined by qRT-PCR,and the expression of these two genes in leaf development were analyzed.The results showed that the expression level of the McMYB10 and McCOP1 gene was positively correlated,and the correlation analysis showed that the expression of McMYB10 gane was positively correlated with anthocyanin content.Consititute light treatment can promote the expression of McMYB10,McCOP1-1 and McCOP1-2,and promotes anthocyanin accumulation in crabapple leaves.Meanwhile,we determined the expression of McMYB10 and its specific E3 ubiquitin ligase,McCOP1,in crabapple leaves during the course of a day.Interestingly,the results showed that the expression of McCOP1 s was higher in daylight than at night,and McMYB10 expression showed a positive correlation with the transcription of McCOP1-1 and McCOP1-2 and anthocyanin accumulation in crabapple cultivar with red-colored leaves.Meanwhile,to detect the potential interaction between the McCOP1 promoter and McMYB10,several MYB transcription factor binding sites of the MYBCORE type were found in the McCOP1-1 and McCOP1-2 promoters by searching the PLANTCARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)database,and we deduced that there may be a relationship between McMYB10 and McCOP1-1 and McCOP1-2 at the transcriptional level.Yeast one hybrid demonstrated that the McMYB10 transcription factor binds specifically to the promoter of McCOP1-1 and McCOP1-2.Moreover,we verified that McMYB10 may directly combine with the MYB recognition site in the promoter of McCOP1-1 and McCOP1-2 by EMSA assay.Furthermore,overexpression or silencing of McMYB10 in crabapple leaves and apple fruits also led to an increase or decrease,respectively,in the expression of McCOP1-1 and McCOP1-2 and in anthocyanin biosynthesis.In total,our results reveal a new self-regulation mechanism in where McMYB10 modulates its own expression by activating McCOP1-1 and McCOP1-2 expression to promote ubiquitination of the McMYB10 protein by McCOP1.Light can induce the expression of McMYB10,then McMYB10 can combine in the promoter of McCOP1 and activating the expression of McCOP1.However,McCOP1 s can degrade McMYB10 protein by ubiquitination,then restrain the excessive accumulation of McMYB10 protein in the dark.