Allelic Variation and Transcriptional Isoforms of Wheat TaMYC1 Gene Regulating Anthocyanin Synthesis in Pericarp

Abstract

Recently the TaMYC1 gene encoding bHLH transcription factor has been isolated from the bread wheat (Triticum aestivum L.) genome and shown to co-locate with the Pp3 gene conferring purple pericarp color. As a functional evidence of TaMYC1 and Pp3 being the same, higher transcriptional activity of the TaMYC1 gene in colored pericarp compared to uncolored one has been demonstrated. In the current study, we present additional strong evidences of TaMYC1 to be a synonym of Pp3. Furthermore, we have found differences between dominant and recessive Pp3(TaMyc1) alleles. Light enhancement of TaMYC1 transcription was paralleled with increased AP accumulation only in purple-grain wheat. Coexpression of TaMYC1 and the maize MYB TF gene ZmC1 induced AP accumulation in the coleoptile of white-grain wheat. Suppression of TaMYC1 significantly reduced AP content in purple grains. Two distinct TaMYC1 alleles (TaMYC1p and TaMYC1w) were isolated from purple- and white-grained wheat, respectively. A unique, compound cis-acting regulatory element had six copies in the promoter of TaMYC1p, but was present only once in TaMYC1w. Analysis of recombinant inbred lines showed that TaMYC1p was necessary but not sufficient for AP accumulation in the pericarp tissues. Examination of larger sets of germplasm lines indicated that the evolution of purple pericarp in tetraploid wheat was accompanied by the presence of TaMYC1p. Our findings may promote more systematic basic and applied studies of anthocyanins in common wheat and related Triticeae crops.

Keywords: Pp3; anthocyanin biosynthesis; bHLH transcription factor; common wheat; purple pericarp.

Figure 1

Molecular analysis of TaMYC1 transcripts and deduced protein. (A) The six transcript isoforms (I–VI) identified for TaMYC1. The number of exons (represented by filled boxes) covered by the six isoforms varied from five (Isoform IV) to nine (Isoform V). (B) Amino acid sequence comparison between TaMYC1 (deduced from Isoform III) and two representative bHLH regulators of anthocyanin biosynthesis, RS from maize and Ra from rice. The three domains (bHLH-MYC_N domain, HLH domain and ACT-like domain) conserved among known bHLH TFs regulating anthocyanin biosynthesis are underlined. The GenBank accession numbers of the three sequences are NP_001106073 (RS), AAC49219 (Ra), or KX867111 (TaMYC1). (C) Localization of TaMYC1-GFP fusion protein in the nucleus of Arabidopsis protoplast. As a control, the free GFP protein was distributed throughout the cytoplasm of the protoplast. The images shown were taken under a confocal microscope using green (for GFP fluorescence) or red (for chlorophyll fluorescence) filters or under bright field (BF). The merged images depicted more clearly the relative positions of GFP and chlorophyll fluorescence in the photographed protoplasts.

Figure 2

Transcriptional characteristics of TaMYC1. (A) Relative transcript levels of TaMYC1 in the different organs/tissues (glume, stem, leaf, pericarp, coleoptile, and root) of Gaoyuan 115 (G) and Opata (O) as assessed using semi-quantitative RT-PCR. The amplification of wheat tubulin gene served as an internal control. (B) Artificial removal of outer and inner glumes induced purple AP accumulation in the developing grains of Gaoyuan 115. Glume removal was conducted at 14 days after flowering, with AP induction becoming visible in the grains (indicated by asterisks) 2 days after the treatment. (C) Relative transcript levels of TaMYC1 and TaDFR in the grains of Gaoyuan 115 and Opata without (−) or with (+) glume removal treatment. The transcript levels were evaluated using semi-quantitative RT-PCR with the amplification of wheat tubulin gene as an internal control. The data displayed are representative of three separate tests.

Figure 3

Induction of anthocyanin biosynthesis by TaMYC1 in combination with ZmC1. The expression constructs of ZmC1, ZmR, and the six transcript isoforms of TaMYC1 (Isoforms I–VI) were delivered into the coleoptile cells of the white-grain wheat Opata with particle bombardment in appropriate combinations or singularly. The presence of red colored cells in the bombarded coleoptiles indicates induction of anthocyanin biosynthesis. Of the six different types of transcripts of TaMYC1, only Isoform III induced anthocyanin biosynthesis in combination with ZmC1. The results shown are typical of three independent assays.

Figure 4

Analysis of the function of TaMYC1 in regulating anthocyanin biosynthesis using virus induced gene silencing. Three recombinant barley stripe mosaic viruses (BSMV:GFP, BSMV:PDSas, and BSMV:TaMYC1as) were used in this experiment. Wheat plants (cv Gaoyuan 115) were inoculated with the three viruses, respectively. The developing grains were used for the experiment 14 days after the flowering. (A) GFP fluorescence was detected in the developing grains of the plants infected by BSMV:GFP. The images shown were taken under a confocal microscope in GFP channel and bright field (BF), respectively, followed by merging. (B) Photo bleaching was observed in the developing grains of the plants infected by BSMV:PDSas because of silencing the expression of phytoene desaturase gene. The bleaching phenotype did not occur in the developing grains of the plants infected by BSMV:GFP. (C) Evaluation of the relative transcript levels of TaMYC1 and BSMV CP gene in the developing grains of mock controls and the plants infected by BSMV:GFP or BSMV:PDSas. The grains were subjected to glume removal, and at 2 days after the treatment, they were collected for this analysis. Purple anthocyanin pigments were induced in the grains of mock controls and the plants infected by BSMV:GFP but not those infected by BSMV:TaMYC1as. Consistent with this finding, TaMYC1 transcripts accumulated in the grains of mock controls and the plants infected by BSMV:GFP, but were undetectable by RT-PCR in the grains of the plants infected by BSMV:TaMYC1as. Successful infection of the developing grains by BSMV:GFP or BSMV:PDSas was confirmed by positive detection of viral CP transcripts. The results depicted are representative of three independent experiments. (D) Comparison of anthocyanin contents among the developing grains of mock controls and the plants infected by BSMV:GFP or BSMV:TaMYC1as. The three sources of grains, as shown in (C), were individually assayed for anthocyanin content, with the averaged values (means ± SE, n = 20) being compared statistically. The means marked by different letters are statistically significant (P < 0.05). The data shown were reproducible in another two separate determinations.

Figure 5

Bioinformatic analysis of a putative, 261 nt cis-regulatory element in the promoter region of TaMYC1. (A) Presence of multiple cis-acting regulatory motifs (boxes) in the 261 nt element as predicted using the PlantCARE software. (B) A diagram illustrating copy number difference of the 261 nt element in the promoter regions of two TaMYC1 alleles (TaMYC1p and TaMYC1w) isolated from purple- and white-grained wheat cultivars, respectively. The 261 nt element had three perfect (261 nts), one nearly intact (260 nts) and two partial (27 or 205 nts) copies in the promoter of TaMYC1p, but was present only once (261 nts) in the corresponding region of TaMYC1w. ATG indicates the start codon of the coding sequence.