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.

Keywords: Litchi chinensis; MYB; anthocyanins; bHLH; interaction; tobacco.

FIGURE 1

Protein sequence alignment of three LcbHLH proteins and the known anthocyanin bHLH regulators in other species. Identical residues are shown in black and conserved residues in dark gray. MYB interaction region, bHLH domain and ACT-like domain are conserved among these bHLH transcription factors.

FIGURE 2

Phylogenetic relationships between LcbHLH1-3 and anthocyanin-related bHLHs in other species. The tree was constructed using MEGA 5, neighboring-joining phylogeny testing, and 1,000 bootstrap replicates. The accession number of these proteins (or translated products) are as follows in the GenBank database:AtTT8, CAC14865.1; AtGL3, NP_680372; AtEGL3, NP_176552; MdbHLH33, ABB84474.1; PhJAF13, AAC39455; IpIVS, BAD18982.1; VvMYCA1, NP_001267954.1; CsMYC2, ABR68793.1; PhAN1, AAG25927; AmDEL, AAA32663; DvIVS, BAJ33515; DvDEL, BAJ33516; InDEL, BAE94393; ZmB, AGO65322.1; ZmLC, NP_001105339.1; InIVS, BAE94394; ZmIN1, AAB03841; MrbHLH1, JX629461; MrbHLH2, JX629462; AtMYC2, NP_174541.1; AtICE1, NM_113586.3; VvCBF1, AFI49627.1; AtPIF3, NM_179295.2; AtBEE1, AY138253.1.

FIGURE 3

Subcellular localization of LcMYB1 and LcbHLHs in N. benthamiana leaf protoplasts. Epidermal cells of N. benthamiana leaves were transiently transformed with LcMYB1–GFP and LcbHLHs–GFP constructs in Agrobacterium tumefaciens strain GV3101. GFP fluorescence was observed with a fluorescence microscope. Images were taken in a dark field for green fluorescence, while the outline of the cell and the merged were photographed in a bright field. Bars, 20 μm.

FIGURE 4

Physical interactions between LcbHLH1-3 proteins and LcMYB1 detected in Y2H assays. (A) The coding regions of full length and partitial sequence of LcMYB1 were cloned into the pGBKT7 (GAL4 DBD) vector to create the DBD-LcMYB1 and DBD-LcMYB1A-D constructs, respectively. (B) Transcriptional activation analysis of LcMYB1 and partitial sequence of LcMYB1. (C) All of the constructs together with the positive control (p-53+T-antigen) and negative control (pGBKT7) were transformed into yeast strain Gold Y2H. Yeast clones transformed with different constructs were grown on SD plates with or without tryptophan, histidine, and adenine but containing 125 μM Aureobasidin A for 3 days at 30°C. Transcription activation was monitored by the detection of yeast growth and a α-galactosidase (α-Gal) assay.

FIGURE 5

Bimolecular fluorescence complementation visualization of the LcbHLH1 and LcbHLHs interaction in N. benthamiana leaf protoplasts. YFP indicates fluorescence of YFP; Merge is digital merge of bright field and fluorescent images. Bars, 20 μm.

FIGURE 6

Expression of three litchi bHLH in relation to anthocyanin accumulation. (A) The transcript patterns of three litchi bHLHs in relation to anthocyanin accumulation among tissues. (B) The developmental transcript patterns of three litchi bHLHs in relation to anthocyanin accumulation in the pericarp of ‘Zinianxi’ and ‘Yamulong’. (C) The developmental transcript patterns of three litchi bHLHs in relation to anthocyanin accumulation in leaves of ‘Zinianxi’ and ‘Yamulong’. The vertical bars represent the standard error of triplicate experiments. Different letters on the top of columns indicate significant difference at p < 0.05.

FIGURE 7

Anthocyanin accumulation in tobacco leaves infiltrated with LcMYB1 and co-infiltrated with LcMYB1 and LcbHLHs. (A) pEAQ-HT empty vector; (B) pEAQ-LcbHLH1; (C) pEAQ-LcbHLH2; (D) pEAQ-LcbHLH3; (E) pEAQ-MYB1; (F) pEAQ-MYB1 with pEAQ-LcbHLH1; (G) pEAQ-MYB1 with pEAQ-LcbHLH2; (H) pEAQ-MYB1 with pEAQ-LcbHLH3. Pictures were taken at 4 days after infiltration. (I) Anthocyanin contents in different transiently transformed tobacco leaf patches. The vertical bars represent the standard error of triplicate experiments. Different letters on the top of columns indicate significant difference at p < 0.05.

FIGURE 8

The biosynthesis of anthocyanins in LcMYB1 or/and LcbHLH3 overexpression tobacco. (A) Color development in leaves of untransformed control and leaves transformation with LcMYB1 or/and LcbHLH3. (B) Anthocyanin contents in untransformed control and transgenic tobacco lines. (C) The expressions of exogenous litchi regulatory genes and tobacco endogenous regulatory and structural gene in the anthocyanin biosynthetic pathway in transgenic lines. The actin gene was used to normalize gene expression of the genes under identical conditions. The vertical bars represent the standard error of triplicate experiments. Different letters on the top of columns indicate significant difference at p < 0.05.

FIGURE 9

In vivo interactions between litchi transcriptional factors and promoters of anthocyanin biosynthetic genes in litchi studied by dual luciferase assay in N. benthamiana leaves. In vivo associations of MYB, bHLHs and anthocyanin biosynthetic gene promoters as revealed by transient assays. The vertical bars represent the standard error of four replicate reactions. Different letters on the top of columns indicate significant difference at p < 0.05.