Glycyrrhiza inflata Batalin is a medicinal licorice species that has been widely used by humans for centuries. Licochalcone A (LCA) is a characteristic flavonoid that accumulates in G. inflata roots with high economical value. However, the biosynthetic pathway and regulatory network of its accumulation remain largely unknown. Here we found that a histone deacetylase (HDAC) inhibitor nicotinamide (NIC) could enhance the accumulation of LCA and total flavonoids in G. inflata seedlings. GiSRT2, a NIC-targeted HDAC was functionally analyzed and its RNAi transgenic hairy roots accumulated much more LCA and total flavonoids than its OE lines and the controls, indicating a negative regulatory role of GiSRT2 in the accumulation of LCA and total flavonoids. Co-analysis of transcriptome and metabolome of RNAi-GiSRT2 lines revealed potential mechanisms in this process. An O-methyltransferase gene, GiLMT1 was up-regulated in RNAi-GiSRT2 lines and the encoded enzyme catalyzed an intermediate step in LCA biosynthesis pathway. Transgenic hairy roots of GiLMT1 proved that GiLMT1 is required for LCA accumulation. Together, this work highlights the critical role of GiSRT2 in the regulation of flavonoid biosynthesis and identifies GiLMT1 as a candidate gene for the biosynthesis of LCA with synthetic biology approaches.

Echinatin and licochalcone A (LCA) are valuable chalcones preferentially accumulated in roots and rhizomes of licorice (Glycyrrhiza inflata). The licorice chalcones (licochalcones) are valued for their anti-inflammatory, antimicrobial, and antioxidant properties and have been widely used in cosmetic, pharmaceutical, and food industries. However, echinatin and LCA are accumulated in low quantities, and the biosynthesis and regulation of licochalcones have not been fully elucidated. In this study, we explored the potential of a R2R3-MYB transcription factor (TF) AtMYB12, a known regulator of flavonoid biosynthesis in Arabidopsis, for metabolic engineering of the bioactive flavonoids in G. inflata hairy roots. Overexpression of AtMYB12 in the hairy roots greatly enhanced the production of total flavonoids (threefold), echinatin (twofold), and LCA (fivefold). RNA-seq analysis of AtMYB12-overexpressing hairy roots revealed that expression of phenylpropanoid/flavonoid pathway genes, such as phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and flavanone 3'-hydroxylase (F3'H), is significantly induced compared to the control. Transient promoter activity assay indicated that AtMYB12 activates the GiCHS1 promoter in plant cells, and mutation to the MYB-binding motif in the GiCHS1 promoter abolished activation. In addition, transcriptomic analysis revealed that AtMYB12 overexpression reprograms carbohydrate metabolism likely to increase carbon flux into flavonoid biosynthesis. Further, AtMYB12 activated the biotic defense pathways possibly by activating the salicylic acid and jasmonic acid signaling, as well as by upregulating WRKY TFs. The transcriptome of AtMYB12-overexpressing hairy roots serves as a valuable source in the identification of potential candidate genes involved in LCA biosynthesis. Taken together, our findings suggest that AtMYB12 is an effective gene for metabolic engineering of valuable bioactive flavonoids in plants.
Copyright © 2022 Wu, Singh, Lyu, Pattanaik, Wang, Li, Yuan and Liu.