Abstract
Anthocyanin biosynthesis requires the MYB-bHLH-WD40 protein complex to activate the late biosynthetic genes. LcMYB1 was thought to act as key regulator in anthocyanin biosynthesis of litchi. However, basic helix-loop-helix proteins (bHLHs) as partners have not been identified yet. The present study describes the functional characterization of three litchi bHLH candidate anthocyanin regulators, LcbHLH1, LcbHLH2, and LcbHLH3. Although these three litchi bHLHs phylogenetically clustered with bHLH proteins involved in anthcoyanin biosynthesis in other plant, only LcbHLH1 and LcbHLH3 were found to localize in the nucleus and physically interact with LcMYB1. The transcription levels of all these bHLHs were not coordinated with anthocyanin accumulation in different tissues and during development. However, when co-infiltrated with LcMYB1, both LcbHLH1 and LcbHLH3 enhanced anthocyanin accumulation in tobacco leaves with LcbHLH3 being the best inducer. Significant accumulation of anthocyanins in leaves transformed with the combination of LcMYB1 and LcbHLH3 were noticed, and this was associated with the up-regulation of two tobacco endogenous bHLH regulators, NtAn1a and NtAn1b, and late structural genes, like NtDFR and NtANS. Significant activity of the ANS promoter was observed in transient expression assays either with LcMYB1-LcbHLH1 or LcMYB1-LcbHLH3, while only minute activity was detected after transformation with only LcMYB1. In contrast, no activity was measured after induction with the combination of LcbHLH2 and LcMYB1. Higher DFR expression was also oberseved in paralleling with higher anthocyanins in co-transformed lines. LcbHLH1 and LcbHLH3 are essential partner of LcMYB1 in regulating the anthocyanin production in tobacco and probably also in litchi. The LcMYB1-LcbHLH complex enhanced anthocyanin accumulation may associate with activating the transcription of DFR and ANS.
Highlights
Among the pigments that confer color to plants, anthocyanins are of particular interest because they are responsible for most of the red, blue, or black color in plants, and for the beneficial effects on plant physiological processes and human health (Winkel, 2006)
Three putative members of the basic helix-loop-helix (bHLH) family of transcription factors were identified from the litchi pericarp transcriptomic (Lai et al, 2015) and genomic database1, denominated as LcbHLH1, LcbHLH2, and LcbHLH3
Three conserved motifs are identified by sequence alignments of LcbHLH1, LcbHLH2, LcbHLH3, and other bHLH transcription factor proteins related to plant anthocyanin biosynthesis (Figure 1)
Summary
Among the pigments that confer color to plants, anthocyanins are of particular interest because they are responsible for most of the red, blue, or black color in plants, and for the beneficial effects on plant physiological processes and human health (Winkel, 2006). Research on model plants has shown that the expression of structural anthocyanin genes, late genes, are orchestrated by a so-called MBW ternary complex, which is composed of MYB and bHLH transcription factors, together with WD40 repeat proteins (Broun, 2005; Koes et al, 2005; Hichri et al, 2011). R2R3 MYBs are considered to be key transcription factors known as the regulators of anthocyanin biosynthesis. MYBs in determining anthocyanin biosynthesis have been well characterized in model plants and fruit trees, such as Arabidopsis (Borevitz et al, 2000), antirrhinum (Schwinn et al, 2006), petunia (Quattrocchio et al, 1999), apple (Ban et al, 2007; Chagne et al, 2007, 2013), pear (Feng et al, 2010), grape (Kobayashi et al, 2002), litchi (Lai et al, 2014), mangosteen (Palapol et al, 2009), and Chinese bayberry (Niu et al, 2010). The R3 domain of MYBs suggests protein–protein interaction, especially with the bHLH co-factor, known as MYC (Grotewold et al, 2000; Zimmermann et al, 2004)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
