DkmiR397 Regulates Proanthocyanidin Biosynthesis via Negative Modulating DkLAC2 in Chinese PCNA Persimmon

Abstract

Persimmon fruits accumulate a large amount of proanthocyanidins (PAs), which makes an astringent sensation. Proanthocyanidins (PAs) are the polymers of flavan-3-ols stored in plant vacuoles under laccase activation. A laccase gene, <i>DkLAC2</i>, is putatively involved in PAs biosynthesis and regulated by microRNA (DkmiR397) in persimmon. However, the polymerization of PAs in association with miRNA397 still needs to be explored in persimmon. Here, we identified pre-DkmiR397 and its target gene <i>DkLAC2</i> in 'Eshi 1' persimmon. Histochemical staining with GUS and dual luciferase assay both confirmed DkmiR397-<i>DkLAC2</i> binding after co-transformation in tobacco leaves. Diverse expression patterns of <i>DkLAC2</i> and DkmiR397 were exhibited during persimmon fruit development stages. Moreover, a contrasting expression pattern was also observed after the combined <i>DkLAC2</i>-miR397 transformation in persimmon leaves, suggesting that DkmiR397 might be a negative regulator of <i>DkLAC2</i>. Similarly, the transient transformation of DkmiR397 in persimmon fruit discs in vitro also reduced PA accumulation by repressing <i>DkLAC2</i>, whereas the up-regulation of <i>DkLAC2</i> increased the accumulation of PAs by short tandem target mimic STTM-miR397. A similar expression pattern was observed when overexpressing of <i>DkLAC2</i> in <i>Arabidopsis</i> wild type (WT) and overexpression of <i>DkLAC2</i>, DkmiR397 in persimmon leaf callus. Our results revealed that the role of DkmiR397 repressed the expression of <i>DkLAC2</i> concerning PA biosynthesis, providing a potential target for the manipulation of PAs metabolism in persimmon.