Abstract
BackgroundThe basic helix-loop-helix (bHLH) transcription factors (TFs), as one of the largest families of TFs, play important roles in the regulation of many secondary metabolites including flavonoids. Their involvement in flavonoids synthesis is well established in vascular plants, but not as yet in the bryophytes. In liverworts, both bisbibenzyls and flavonoids are derived through the phenylpropanoids pathway and share several upstream enzymes.ResultsIn this study, we cloned and characterized the function of PabHLH1, a bHLH family protein encoded by the liverworts species Plagiochasma appendiculatum. PabHLH1 is phylogenetically related to the IIIf subfamily bHLHs involved in flavonoids biosynthesis. A transient expression experiment showed that PabHLH1 is deposited in the nucleus and cytoplasm, while the yeast one hybrid assay showed that it has transactivational activity. When PabHLH1 was overexpressed in P. appendiculatum thallus, a positive correlation was established between the content of bibenzyls and flavonoids and the transcriptional abundance of corresponding genes involved in the biosynthesis pathway of these compounds. The heterologous expression of PabHLH1 in Arabidopsis thaliana resulted in the activation of flavonoids and anthocyanins synthesis, involving the up-regulation of structural genes acting both early and late in the flavonoids synthesis pathway. The transcription level of PabHLH1 in P. appendiculatum thallus responded positively to stress induced by either exposure to UV radiation or treatment with salicylic acid.ConclusionPabHLH1 was involved in the regulation of the biosynthesis of flavonoids as well as bibenzyls in liverworts and stimulated the accumulation of the flavonols and anthocyanins in Arabidopsis.
Highlights
The basic helix-loop-helix transcription factors (TFs), as one of the largest families of TFs, play important roles in the regulation of many secondary metabolites including flavonoids
A phylogenetic analysis involving the sub-group IIIf of plant basic helix-loop-helix (bHLH) divided the proteins into two clades (Fig. 3): clade I included AtTT8, maize IN1, grapevine MYC1 and rice Rc, all of which are involved in the regulation of flavonoids biosynthesis, while clade II included transcription factors Petunia JAF13, Arabidopsis GL3, Snapdragon Delila (Del) and Maize Lc
The increase in flavonoid content induced by PabHLH1 over-expression was accompanied by a significant up-regulation of the genes encoding the flavonoid synthesis enzymes Chalcone isomerase (CHI) and Chalcone synthase (CHS)
Summary
The basic helix-loop-helix (bHLH) transcription factors (TFs), as one of the largest families of TFs, play important roles in the regulation of many secondary metabolites including flavonoids. Their involvement in flavonoids synthesis is well established in vascular plants, but not as yet in the bryophytes. The structural genes responsible for flavonoids biosynthesis are regulated largely at the transcriptional level, controlled by two classes of transcription factors (TF) (MYB and bHLH) along with the WD40 proteins. The products of Rc and Rd are bHLH transcription factors which control proanthocyanidins synthesis in the grain pericarp, while the tobacco genes NtAn1a and NtAn1b encode enhancers of anthocyanin accumulations in the flower [21]. The two grapevine bHLH proteins VvMYC1 and VvMYCA1 are required for the production of, respectively, anthocyanins and proanthocyanidins [24, 25]
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