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

Abstract

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.

Functional Differentiation of Duplicated Flavonoid 3-O-Glycosyltransferases in the Flavonol and Anthocyanin Biosynthesis of Freesia hybrida

“…‘Red River ® ’ and ‘Ambiance’, cultivars of F. hybrida with red and white flowers, were grown in a greenhouse with a 14-h:10-h (light/dark) photoperiod at about 15°C. For detecting spatiotemporal expression profiles, five floral developmental stages and eight kinds of tissues or organs of Red River ® were sampled as mentioned in earlier studies (Sui et al, 2011; Sun et al, 2015; Sun et al, 2016; Sun et al, 2017; Li, Y. et al, 2016; Li et al, 2017; Gao et al, 2018; Ju et al, 2018; Li et al, 2019). For gene expression analysis between Red River ® and Ambiance, flowers fully bloomed on the first day were collected.…”

“…To mine the potential Fh3GT -like genes, the amino acid sequence of the earlier characterized Fh3GT1 (GenBank: ADK75021.1) was used as a bait sequence to conduct in situ TBLASTN screen against the Freesia transcriptomic database mentioned in our earlier studies (Li, Y. et al, 2016; Li et al, 2019). Manual BLASTX search of the National Center for Biotechnology Information (NCBI) was then carried out for the candidate gene screen.…”

“…Till now, a versatile transient protoplast transfection assay was established based on the protoplasts isolated from Freesia callus (Shan et al, 2019a). Moreover, four regulatory genes and seven anthocyanin biosynthetic genes, including one glycosyltransferase gene Fh3GT1 shown to prefer UDP-glucose as sugar donor to glycosylate anthocyanidins and flavonols, were cloned and characterized (Sui et al, 2011; Sun et al, 2015; Sun et al, 2016, Sun et al, 2017; Li, Y. et al, 2016; Li et al, 2017; Li et al, 2019; Ju et al, 2018; Shan et al, 2019b). However, the expression patterns and enzymatic characteristics of Fh3GT1 could not coincide well with flavonol accumulations during flower development, which suggested the existence of other potentially uncharacterized UF3GT genes preferentially linked to flavonol glycosylation.…”

The proanthocyanin-related transcription factors MYBC1 and WRKY44 regulate branch points in the kiwifruit anthocyanin pathway

“…VvMYB5b was also able to activate the promoters of structural genes involved in the anthocyanin and PA pathways such as VvLAR1, VvANS, VvANR and VvF3′5′H, suggesting a regulatory role in different branches of the phenylpropanoid pathway. Tea CsMYB5a and CsMYB5e and freesia FhMYB5 all demonstrated regulatory roles in the anthocyanin www.nature.com/scientificreports/ and PA biosynthesis pathway 33,34 . Recently, in red-centred kiwifruit A. chinensis 'Hongyang' , a TT2 type R2R3 MYB TF, AcMYB123 and a bHLH TF, AcbHLH42 were identified to be involved in the inner pericarp-specific accumulation of anthocyanins by activating the expression of AcANS and AcF3GT 42 .…”

“…In poplar, MYB6 encodes for a R2R3 MYB TF that is homologous to VvMYB5, and regulates anthocyanin and PA biosynthesis as well as lignin biosynthesis, a side branch of the general phenylpropanoid pathway 32 . In Freesia hybrida , the R2R3 MYB TF, FhMYB5, clusters with VvMYB5b and MYBs associated with PA regulators and not with the anthocyanin-related subgroup 6 33 . The spatio-temporal expression of FhMYB5 correlates with both anthocyanin and PA accumulation, and its expression significantly up-regulates the expression of F3′H , F3′5′H , DFR , LDOX , in the presence of bHLH partners.…”