microRNA858 represses the transcription factor gene SbMYB47 and regulates flavonoid biosynthesis in Scutellaria baicalensis

Abstract MicroRNAs (miRNAs) are noncoding endogenous single-stranded RNAs that regulate target gene expression by reducing their transcription and translation. Several miRNAs in plants function in secondary metabolism. The dried root of Scutellaria baicalensis Georgi is a traditional Chinese medicine that contains flavonoids (baicalin, wogonoside, and baicalein) as its main active ingredients. Although the S. baicalensis genome sequence has been published, information regarding its miRNAs is lacking. In this study, 12 small RNA libraries of different S. baicalensis tissues were compiled, including roots, stems, leaves, and flowers. A total of 129 miRNAs were identified, including 99 miRNAs from 27 miRNA families and 30 predicted miRNAs. Furthermore, 46 reliable target genes of 15 miRNA families were revealed using psRNATarget and confirmed by degradome sequencing. It was speculated that the microRNA858 (miR858)–SbMYB47 module might be involved in flavonoid biosynthesis. Transient assays in Nicotiana benthamiana leaves indicated that miR858 targets SbMYB47 and suppresses its expression. Artificial miRNA-mediated knockdown of miR858 and overexpression of SbMYB47 significantly increased the flavonoid content in S. baicalensis hairy roots, while SbMYB47 knockdown inhibited flavonoid accumulation. Yeast one-hybrid and dual-luciferase assays indicated that SbMYB47 directly binds to and activates the S. baicalensis phenylalanine ammonia-lyase 3 (SbPAL-3) and flavone synthase II (SbFNSⅡ-2) promoters. Our findings reveal the link between the miR858–SbMYB47 module and flavonoid biosynthesis, providing a potential strategy for the production of flavonoids with important pharmacological activities through metabolic engineering.