COMT, CRTZ, and F3'H regulate glycyrrhizic acid biosynthesis in Glycyrrhizauralensis hairy roots

Abstract

Abstract Glycyrrhiza uralensis Fisch. is prescribed as one of the original plants of licorice in Chinese Pharmacopoeia. This herbal medicine possesses numerous important pharmacological activities and has been used in clinic in China since ancient times. Glycyrrhizic acid (GA) is a triterpenoid compound isolated from G. uralensis and also one of the marker components for its quality control. Based on our pervious transcriptome study, three genes, the caffeic acid 3-O-methyltransferase gene (COMT), the β-carotene 3-hydroxylase gene (CRTZ), and the flavonoid 3'-monooxygenase gene (F3'H), were selected as our target genes due to a high correlation of their expression levels with GA biosynthesis. In this study, we investigated the regulatory effects of these genes on GA biosynthesis through gene editing and overexpression in G. uralensis hairy roots. We observed that neither knockout nor overexpression of any of the genes affects the viability of the transgenic hairy roots, indicating that these genes are not essential for survival of hairy roots. However, compared with the wild type and negative control hairy roots, GA contents were significantly lower in hairy roots overexpressing COMT, CRTZ, and F3'H, while higher in those knocking out the three genes. Our findings demonstrate that the three genes, COMT, CRTZ, and F3'H, all negatively regulate the GA biosynthesis.