PlgMYBR1, an R2R3-MYB transcription factor, plays as a negative regulator of anthocyanin biosynthesis in Platycodon grandiflorus

Abstract

Floral color plays a major role in pollinator specificity, and changes in color may result in pollinator shifts and pollinator-mediated speciation. In the purple flowers of Platycodon grandiflorus, anthocyanins are the major pigment metabolites, whereas white flowers result due to the absence of anthocyanins. The lack of anthocyanins may be due to the inhibition of the anthocyanin biosynthesis pathway. However, the molecular mechanism of anthocyanin biosynthesis in P. grandiflorus is not fully understood. Hence, we identified R2R3-MYB transcription factor, PlgMYBR1, as a negative regulator for anthocyanin biosynthesis using sequence homology and tissue-specific expression pattern analyses. A heterologous co-expression assay suggested that PlgMYBR1 inhibited the function of AtPAP1 (Arabidopsis thaliana production of anthocyanin pigment 1), indicating that PlgMYBR1 plays as a repressor of anthocyanin biosynthesis in P. grandiflorus. Our results provide a foundation for future efforts to understand the anthocyanin biosynthesis in P. grandiflorus and, thereby, to improve flower color through genetic engineering.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-023-03490-6.

Keywords: Anthocyanin; Flower color; Platycodon grandiflorus; R2R3-MYB transcription factor.

Fig. 1

Change in the color, anthocyanin content, and chlorophyll (Chl) content during flower development of P. grandiflorus. A The images of the flowers of P. grandiflorus varieties. S1: < 1 cm diameter, S2: 1–2 cm diameter, S3: 2–3 cm diameter, and S4: fully open. Analyses of anthocyanin accumulation (B) and total Chl contents (C) during the flower development in both varieties. The data are representative of three independent experiments (mean ± SE). *p < 0.05, **p < 0.01, and ***p < 0.001, as compared with those of purple petals

Fig. 2

The expression levels of genes involved in anthocyanin biosynthesis in the petal of two P. grandiflorus varieties. The expression levels of genes from the white petals were compared with those of the purple petals. The data are representative of three independent experiments (mean ± SE). *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with those of the purple petals. CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone hydroxylase; DFR, dihydroflavanol-4-reductase; ANS, anthocyanin synthase; UFGT, flavonoid-3-O-glucosyltransferase

Fig. 3

Comparison of putative PlgMYB repressors with other plant R2R3 type MYB repressors. Motif distributions were investigated using the MEME web server

Fig. 4

Expression pattern of putative PlgMYB repressors (PlgMYBRs). A Tissue-specific expression pattern of PlgMYBRs. Data represent FPKM values of RNA-Seq data generated from eight different tissues of P. grandiflorus with white flower. B The expression patterns of the selected PlgMYBRs were analyzed using qRT-PCR. The expression levels of genes from the white petals were compared with those of the purple petals. *p < 0.05. FPKM, fragments per kilobase of transcript per million mapped reads

Fig. 5

Transient activation of PlgMYBR1 in tobacco leaves. A Transient expression of PlgMYBR1-expressing constructs in tobacco introduced through agro-infiltration. AtPAP1 infiltration led to high accumulation of anthocyanin, whereas PlgMYBR1 inhibited the accumulation of AtPAP1-induced anthocyanin. B Effect of PlgMYBR1 on the expression of AtPAP1-induced genes involved in the anthocyanin biosynthetic pathway. The level of expression is represented as log2 ratio. Data are means (± SE) of three biological replicates per construct. Different letters indicate statistically significant differences between the samples by Duncan multiples: **p < 0.01, ***p < 0.001