miRNA858b Inhibits Proanthocyanidin Accumulation by Repression of DkMYB19 and DkMYB20 in Persimmon

Persimmon proanthocyanidin (PA) biosynthetic had been reported to be regulated by several transcription factors, but the miRNAs function involved in this process was poorly understood. We identified a miRNA858b that putatively targeted two R2R3-MYB transcription factors, DkMYB19/DkMYB20. Transcript accumulation of DkMYB19/DkMYB20 and miRNA858b showed contrasting divergent expression patterns during fruit development. DkMYB19/DkMYB20 were confirmed to be localized in the nucleus. The interaction between miRNA858b and DkMYB19/DkMYB20 were experimentally validated by 59 RNA ligase-mediated RACE and LUC enzyme activity detection. Overexpression of miRNA858b led to the down-regulation of DkMYB19/DkMYB20 which reduced the accumulation of PA, whereas the reduced miRNA858b activity that up-regulated the DkMYB19/DkMYB20 resulted in high levels of PA in STTM858b transient expression in leaves in vivo. Similarly, the transient transformation of miRNA858b in fruit wafers in vitro also reduced the accumulation of PA by repressing the DkMYB19/DkMYB20, while the up-regulation of DkMYB19/DkMYB20 enhanced the accumulation of PA in STTM858b or DkMYB19/DkMYB20 transient transformation in fruit wafers. PA content decreased after overexpression of miRNA858b in Arabidopsis wild type and DkMYB19/DkMYB20 in persimmon leaf callus consisted with the above results. These findings suggested that miRNA858b repressed the expression of DkMYB19/DkMYB20 which contribute to PA accumulation in persimmon.