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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.

Best known as licorice, Glycyrrhiza Linn., a genus of herbaceous perennial legume, has been used as a traditional herbal medicine in Asia and a flavoring agent for tobacco and food industry in Europe and America. Abiotic stresses and hormonal treatments can significantly impact the development and metabolism of secondary metabolites in Glycyrrhiza. To better understand the biosynthesis of the trace-amount bioactive compounds, we first screened for the suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) analysis in Glycyrrhiza. The expression profiles of 14 candidate reference genes, including Actin1 (ACT), Clathrin complex AP1 (CAC), Cyclophilin (CYP), Heat-shock protein 40 (DNAJ), Dehydration responsive element binding gene (DREB), Translation elongation factor1 (EF1), Ras related protein (RAN), Translation initiation factor (TIF1), &beta, Tubulin (TUB), Ubiquitin-conjugating enzyme E2 (UBC2), ATP binding-box transpoter 2 (ABCC2), COP9 signal compex subunit 3 (COPS3), Citrate synthase (CS), and R3H domain protein 2 (R3HDM2) from two congeneric species, Glycyrrhiza uralensis F. and Glycyrrhiza inflata B., were examined under abiotic stresses (osmotic and salinity) and hormonal treatments (Abscisic acid (ABA) and methyl jasmonic acid (MeJA)) using a panel of software, including geNorm, NormFinder, BestKeeper, and Delta CT. The overall stability, however, was provided by RefFinder, a comprehensive ranking system integrating inputs from all four algorithms. In G. uralensis, the most stable reference genes under osmotic stress, salt stress, ABA treatment, and MeJA treatment were TIF1, DNAJ, CS, and ABCC2 for leaves and DNAJ, DREB, CAC, and CAC for roots, respectively. In comparison, the top ranked genes were TUB, CAC, UBC2, and RAN for leaves and TIF1, ABCC2, CAC, and UBC2 for roots, respectively, under stress and hormonal treatments in G. inflata. ACT and TIF1, on the other hand, were the least stable genes under the most experimental conditions in the two congeneric species. Finally, our survey of the reference genes in legume shows that EF, ACT, UBC2, and TUB were the top choices for the abiotic stresses while EF, UBC2, CAC, and ABCC2 were recommended for the hormonal treatments in Leguminosae. Our combined results provide reliable normalizers for accurate gene quantifications in Glycyrrhiza species, which will allow us to exploit its medicinal potential in general and antiviral activities in particular.