A functional homologue of Arabidopsis TTG1 from Freesia interacts with bHLH proteins to regulate anthocyanin and proanthocyanidin biosynthesis in both Freesia hybrida and Arabidopsis thaliana.

The MBW complex, consisting of MYB, basic helix-loop-helix (bHLH) and WD40 proteins, regulates multiple traits in plants, such as anthocyanin and proanthocyanidin biosynthesis and cell fate determination. The complex has been widely identified in dicot plants, whereas few studies are concentrated on monocot plants which are of crucial importance to decipher its functional diversities among angiosperms during evolution. In present study, a WD40 gene from Freesia hybrida, designated as FhTTG1, was cloned and functionally characterized. Real-time PCR analysis indicated that it was expressed synchronously with the accumulation of both proanthocyanidins and anthocyanins in Freesia flowers. Transient protoplast transfection and biomolecular fluorescence complementation (BiFC) assays demonstrated that FhTTG1 could interact with FhbHLH proteins (FhTT8L and FhGL3L) to constitute the MBW complex. Moreover, the transportation of FhTTG1 to nucleus was found to rely on FhbHLH factors. Outstandingly, FhTTG1 could highly activate the anthocyanin or proanthocyanidin biosynthesis related gene promoters when co-transfected with MYB and bHLH partners, implying that FhTTG1 functioned as a member of MBW complex to control the anthocyanin or proanthocyanidin biosynthesis in Freesia hybrida. Further ectopic expression assays in Arabidopsis ttg1-1 showed the defective phenotypes of ttg1-1 were partially restored. Molecular biological assays validated FhTTG1 might interact with the endogenous bHLH factors to up-regulate genes responsible for anthocyanin and proanthocyanidin biosynthesis and trichome formation, indicating that FhTTG1 might perform exchangeable roles with AtTTG1. These results will not only contribute to the characterization of FhTTG1 in Freesia but also shed light on the establishment of flavonoid regulatory system in monocot plants, especially in Freesia hybrida.

The R2R3-MYB Factor FhMYB5 From Freesia hybrida Contributes to the Regulation of Anthocyanin and Proanthocyanidin Biosynthesis.

The flavonoids are important and nourishing compounds for plants and human. The transcription regulation of anthocyanin and proanthocyanidin (PA) biosynthesis was extensively studied in dicot compared with monocot plants. In this study, we characterized the functionality of an R2R3-MYB gene FhMYB5 from the monocotyledonous flowering plant of Iridaceae, Freesia hybrida. Multiple sequence alignment and phylogenetic analysis implied that FhMYB5 was clustered into grapevine VvMYB5b subclade. Correlation analysis indicated that the spatio-temporal expression patterns of FhMYB5 coincided well with anthocyanin and PA accumulations in Freesia per se. Furthermore, transient transfection assays in Freesia protoplasts revealed that the late flavonoid biosynthetic genes (e.g., DFR and LDOX) were slightly up-regulated by FhMYB5 alone, whereas both early and late biosynthetic genes were significantly activated when FhMYB5 were co-infected with either of the two IIIf clade bHLH genes, FhTT8L and FhGL3L. Moreover, these results were further confirmed by co-transfection of FhMYB5 with either of the bHLH genes aforementioned into protoplasts expressing GUS reporter gene driven by Freesia promoters. In addition, the overexpression of FhMYB5 in tobacco and Arabidopsis could also significantly up-regulate the expression of genes participating in the general flavonoid pathway. In conclusion, FhMYB5 was proved to function in the general flavonoid pathway in Freesia. The results implied a function conservation of flavonoid biosynthesis related MYB regulators in angiosperm plants.