Two transcription factors TaPpm1 and TaPpb1 co-regulate anthocyanin biosynthesis in purple pericarps of wheat

Abstract

Purple pericarps of bread wheat (Triticum aestivum L.) are a useful source of dietary anthocyanins. Previous mapping results indicated that the purple pericarp trait is controlled by two complementary genes located on chromosomes 7D and 2A. However, the identity of the genes and the mechanisms by which they regulate the trait are unknown. In this study, two transcription factors were characterised as anthocyanin activators in purple pericarps: TaPpm1 (purple pericarp-MYB 1) and TaPpb1 (purple pericarp-bHLH 1). Three non-functional variants were detected in the coding sequence of TaPpm1 from non-purple seed lines, in which the function of TaPpm1 was destroyed either by insertion-induced frame shifts or truncated peptides. There were six 261-bp tandem repeats in the promoter region of TaPpb1 in the purple-grained varieties, while there was only one repeat unit present in the non-purple varieties. Furthermore, using yeast two-hybrid, dual luciferase, yeast one-hybrid, and transient assays, we were able to demonstrate that the interaction of TaPpm1 and TaPpb1 co-regulates the synthesis of anthocyanin. Overall, our results provide a better understanding of the molecular basis of anthocyanin synthesis in the wheat pericarp and indicate the existence of an integrated regulatory mechanism that controls production.

Fig. 1.

Pericarp samples of variety H76 at different stages under normal and high-light conditions. H-10d, 10 d after pollination (DAP) under normal conditions; H-10p, 10 DAP directly exposed in sunlight for 12 h; H-17d, 17 DAP under normal conditions. Both H-10p and H-17d had anthocyanin accumulation in the pericarps.

Fig. 2.

Phylogenetic analyses of the transcription factors (TFs) MYB and bHLH, and validation of the expression levels of four TFs related to anthocyanin synthesis in wheat. (A) A total of 26 TaMYBs (top) and 24 TabHLHs (bottom) protein sequences derived from wheat transcriptome data are labelled with dots. Proteins labelled with red dots belong to the clades of anthocyanin synthesis. (B) qRT-PCR validation of the four unigenes c53583_g3_i3, c53583_g3_i2, c53583_g3_i1, and c53583_g8_i1 (designated as TaPpm1, TaPpm2, TaPpm3, and TaPpb1, respectively). Expression is relative to that of TaActin.

Fig. 3.

Expression patterns of the three wheat TaPpms and TaPpb1. (A) Relative expression of transcription factor (TF) genes in different tissues of the two wheat lines, A14 (white pericarp) and H76 (purple pericarp). (B) Relative expression of TF genes at four physiological stages (days after pollination) during pericarp development. Data are means (±SD) of three biological replicates. Expression is relative to that of TaActin.

Fig. 4.

The variants of TaPpm1 and TaPpb1 and their distribution in the 34 wheat lines examined. (A) The genetic and transcript structures of the four variants of TaPpm1. (B) The genetic structures and the rearranged promoters of two TaPpb1 variants (TaPpb1a and TaPpb1b). The purple boxes indicate the duplicated 261-bp units in the promoter region of TaPpb1a or TaPpb1b. In the fourth 261-bp unit, the 259-bp section belongs to the insertion region. The ‘C’ base, highlighted in red, in the fourth 261-bp unit is a transition from the ‘T’ base compared with other identical 261-bp units. (C) Different allelic variants of TaPpm1 and TaPpb1 were distinguished in 34 wheat varieties using specific markers. Varieties highlighted in purple have purple pericarps. The specific primer set 7D02 could distinguish TaPpm1c and TaPpm1d from the other two allelic variants (TaPpm1a and TaPpm1b). Amplicons of the dCAPS primer set 7D03 were digested by the DraIII enzyme to recognize TaPpm1a and TaPpm1b genotypes. TaPpb1a and TaPpb1b variants could be distinguished by 2APRO1.

Fig. 5.

Association analysis between genotypes of TaPpm1 and TaPpb1 and the pericarp colours in F₂ individuals. TaPpm1a and TaPpb1a highlighted in red are dominant to TaPpm1d and TaPpb1b, respectively. (A) Genotypes of TaPpm1 and TaPpb1 in different-coloured F₂ progenies of the ‘A14×H76’ population. TaPpm1a presents a 967-bp sequence while TaPpm1d presents a 2960-bp fragment amplified with the specific primer set 7D02. TaPpb1a yields a 3417-bp fragment and TaPpb1b presents only a short 2143-bp fragment using 2APRO1. A 2143-bp and a 1996-bp fragment were amplified from heterozygous TaPpb1 (TaPpb1a/TaPpb1b) by the primer sets 2APRO1 and 2APRO2, respectively. (B) The expression of TaPpm1, TaPpb1, and six structural genes was detected in F₂ individuals with diverse genotypes of TaPpm1 and TaPpb1. Expression is relative to that of TaActin.

Fig. 6.

Detection of the interactions between TaPpm1s and TaPpb1 via yeast two-hybrid screening. (A) Interaction of TaPpm1s (TaPpm1a, TaPpm1b, TaPpm1c, and TaPpm1d) with truncated TaPpb1 protein in the yeast cells. TaPpm1s were fused to the GAL4 activation domain whereas a truncated form of TaPpb1 (1–642 bp, encoding 214 amino acids) was fused to the GAL4 binding domain. Yeast clones transformed with different constructs were grown on DDO (SD/–Leu/–Trp), QDO (SD/–Leu/–Trp/–Ade/–His) and QDO/X/A (QDO supplemented with ABA and X-α-Gal) plates for 5 d. Yeast cells transformed with the AD-T-antigen and BD-Lamin were used as a negative control, while yeast cells transformed with the AD-T-antigen and BD-P53 served as a positive control. (B) Serial dilutions (10^–1, 10^–2) of the yeast cells with different combinations (AD-TaPpm1a with BD-TaPpb1₍₆₄₂₎ or AD-TaPpm1b with BD-TaPpb1₍₆₄₂₎) were cultured on DDO, QDO, or QDO/X/A for 1 d to detect their interaction abilities. (This figure is available in colour at JXB online.)

Fig. 7.

Transient expression of TaPpm1s and TaPpb1 genes in wheat calluses and detection of their activation features by dual-luciferase assays. (A) The phenotypes of calluses bombarded with different configurations of TaPpm1s, TaPpb1, and the empty vector pCXSN. (B) The expression of TaPpm1, TaPpb1, and seven structural genes in calluses bombarded with TaPpm1s and TaPpb1. All structural genes were selected from unigenes of the transcriptome data with high expression in purple samples. The expression of transcription factor genes and structural genes are distinguished with black and grey bars, respectively. Expression is relative to that of TaActin. (C) Promoter activation assays in Nicotiana benthamiana leaves using a dual-luciferase assay. Firefly luciferase activity was normalized to the Renilla internal control (LUC/REN). Data are means (±SD) determined from four replicates.