Changing Carrot Color: Insertions in DcMYB7 Alter the Regulation of Anthocyanin Biosynthesis and Modification

Abstract

The original domesticated carrots (Daucus carota) are thought to have been purple, accumulating large quantities of anthocyanins in their roots. A quantitative trait locus associated with anthocyanin pigmentation in purple carrot roots has been identified on chromosome 3 and includes two candidate genes, DcMYB6 and DcMYB7 Here, we characterized the functions of DcMYB6 and DcMYB7 in carrots. Overexpression of DcMYB7, but not DcMYB6, in the orange carrot 'Kurodagosun' led to anthocyanin accumulation in roots. Knockout of DcMYB7 in the solid purple (purple periderm, phloem, and xylem) carrot 'Deep Purple' using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system resulted in carrots with yellow roots. DcMYB7 could activate the expression of its DcbHLH3 partner, a homolog of the anthocyanin-related apple (Malus × domestica) bHLH3, and structural genes in the anthocyanin biosynthetic pathway. We determined that the promoter sequence of DcMYB7 in nonpurple carrots was interrupted either by DcMYB8, a nonfunctional tandem duplication of DcMYB7, or by two transposons, leading to the transcriptional inactivation of DcMYB7 in nonpurple carrot roots. As a result, nonpurple carrots fail to accumulate anthocyanins in their roots. Our study supports the hypothesis that another genetic factor suppresses DcMYB7 expression in the phloem and xylem of purple peridermal carrot root tissues. DcMYB7 also regulated the glycosylation and acylation of anthocyanins by directly activating DcUCGXT1 and DcSAT1 We reveal the genetic factors conditioning anthocyanin pigmentation in purple versus nonpurple carrot roots. Our results also provide insights into the mechanisms underlying anthocyanin glycosylation and acylation.

Figure 1.

Expression patterns of DcMYB6 and DcMYB7 in carrot roots. A, Four-month-old purple and nonpurple carrots of the 11 cultivars used in this study. Cultivar abbreviations are defined in “Results.” Separate images are shown as a composite for comparison. B, Relative transcript levels of DcMYB6 and DcMYB7 in different root tissues of 11 purple and nonpurple carrot cultivars. Data are means of biological triplicate RT-qPCRs ± sd.

Figure 2.

Functional detection of DcMYB6 and DcMYB7 in carrots. A and B, Overexpression of DcMYB6 (A) and DcMYB7 (B) in orange KRD carrots. Separate images are shown as composites for comparison. C, Total anthocyanin contents in roots of untransformed, 35S:DcMYB6, and 35S:DcMYB7 transgenic KRD carrots. Values are means of three biological replicates with error bars representing sd. D, Untransformed and DcMYB7-knockout DPP carrots. Separate images are shown as a composite for comparison. E, Total anthocyanin contents in roots of untransformed and DcMYB7-knockout DPP carrots. Data are means of three technical replicates ± sd. fw, Fresh weight.

Figure 3.

Role of DcMYB7 in regulating DcbHLH3 and structural genes in the anthocyanin pathway. A, Yeast two-hybrid assays validating the interaction of DcMYB7 with DcbHLH3. SD, Synthetic defined; X-α-Gal, 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside. B, Relative transcript levels of DcbHLH3 and structural genes in roots of untransformed and 35S:DcMYB7 transgenic KRD carrots. Data represent means of biological triplicate RT-qPCRs ± sd. C, Relative transcript levels of DcbHLH3 and structural genes in roots of untransformed and DcMYB7-knockout DPP carrot lines. Data are means of three technical replicates with error bars representing sd.

Figure 4.

Functional investigation of DcMYB7 gDNA sequences from various carrot cultivars. A, PCR amplification of DcMYB7 gDNA from 11 different carrot cultivars. B, Schematic representation of full-length DcMYB7 from 11 different carrot cultivars having three exons (black boxes) and two introns (lines between exons). C, Overexpression of DcMYB7 gDNA from different carrot cultivars in the orange KRD carrot under the control of the CaMV 35S promoter. KRD explants transformed with the pCAMBIA 1301 vector were used as the control.

Figure 5.

Functional investigation of the DcMYB7 promoter from various carrot cultivars. A, Physical locations of DcMYB6, DcMYB7, and DcMYB8 on chromosome 3 of the DH1 orange carrot genome. B, PCR amplification of the sequence upstream of DcMYB7 from 11 different carrot cultivars. C, Schematic representation of DcMYB6, DcMYB7, and DcMYB8 from 11 different carrot cultivars. D, Functional investigation of the DcMYB7 promoter from different carrots in KRD and SHLC carrots.

Figure 6.

Anthocyanin modifications in carrots. A, Anthocyanin composition profiles from roots of DPP carrots and 35S:DcMYB7 transgenic KRD carrots (line 1). B, Schematic of the proposed biosynthetic pathway of Cy3XSGG. UCGXT and SAT were identified in this study.

Figure 7.

Role of DcMYB7 in regulating DcUCGXT1 and DcSAT1. A, Relative transcript levels of DcUCGXT1 and DcSAT1 in roots of untransformed and 35S:DcMYB7 transgenic KRD (top) as well as untransformed and DcMYB7-knockout DPP (bottom) carrot lines. Data are means of three biological (top) or technical (bottom) replicates with error bars representing sd. B, Yeast one-hybrid assays showing that DcMYB7 binds to the promoter fragments of DcUCGXT1 and DcSAT1. 3-AT, 3-amino-1,2,4-triazole. C, Transient expression assays showing that DcMYB7 promotes the expression of DcUCGXT1 and DcSAT1. D, Promoter activities of DcUCGXT1 and DcSAT1 expressed as a ratio of firefly LUC to Renilla luciferase (REN) activity. Data are means of six replicate reactions with error bars representing sd.