A bHLH transcription factor, DvIVS, is involved in regulation of anthocyanin synthesis in dahlia (Dahlia variabilis)

Abstract

Dahlias (Dahlia variabilis) exhibit a wide range of flower colours because of accumulation of anthocyanin and other flavonoids in their ray florets. Two lateral mutants were used that spontaneously occurred in 'Michael J' (MJW) which has yellow ray florets with orange variegation. MJOr, a bud mutant producing completely orange ray florets, accumulates anthocyanins, flavones, and butein, and MJY, another mutant producing completely yellow ray florets, accumulates flavones and butein. Reverse transcription-PCR analysis showed that expression of chalcone synthase 1 (DvCHS1), flavanone 3-hydroxylase (DvF3H), dihydroflavonol 4-reductase (DvDFR), anthocyanidin synthase (DvANS), and DvIVS encoding a basic helix-loop-helix transcription factor were suppressed, whereas that of chalcone isomerase (DvCHI) and DvCHS2, another CHS with 69% nucleotide identity with DvCHS1, was not suppressed in the yellow ray florets of MJY. A 5.4 kb CACTA superfamily transposable element, transposable element of Dahlia variabilis 1 (Tdv1), was found in the fourth intron of the DvIVS gene of MJW and MJY, and footprints of Tdv1 were detected in the variegated flowers of MJW. It is shown that only one type of DvIVS gene was expressed in MJOr, whereas these plants are likely to have three types of the DvIVS gene. On the basis of these results, the mechanism regulating the formation of orange and yellow ray florets in dahlia is discussed.

Fig. 1.

Simplified flavonoid synthesis pathways. Only compounds detected in ‘Michael J’ are shown. ANS, anthocyanidin synthase; CH3H, chalcone 3-hydroxylase; CHI, chalcone isomerase; CHR, chalcone reductase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; FNS, flavone synthase.

Fig. 2.

Inflorescence phenotypes of ‘Michael J’ (MJW) and its bud mutants: (A) MJW, (B) MJOr, and (C) MJY. (A) MJW ray florets are bright yellow with a small number of brilliant orange markings. (B) MJOr ray florets are brilliant orange; this colour is mainly derived from anthocyanins and butein. (C) MJY ray florets are bright yellow; this colour is mainly derived from butein.

Fig. 3.

Temporal expression of anthocyanin synthesis pathway genes and transcription factors. (A) Developmental stages of ray florets. Developmental stages of MJOr ray florets were defined on the basis of the degree of colouration. (B) Semi-quantitative RT-PCR analysis of temporal expression of anthocyanin synthesis genes and transcription factors in MJOr and MJY. The constitutively expressed gene for actin in Dahlia variabilis was used as an internal control. (C) Relative expression levels of DvCHS1, DvCHS2, and DvIVS in MJOr compared with those in MJY at each ray floret developmental stage. The vertical bars are the standard error (±SE) of the means of three biological replicates. The constitutively expressed gene for actin in D. variabilis was used as an internal control.

Fig. 4.

Phylogenetic analysis of DvIVS. (A) Amino acid comparison of the bHLH domain of DvIVS, InIVS, PhAN1, and AtTT8. Numbers (*) indicate amino acids that are fully conserved in each of the proteins. (B) Phylogenetic tree for bHLH transcription factors associated with anthocyanin synthesis pathways. The entire amino acid sequences were aligned using ClustalW, and the tree was constructed by the Neighbor–Joining method. Bootstrap values of 1000 retrials are indicated on each branch, and the scale shows 0.1 amino acid substitutions per site. The abbreviations shown in front of each protein indicate the plant species: Dv, Dahlia variabilis; Am, Antirrhinum majus; At, Arabidopsis thaliana; In, Ipomoea nil; Pf, Perilla frutescens; Ph, Petunia hybrida; Zm, Zea mays. The accession number of each protein is: DvIVS (BAJ33515), DvDEL (BAJ33516), AmDEL (AAA32663), AtEGL3 (NP_176552), AtGL3 (NP_680372), AtTT8 (CAC14865), InDEL (BAE94393), InIVS (BAE94394), PfF3G1 (BAC56998), PhAN1 (AAG25927), PhJAF13 (AAC39455), and ZmIN1 (AAB03841).

Fig. 5.

Structure of the DvIVS gene. (A) Structure of the DvIVS gene and genomic PCR. Rectangles with roman figures indicate exons. The bHLH domain is located in the eighth exon. Tdv1 is inserted in the fourth intron in the antisense direction of the MJY genome. Arrows indicate the primer set used for genomic PCR. M indicates a 1 kb marker. (B) Footprints of Tdv1. The uppermost sequence is assumed to be the standard sequence. Sequences in the black box indicate the Tdv1 sequence. With respect to MJW, Tdv1-inserted sequences were not analysed. (C) Truncated transcripts of DvIVS in MJOr and MJY. Grey rectangles indicate odd numbered exons and black rectangles indicate even numbered exons. Arrows indicate primer sets used for RT-PCR.

Fig. 6.

The putative model of anthocyanin and butein synthesis in MJOr and MJY. For simplicity, the 3′ hydroxylation pathway is abbreviated. (A) The model for MJOr. DvIVS can function, then DvCHS1, DvF3H, DvDFR, and DvANS are activated, and anthocyanidin, flavone, and butein are synthesized. (B) The model for MJY. Because of Tdv1 insertion, only truncated DvIVS mRNA is expressed and transcriptional regulation cannot function. Although DvCHS1, DvF3H, DvDFR, and DvANS are not activated, DvCHS2 and DvCHI are not affected by DvIVS, resulting in accumulation of flavone and butein.