The miR156/SPL9 interaction mode regulates the anthocyanin accumulation in potato

Anthocyanins are an essential class of flavonoids that represent a large group of plant secondary metabolites. microRNAs (miRNAs) can target TFs related to anthocyanin synthesis and inhibit their expression, thereby affecting the expression level of key downstream structural genes and ultimately regulating the synthesis and accumulation of anthocyanins in plants. Nevertheless, the regulatory function of miR156a in anthocyanin synthesis, especially in potatoes, has yet to be investigated. In this study, the miR156a and its target gene StSPL9 were screened and analyzed from small RNA sequencing, degradome sequencing and transient expression assay. The function of miR156a in anthocyanin synthesis of potato tuber was investigated. Overexpression of miR156a (OE-miR156a) in potato tubers promoted anthocyanin synthesis while concurrently reducing flavonoid synthesis compared with wild-type (WT) potatoes. The miR156a-silenced tubers (STTM-miR156a) achieved using short tandem target mimics (STTM) contained significantly lower anthocyanin content and increased flavonoid accumulation compared to WT and OE-miR156a. Notably, overexpression of StSPL9 in potato yielded identical results. The relative expression levels of the anthocyanin-related structural genes PAL , 4CL , CHS , CHI , F3H , DFR , and UFGT in STTM-miR156a transgenic potato tubers showed the opposite trend to that observed in OE-miR156a potato tubers, as demonstrated by RNA sequencing and quantitative real-time PCR (qRT-PCR). The integrated analysis of the transcriptome and metabolome revealed that the accumulation of flavonoids and flavonols in STTM-miR156a was significantly increased than in the wild type, whereas the contents of anthocyanins and phenolic acids were notably reduced. Further analysis showed that the expression of CHS , FG2 , BEATH , and CYP75A were up-regulated in STTM-miR156a, and HCT was downregulated, which resulted in a reduced accumulation of methyl 4-caffeoylquinate, a phenolic acid metabolite. Therefore, it is demonstrated that miR156a indirectly modulates the expression of CHS , BEATH , CYP75A , HCT , and FG2 by targeting and degrading its target gene StSPL9 and other SPL genes, thus influencing anthocyanin accumulation in potato tuber. This study provides valuable insights into the complex regulatory network governing anthocyanin biosynthesis in Solanum species.