microRNA172 targets APETALA2 to regulate flavonoid biosynthesis in apple (Malus domestica)

MicroRNA172 (miR172) plays a role in regulating a diverse range of plant developmental processes, including flowering, fruit development and nodulation. However, its role in regulating flavonoid biosynthesis is unclear. In this study, we show that transgenic apple plants over-expressing miR172 show a reduction in red coloration and anthocyanin accumulation in various 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 qRT-PCR analyses. 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 flavonoid (including anthocyanins and flavonols) concentration and RNA transcript abundance of flavonoid 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 flavonoid biosynthesis through suppressing the expression of an AP2 transcription factor that positively regulates MdMYB10.