microRNA172 targets APETALA2 to regulate anthocyanin biosynthesis

MicroRNA172 (miR172) plays a role in regulating a diverse range of plant developmental processes. However, its role in regulating anthocyanin biosynthesis is unclear. In this study, we show that transgenic apple plants over-expressing miR172 show a reduction in red colouration and anthocyanin accumulation in multiple tissue types. This reduction was consistent with decreased expression of APETALA2 homolog MdAP2_1a (a miR172 target gene), MdMYB10, and targets of MdMYB10, as demonstrated by both RNA-seq and RT-PCR analyses. During the onset of anthocyanin biosynthesis in apple fruit skin, miR172 transcript abundance was negatively correlated and MdAP2_1a mRNA concentration was positively correlated with fruit skin red-colouration. The positive role of MdAP2_1a in regulating anthocyanin biosynthesis was supported by the enhanced petal anthocyanin accumulation in transgenic tobacco plants overexpressing MdAP2_1a, and by the reduction in anthocyanin accumulation in apple and cherry fruits transfected with an MdAP2_1a virus-induced-gene-silencing construct. We demonstrated that MdAP2_1a could bind directly to the promoter and protein sequences of MdMYB10 in yeast and tobacco, and enhance MdMYB10 promotor activity. In Arabidopsis, over-expression of miR172 reduced anthocyanin concentration and RNA transcript abundance of anthocyanin genes in plantlets cultured on medium containing 7% sucrose. The anthocyanin content and RNA abundance of anthocyanin genes could be partially restored by using a synonymous mutant of MdAP2_1a, which had lost the miR172 target sequences at mRNA level, but not restored by using a WT MdAP2_1a. These results indicate that miR172 inhibits anthocyanin biosynthesis through suppressing the expression of an AP2 transcription factor that positively regulates MdMYB10. Overall design: Three biological replicates were collected from whole flowers at two stages of flower development (BL, balloon flower; OF, open flower) and from mature fruit skin (FS) of wide type (WT) and miR172 transgenic 'Royal Gala'.