Two LcbHLH Transcription Factors Interacting with LcMYB1 in Regulating Late Structural Genes of Anthocyanin Biosynthesis in Nicotiana and Litchi chinensis During Anthocyanin Accumulation

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.

Identification of MicroRNAs and Their Target Genes Related to the Accumulation of Anthocyanins in Litchi chinensis by High-Throughput Sequencing and Degradome Analysis

“…Based on our previously published results, LcMYB1 might play major roles in the MBW complex determining anthocyanin accumulation in litchi (Lai et al, 2014). Recently, two LcbHLH TFs interacting with LcMYB1 in regulating late structural genes of anthocyanin biosynthesis in litchi during anthocyanin accumulation has been reported (Lai et al, 2016). In the present study, to explore interaction of LcSPL1/2 with LcMYB1 or LcbHLH1/2/3, the Y2H has been employed.…”

“…However, there was no interaction between LcSPL1/2 and LcbHLL1/2/3 ( Figure 6 ). Recently, LcbHLH1/3 interacting with LcMYB1 in regulating late structural genes of anthocyanin biosynthesis in Nicotiana and Litchi has been reported (Lai et al, 2016). In previous study, SPL9 was reported to destabilize the MBW complex to repress anthocyanin biosynthesis (Gou et al, 2011).…”

“…The entire coding sequences of LcSPL1 / 2 and LcMYB1 (without their stop codons) were amplified and subcloned into the pEAQ-CYFP and pEAQ-NYFP vectors described by Lai et al (2016). The expression of target genes alone was used as negative controls.…”

“…Apart from the delicious taste, attractive red skin of litchi is another important aspect of the fruit quality. Recently, the gene involved in anthocyanin biosynthesis and sequestration from litchi has been reported (Wei et al, 2011; Lai et al, 2014, 2015, 2016; Li et al, 2015; Hu et al, 2016). However, the mechanism of miRNAs regulating anthocyanin biosynthesis in litchi has not been reported.…”

Novel R2R3-MYB transcription factors fromPrunus americanaregulate differential patterns of anthocyanin accumulation in tobacco and citrus

“…In contrast the vegetative growth and seed set of the MoroMybA as well as the PamMybA transgenic lines were not noticeably different than wild type suggesting that they are suitable candidates for biotech applications. These results further indicate that there is no requirement for the presence of an exogenous bHLH partner for the plum MYBA.1 and MYBA.5 transcription factors to achieve full functionality in this heterologous system, unlike other MYBs from cherry plum, litchi and apple (Lai et al., 2016; Espley et al., 2007; Li et al., 2007; Takos et al., 2006). …”