Abstract
Polyphenols are the main active components of the anti-inflammatory compounds in dandelion, and chlorogenic acid (CGA) is one of the primary polyphenols. However, the molecular mechanism underlying the transcriptional regulation of CGA biosynthesis remains unclear. Hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT2) is the last rate-limiting enzyme in chlorogenic acid biosynthesis in Taraxacum antungense. Therefore, using the TaHQT2 gene promoter as a probe, a yeast one-hybrid library was performed, and a basic helix-loop-helix (bHLH) transcription factor, TabHLH1, was identified that shared substantial homology with Gynura bicolor DC bHLH1. The TabHLH1 transcript was highly induced by salt stress, and the TabHLH1 protein was localized in the nucleus. CGA and luteolin concentrations in TabHLH1-overexpression transgenic lines were significantly higher than those in the wild type, while CGA and luteolin concentrations in TabHLH1-RNA interference (RNAi) transgenic lines were significantly lower. Quantitative real-time polymerase chain reaction demonstrated that overexpression and RNAi of TabHLH1 in T. antungense significantly affected CGA and luteolin concentrations by upregulating or downregulating CGA and luteolin biosynthesis pathway genes, especially TaHQT2, 4-coumarate-CoA ligase (Ta4CL), chalcone isomerase (TaCHI), and flavonoid-3′-hydroxylase (TaF3′H). Dual-luciferase, yeast one-hybrid, and electrophoretic mobility shift assays indicated that TabHLH1 directly bound to the bHLH-binding motifs of proTaHQT2 and proTa4CL. This study suggests that TabHLH1 participates in the regulatory network of CGA and luteolin biosynthesis in T. antungense and might be useful for metabolic engineering to promote plant polyphenol biosynthesis.
Highlights
Dandelions (Taraxacum spp.) have been used as medicinal herbs and functional foods for several centuries[1,2]
Isolation and characterization of TabHLH1 To identify basic helix-loop-helix (bHLH) transcription factor (TF) involved in chlorogenic acid (CGA) biosynthesis of
The results showed that pMutantTaHQT2 interacted with the isolated gene coding protein
Summary
Dandelions (Taraxacum spp.) have been used as medicinal herbs and functional foods for several centuries[1,2]. In the first three steps of polyphenol biosynthesis, the key enzymes are phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), and cinnamate 4-hydroxylase (C4H), which catalyze the synthesis of p-coumaroyl-CoA from phenylalanine[19]. PAL, CHS, CHI, and F3′H increase CGA and luteolin concentrations in L. japonica, whereas in Fagopyrum tataricum, the rutin concentration is increased by upregulation of FLS and UFGT gene expression[25,26]. Identification of these polyphenol biosynthesis pathway genes will lay the foundation for further genetic engineering research in T. antungense (Fig. 1)
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