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 3 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. Carrot (Daucus carota ssp. sativus; 2n 5 2x 5 18) provides rich health-promoting nutrients to humans. Carrots are classified into two groups according to exact botanical determination: the carotene group (variety sativus) and the anthocyanin group (variety atrorubens; Kammerer et al., 2004). Carotene group members, also known as nonpurple carrots, accumulate massive amounts of carotenoids in their roots (Clotault et al., 2008; Arscott and Tanumihardjo, 2010); anthocyanin group members, also known as purple carrots, accumulate high

DcMYB113, a root‐specific R2R3‐MYB, conditions anthocyanin biosynthesis and modification in carrot

“…Iorizzo et al identified an R2R3‐MYB gene, DcMYB7 , within the P 3 region as a candidate for the gene controlling anthocyanin biosynthesis in carrot with a purple root and petiole (Iorizzo et al , ). Recently, we confirmed that DcMYB7 is P 3 (Xu et al , ). However, the key gene within the P 1 region that controls anthocyanin biosynthesis in carrot with a purple root and nonpurple petiole is still unknown.…”

“…Cavagnaro et al found several QTLs associated with variations in anthocyanin glycosylation and acylation, but did not identify any candidate genes (Cavagnaro et al , ). Following on from that important work, DcUCGXT1 and DcSAT1 , encoding a glycosyltransferase and an acyltransferase, respectively, were found within two QTLs (Xu et al , ). In this study, we confirmed that DcUCGXT1 catalysed the synthesis of Cy3XG by adding xylose to Cy3G and DcSAT1 catalysed the addition of sinapoylglucose to anthocyanins.…”

“…However, the genes responsible for further glycosylation and acylation of anthocyanins in carrot were not identified or characterized in their study. Recently, we found two genes encoding a glycosyltransferase and an acyltransferase, respectively, within two QTLs associated with the content of glycosylated and acylated anthocyanins (Xu et al , ). But their glycosyltransferase or acyltransferase activities were not determined.…”

Insights into long non-coding RNA regulation of anthocyanin carrot root pigmentation

“…As expected, we identify several differentially expressed genes (DEG) between the two genotypes known to be involved in carrot root anthocyanin biosynthesis 21,23–26 . Most of the known genes of the pathway and their main regulators were differentially expressed between the two genotypes (Supplementary Table S4).…”

“…DcbHLH3 , DcUCGXT1 and DcSAT1 ) also displayed no tissue specificity. DcbHLH3 was described as a co-regulator in anthocyanin biosynthesis, while DcUCGXT1 and DcSAT1 participate in anthocyanin glycosylation and acylation, respectively 26,47 . Additionally, seven TFs showed xylem preferential expression-specificity, while only one was preferentially expressed specifically in phloem.…”