Table 1.
Primers used in this research.
Fig 1.
Phylogenetic analysis of 178 bHLH members including 162 from Arabidopsis, 14 from other plants, and 2 from chrysanthemum.
The two bHLHs from chrysanthemum are marked with solid circles, while those related to anthocyanin biosynthesis regulation in Arabidopsis and other species are marked with open circles. Genes IDs are as follows: NtAn1b (Nicotiana tabacum, GenBank accession number AEE99258), NtAN1-like (N. tomentosiformis, AEE99260), NtAn1a (N. tabacum, AEE99257), AN1 (Petunia hybrid, AF260918), MrbHLH1 (Myrica rubra, JX629461), LjTT8 (Lotus japonicus, BAH28881), MdbHLH3 (Malus domestica, HM122458), DvIVS (Dahlia variabilis, AB601005), AmDELILA (Antirrhinum majus, AAA32663), R-S (Zea mays, X15806), L-c (Z. mays, NM001111869), MrbHLH2 (M. rubra, JX629462), MdbHLH33 (M. domestica, DQ266451), VvMYC (Vitis vinifera, EU447172).
Fig 2.
The anthocyanin contents of flowers of three different chrysanthemum cultivars.
(a) The photographs of three chrysanthemum cultivars, named ‘Z1’, ‘Z2’ and ‘Z3’, respectively, where the bar represents 1 cm. (b) Anthocyanin contents in flower petals from Z1, Z2 and Z3. The vertical bars represent S.E. of three biological replicates.
Fig 3.
The transcript levels of seven anthocyanin biosynthetic genes and three TFs in the ray florets of three chrysanthemum cultivars used in this study.
The CmACT gene was used to normalize expression of the genes. Non-template reactions were set as the negative control for each gene. The vertical bars represent S.E. of three biological replicates.
Fig 4.
In vivo interactions between CmMYB6, CmbHLHs and the CmDFR promoter were revealed by dual luciferase assays in tobacco leaves.
Error bars are the S.E. of three independent experiments with at least four replicates.
Fig 5.
Characterization of the interaction of three TFs with the CmDFR promoter by yeast one-hybrid assays.
P53 and its promoter supplied with the kit were used as a positive control to verify the stability of these assays. The auto-activity of CmDFR promoter bait stain was tested on SD media lacking Ura in presence of AbA. CmbHLH1, CmbHLH2 and CmMYB6 were fused into pGADT7 (AD) and transformed separately into the bait stain. The binding was screened on SD media lacking Leu in presence of AbA175.
Fig 6.
Protein-protein interactions between CmbHLHs and CmMYB6 studied by yeast two-hybrid assay.
The yeast strain was co-transformed with the indicated combinations of CmbHLH1 or CmbHLH2 fused into pGBKT7 (BD) and CmMYB6 fused into pGADT7 (AD). BD-p53 and AD-T were used as positive controls, while BD-Lam and AD-T were used as negative controls. Protein-protein interactions were detected on DDO (SD media lacking Leu and Trp) and QDO/X/A (SD media lacking Leu, Trp, His and Ade, with AbA and X-α-gal) media.
Fig 7.
Transient over-expression of CmMYB6 and CmbHLH1 or CmbHLH2 carried out in tobacco leaves.
(a) Photographs taken 8 days after the infiltration. (b) Analysis of anthocyanin contents of tobacco leaves 8 days after infiltration with the TFs. Each experiment was carried out with three biological repeats and the error bars represent the S.E. of these replicate reactions.