Identification of transcription factor genes involved in anthocyanin biosynthesis in carrot (Daucus carota L.) using RNA-Seq

Abstract

Background: Anthocyanins are water-soluble colored flavonoids present in multiple organs of various plant species including flowers, fruits, leaves, stems and roots. DNA-binding R2R3-MYB transcription factors, basic helix-loop-helix (bHLH) transcription factors, and WD40 repeat proteins are known to form MYB-bHLH-WD repeat (MBW) complexes, which activates the transcription of structural genes in the anthocyanin pathway. Although black cultivars of carrots (Daucus carota L.) can accumulate large quantities of anthocyanin in their storage roots, the regulatory genes responsible for their biosynthesis are not well characterized. The current study aimed to analyze global transcription profiles based on RNA sequencing (RNA-Seq), and mine MYB, bHLH and WD40 genes that may function as positive or negative regulators in the carrot anthocyanin biosynthesis pathways.

Results: RNA was isolated from differently colored calli, as well as tissue samples from taproots of various black carrot cultivars across the course of development, and gene expression levels of colored and non-colored tissue and callus samples were compared. The expression of 32 MYB, bHLH and WD40 genes were significantly correlated with anthocyanin content in black carrot taproot. Of those, 11 genes were consistently up- or downregulated in a purple color-specific manner across various calli and cultivar comparisons. The expression of 10 out of these 11 genes was validated using real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).

Conclusions: The results of this study provide insights into regulatory genes that may be responsible for carrot anthocyanin biosynthesis, and suggest that future focus on them may help improve our overall understanding of the anthocyanin synthesis pathway.

Keywords: Anthocyanin; Daucus carota L.; Differential expression analyses; RNA-Seq; Transcription factors.

Fig. 1

A Calli induced on a) CH5544 explants (purple), b) CH5544 explants (white), c) Danvers. B Cross section of carrot taproot for a) CH5544 (6 weeks after sowing), b) CH5544 (10 weeks after sowing), c) CH5544 (12 weeks after sowing), d) Nightbird, and e) Superblack. The black bar indicates 1 cm

Fig. 2

Venn diagram of the differentially expressed genes in carrot calli and taproots. a Venn diagram showing the overlap between the differentially expressed genes (DEGs) in the purple, white and orange calli. PC_WC, PC_OC and WC_OC indicate the comparison between purple and white, purple and orange, and white and orange calli, respectively. b Venn diagram of the DEGs detected in the comparison between the purple outer and white middle tissue of the CH5544 taproot at the age of 6, 10 and 12 weeks old after sowing

Fig. 3

A heatmap of average logFC detected by DESeq2, EdgeR and Limma for 11 transcription factor genes (rows) for which its gene count is significantly correlated with anthocyanin content. Tissue1 and Tissue2 indicate the color of the callus/tissue sample used to perform differential expression analyses. The abbreviation, 44, NB and SB, indicate the cultivar CH5544, Nightbird and Superblack, respectively. For CH5544, the age of the samples are indicated as 6wks, 10wks, and 12wks, indicating 6, 10 and 12 weeks after sowing, respectively. For taproots of each cultivar, the results are summarized as outer/middle and inner/middle tissue comparisons, except for Superblack (SB), in which only outer and inner tissues were compared. A positive or negative number in each cell indicates that a gene was up- or downregulated in purple/slightly green tissue or calli. NA indicates that genes were not differentially expressed by any of the Bioconductor packages used in this study

Fig. 4

Correlation between the transcriptome abundance and total anthocyanin content for genes differentially expressed in a purple color-specific manner. The red-lines and grey-area represent estimated fit-line and 95% confidence region, respectively. The correlations are showed for: a bHLH-A (LOC108204485), b MYB3-like (LOC108208100), c MYB113-like (LOC108213488), d TRANSPARENT TESTA GLABRA 1 (LOC108224236).

Fig. 5

Validation of differentially regulated genes associated with the anthocyanin content by qRT-PCR. Data were normalized to the expression of glyceraldehylde 3-phosphate dehydrogenase (G3PDH) for each sample. P vs W indicates the comparison between purple and white calli, and P vs O indicates the comparison between purple and orange calli. 6WO, 10WO and 12WO indicate the comparison between outer and middle tissue for 6-, 10- and 12-week-old taproots, respectively. Similarly, 6WC, 10WC and 12WC indicate the comparison between center and middle section for 6-, 10- and 12-week-old taproots, respectively. The results are showed for the following genes: a bHLH-A (LOC108204485), b MYB3-like (LOC108208100), c MYB113-like (LOC108213488), d TRANSPARENT TESTA GLABRA 1 (LOC108224236).