Enhancing Flower Color through Simultaneous Expression of the B-peru and mPAP1 Transcription Factors under Control of a Flower-Specific Promoter

Abstract

Flower color is a main target for flower breeding. A transgenic approach for flower color modification requires a transgene and a flower-specific promoter. Here, we expressed the B-peru gene encoding a basic helix loop helix (bHLH) transcription factor (TF) together with the mPAP1 gene encoding an R2R3 MYB TF to enhance flower color in tobacco (Nicotiana tabacum L.), using the tobacco anthocyanidin synthase (ANS) promoter (PANS) to drive flower-specific expression. The transgenic tobacco plants grew normally and produced either dark pink (PANSBP_DP) or dark red (PANSBP_DR) flowers. Quantitative real time polymerase chain reaction (qPCR) revealed that the expression of five structural genes in the flavonoid biosynthetic pathway increased significantly in both PANSBP_DP and PANSBP_DR lines, compared with the non-transformed (NT) control. Interestingly, the expression of two regulatory genes constituting the active MYB-bHLH-WD40 repeat (WDR) (MBW) complex decreased significantly in the PANSBP_DR plants but not in the PANSBP_DP plants. Total flavonol and anthocyanin abundance correlated with flower color, with an increase of 1.6-43.2 fold in the PANSBP_DP plants and 2.0-124.2 fold in the PANSBP_DR plants. Our results indicate that combinatorial expression of B-peru and mPAP1 genes under control of the ANS promoter can be a useful strategy for intensifying flower color without growth retardation.

Keywords: anthocyanin; flower color; flower-specific promoter; tobacco; transcription factor.

Figure 1

Pigmentation phenotypes of PANSBP transgenic tobacco plants. Representative pigmentation phenotypes of NT and PANSBP transgenic tobacco plants are shown. NT tobacco flowers were pink due to anthocyanin accumulation. The PANSBP transgenic tobacco plants displayed a range of flower colors from dark pink to dark red due to enhanced anthocyanin accumulation in petals (A); Leaves lack visible anthocyanin pigmentation in NT and PANSBP transgenic tobacco plants (B).

Figure 2

Flower color phenotype of PANSBP transgenic tobacco plants. (A) Color comparison of mature flowers from two PANSBP transgenic lines and NT tobacco plants; (B) Flowers were hand-sectioned longitudinally to display internal components; (C) Flower color variation among phenotypes throughout the three flower developmental stages, S1 to S3.

Figure 3

Expression of B-peru and mPAP1 in transgenic tobacco plants transformed with PANSBP. qPCR analysis of the expression levels of B-peru and mPAP1 transgenes in various different tissues of PANSBP-DR line. All results represent mean values ± SD from three biological replicates.

Figure 4

Expression profiles of anthocyanin structural and regulatory genes in NT and PANSBP transgenic tobacco petals. (A) Transcript abundance of the B-peru and mPAP1 transgenes and anthocyanin structural genes. The biosynthetic pathway genes evaluated include those encoding chalcone synthase (NtCHS), chalcone isomerase (NtCHI), flavanone 3-hydroxylase (NtF3H), flavonoid 3′-hydroxylase (NtF3′H), flavonoid 3′5′-hydroxylase (NtF3′5′H), flavonol synthase (NtFLS), dihydroflavonol 4-reductase (NtDFR), anthocyanidin synthase (NtANS), and UDP-glucose: flavonoid 3-O-glucosyltransferase (NtUFGT) as well as the upstream enzyme phenylalanine ammonia-lyase (NtPAL) and 4-coumarate-CoA ligase (Nt4CL); (B) T ranscript abundances of endogenous anthocyanin regulators. The tobacco glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used to normalize the total amounts of RNA for each assay. Bar colors are indicative of flower color phenotypes, pink represents NT; dark pink, PANSBP_DP; dark red, PANSBP_DR. All results represent mean values ± SD from three biological replicates. *, ** and *** indicate values that differ significantly from NT at p < 0.05, p < 0.01, and p < 0.001, respectively, according to a Student’s paired t-test.

Figure 5

Analysis of dihydroflavonol and flavonol levels in the petals of non-transformed (NT) and PANSBP transgenic tobacco. (A) Dihydroflavonol levels in NT and transgenic lines (DHQ, dihydroquercetin; DHK, dihydrokaempferol); (B) Flavonols levels in NT and transgenic lines (Q, quercetin; K, kaempferol). All results represent mean values ± SD from three biological replicates. *, ** and *** indicate values that differ significantly from NT at p < 0.05, p < 0.01 and at p < 0.001, respectively, according to a Student’s paired t-test.

Figure 6

Analysis of anthocyanin levels in the petals of non-transformed (NT) and PANSBP transgenic lines. (A) UPLC chromatogram of anthocyanins extracted from the petals of PANSBP_DR line; (B) TOF (quadrupole time of flight) mass spectra of the peak 1 and peak 2; (C) Anthocyanin levels in NT and transgenic lines (Dp, delphinidin derivative; Cy3R, cyanidin-3-O-rutinoside). The result of (C) represents mean values ± SD from three biological replicates. *** Indicate values that differ significantly from NT at p < 0.001 according to a Student’s paired t-test.