Functional characterization of a liverworts bHLH transcription factor involved in the regulation of bisbibenzyls and flavonoids biosynthesis

Background 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. In liverworts, both bisbibenzyls and flavonoids are derived through the phenylpropanoids pathway and share several upstream enzymes. Results In 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. Conclusion PabHLH1 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.

The Heterologous Expression of a Chrysanthemum nankingense TCP Transcription Factor Blocks Cell Division in Yeast and Arabidopsis thaliana

Both the presence of, and the important contribution to growth and development made by TCP transcription factors, have been established in various plant species. Here, a TCP4 homolog isolated from Chrysanthemum nankingense was shown to be more strongly transcribed in the diploid than in the autotetraploid form of the species. CnTCP4 was shown to encode a member of the class II TCP family and to be transcribed most strongly in the leaf and ligulate flowers. Its transcription was found to be substantially inhibited by spraying the plant with the synthetic cytokinin 6-benzylaminopurine. The transient expression of CnTCP4 in onion epidermal cells showed that its product localized to the nucleus, and a yeast one hybrid assay suggested that its product had transcriptional activation ability. The constitutive expression of CnTCP4 in fission yeast suppressed cell proliferation, inducing the formation of longer and a higher frequency of multinuclated cells. Its constitutive expression in Arabidopsis thaliana reduced the size of the leaves. The presence of the transgene altered the transcription of a number of cell division-related genes. A yeast one hybrid assay identified a second TCP gene (CnTCP2) able to interact with the CnTCP4 promoter. A transient expression experiment in Nicotiana benthamiana leaves showed that CnTCP2 was able to activate the CnTCP4 promoter. Like CnTCP4, CnTCP2 was shown to encode a member of the class II TCP family, to be transcribed most strongly in the leaf and ligulate flowers, and to be suppressed by exogenous 6-benzylaminopurine treatment. The CnTCP2 protein also localized to the nucleus, but had no transcriptional activation ability. Its constitutive expression in A. thaliana had similar phenotypic consequences to those induced by CnTCP4.