Identification and characterization of DcUCGalT1, a galactosyltransferase responsible for anthocyanin galactosylation in purple carrot (Daucus carota L.) taproots

Abstract

Purple carrots (Daucus carota ssp. sativus var. atrorubens Alef.) accumulate large amounts of cyanidin-based anthocyanins in their taproots. Cyanidin can be glycosylated with galactose, xylose, and glucose in sequence by glycosyltransferases resulting in cyanidin 3-xylosyl (glucosyl) galactosides in purple carrots. The first step in the glycosylation of cyanidin is catalysis by UDP-galactose: cyanidin galactosyltransferase (UCGalT) transferring the galactosyl moiety from UDP-galactose to cyanidin. In the present study, a gene from ‘Deep purple’ carrot, DcUCGalT1, was cloned and heterologously expressed in E. coli BL21 (DE3). The recombinant DcUCGalT1 galactosylated cyanidin to produce cyanidin-3-O-galactoside and showed optimal activity for cyanidin at 30 °C and pH 8.6. It showed lower galactosylation activity for peonidin, pelargonidin, kaempferol and quercetin. It accepted only UDP-galactose as a glycosyl donor when cyanidin was used as an aglycone. The expression level of DcUCGalT1 was positively correlated with anthocyanin biosynthesis in carrots. The enzyme extractions from ‘Deep purple’ exhibited galactosylation activity for cyanidin, peonidin and pelargonidin, while those from ‘Kuroda’ (a non-purple cultivar) did not.

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

“…In addition, purple carrot root pigmentation extensively varies across different carrot genotypes, ranging from the purple peridermal carrot type (purple periderm but nonpurple phloem and xylem) to the solid purple carrot type (purple periderm, phloem, and xylem). The genetic control of anthocyanin pigmentation in purple carrots has been investigated (Simon, 1996;Yildiz et al, 2013;Cavagnaro et al, 2014;Xu et al, 2014Xu et al, , 2016Xu et al, , 2017Kodama et al, 2018;Iorizzo et al, 2019). Two genes that condition the anthocyanin pigmentation of carrot roots from different genetic backgrounds, P 1 and P 3 , have been identified and genetically mapped within 28.2-and 12-centimorgan regions, respectively, on chromosome 3, supporting the theory of two independent mutation and human selection events during the domestication of purple carrots (Cavagnaro et al, 2014).…”

“…In purple carrots, cyanidin-based anthocyanins are almost exclusively responsible for the purple pigment, while trace amounts of derivatives of peonidin-and pelargonidin-based anthocyanins are also present in some purple carrot cultivars (Kammerer et al, 2004;Montilla et al, 2011). In carrot, UCGalT1 catalyzes the first glycosylation step of anthocyanidins, generating stable anthocyanins (Xu et al, 2016). Anthocyanins further undergo several glycosylation and acylation steps (Glässgen et al, 1998;Cavagnaro et al, 2014).…”

Carrot Anthocyanins Genetics and Genomics: Status and Perspectives to Improve Its Application for the Food Colorant Industry

“…LBGs include genes coding for modification enzymes, such as glycosyltransferases (GTs) and acyltransferases (ATs), which catalyze the addition of sugar moieties and acyl groups, respectively, resulting in specific decoration patterns that greatly influence their function and stability. Since the carrot genome was released in 2016, curated annotation identified 159 potential structural anthocyanin genes, either located within an anthocyanin-related QTL or differentially expressed between purple and non-purple tissues [ 34 , 53 , 56 , 65 , 66 , 67 ] ( Supplementary Table S4 ). These include 8 GPMGs, 8 EBGs and 139 LBGs, including 73 GTs genes, 61 ATs genes and 1 O-methyltransferase (OMT) gene, coding for enzymes involved in anthocyanin glycosylation, acylation and methylation, respectively ( Supplementary Table S4 ).…”

“…Among the annotated carrot anthocyanin structural genes, five were functionally characterized: DcF3H1 [ 83 ], DcUCGalT1 [ 65 ], DcUCGXT1 [ 72 ], DcSCPL1 [ 72 ] and DcUSAGT [ 66 ] ( Figure 3 , Table 3 and Supplementary Table S4 ). The knockout of DcF3H1 ( DCAR_009483 ) using the CRISP/Cas9 system caused the discoloration of calli, which validated the function of this gene in the biosynthesis of anthocyanin in carrot, as well as demonstrating the successful application of CRISPR/Cas9 in carrots.…”

“…The knockout of DcF3H1 ( DCAR_009483 ) using the CRISP/Cas9 system caused the discoloration of calli, which validated the function of this gene in the biosynthesis of anthocyanin in carrot, as well as demonstrating the successful application of CRISPR/Cas9 in carrots. DcUCGalT1 ( DCAR_009912 ) has been shown to catalyze the formation of cyanidin 3-galactoside (Cy3G) in vitro, and DcUSAGT ( DCAR_029082 ) was shown to catalyze the transfer of a glucose moiety to the carboxyl group of sinapic acid, thereby forming 1-O-sinapoylglucose [ 65 , 66 ]. 1-O-sinapoylglucose serves as an acyl donor in the acylation of cyanidin-3-(2″-xylose-6-glucose-galactoside) (Cy3XGG) into its acylated counterpart, cyanidin-3-(2″-xylose-6″-sinapoyl-glucose-galactoside) (Cy3XSGG), which helps stabilize the accumulation of anthocyanins in purple carrots.…”