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

Abstract

Purple carrots, the original domesticated carrots, accumulate highly glycosylated and acylated anthocyanins in root and/or petiole. Previously, a quantitative trait locus (QTL) for root-specific anthocyanin pigmentation was genetically mapped to chromosome 3 of carrot. In this study, an R2R3-MYB gene, namely DcMYB113, was identified within this QTL region. DcMYB113 expressed in the root of 'Purple haze', a carrot cultivar with purple root and nonpurple petiole, but not in the roots of two carrot cultivars with a purple root and petiole (Deep purple and Cosmic purple) and orange carrot 'Kurodagosun', which appeared to be caused by variation in the promoter region. The function of DcMYB113 from 'Purple haze' was verified by transformation in 'Cosmic purple' and 'Kurodagosun', resulting in anthocyanin biosynthesis. Transgenic 'Kurodagosun' carrying DcMYB113 driven by the CaMV 35S promoter had a purple root and petiole, while transgenic 'Kurodagosun' expressing DcMYB113 driven by its own promoter had a purple root and nonpurple petiole, suggesting that root-specific expression of DcMYB113 was determined by its promoter. DcMYB113 could activate the expression of DcbHLH3 and structural genes related to anthocyanin biosynthesis. DcUCGXT1 and DcSAT1, which were confirmed to be responsible for anthocyanins glycosylation and acylation, respectively, were also activated by DcMYB113. The WGCNA identified several genes co-expressed with anthocyanin biosynthesis and the results indicated that DcMYB113 may regulate anthocyanin transport. Our findings provide insight into the molecular mechanism underlying root-specific anthocyanin biosynthesis and further modification in carrot and even other root crops.

Keywords: DcMYB113; WGCNA; anthocyanin; carrot; modification; root-specific.

Figure 1

Identification of DcMYB113 in carrot. (a) Site of P₁ locus on chromosome 3 of carrot. (b) Four different carrot cultivars used in this study at 90‐day‐old stage: PPHZ, ‘Purple haze’; DPP, ‘Deep purple’; CPP, ‘Cosmic purple’; and KRD, ‘Kurodagosun’. (c) Total anthocyanin contents in various root tissues of different carrot cultivars. Values are means of three independent experiments ± SDs. (d) Relative transcript levels of DcMYB113 in different root tissues of various carrot cultivars. Data are means of biological triplicate qRT‐PCRs ± SD.

Figure 2

Functional test of DcMYB113 in carrots. (a) Schematic representing DcMYB113 from PPHZ with exons (black boxes) and introns (black lines). (b) Purple calli produced from DPP explants. (c) Calli generated from CPP explants without transformation or transformed with 35S:DcMYB113. (d) Overexpression of 35S:DcMYB113 in KRD. (e) Total anthocyanin contents in roots of two 35S:DcMYB113 transgenic KRD lines. Values are means of three independent experiments ± SDs.

Figure 3

Complementation of CPP and KRD with DcMYB113 from PPHZ. (a) Alignment analysis of promoter sequences of DcMYB113 cloned from PPHZ (Pro‐PPHZ) and found in the KRD genome (Pro‐KRD). Identical sequences are shaded in black. (b) Calli generated from CPP explants transformed with Pro_PPHZ:DcMYB113 or pCAMBIA 1301 vector (as control). (c) The KRD lines expressing Pro_PPHZ:DcMYB113. (d) Total anthocyanin contents in different root tissues of three Pro_PPHZ:DcMYB113 transgenic KRD lines. Data are means of three biological replicates ± SDs. (e) The expression level (FPKM) of DcMYB113 in root tissues of three Pro_PPHZ:DcMYB113 transgenic KRD lines according to transcriptome data.

Figure 4

The role of DcMYB113 on the expression of DcbHLH3 and structural genes related to anthocyanin biosynthesis. (a) Relative transcript levels of DcbHLH3 in different root tissues of various carrot cultivars and two 35S:DcMYB113 transgenic KRD lines. Data are means of biological triplicate qRT‐PCR ± SD. (b) Interaction between DcMYB113 with DcbHLH3 determined by yeast two‐hybrid assays. (c) Relative transcript levels of anthocyanin pathway structural genes in 90‐day‐old old roots of untransformed KRD and two 35S:DcMYB113 transgenic KRD lines. Data represents means of three biological replicates ± SDs.

Figure 5

Role of DcMYB113 in anthocyanin glycosylation and acylation. (a) Anthocyanin composition profile in 35S:DcMYB113 (line 1) and Pro_PPHZ:DcMYB113 (line 1) transgenic KRD roots. (b) Proposed enzymes participating in Cy3XSGG biosynthesis. Enzymes identified in this study are marked in blue. (c) HPLC‐MS analysis of products after incubation of cyanidin‐3‐O‐galactoside and UDP‐xylose with rDcUCGXT1 or crude protein (control) extracted from E. coli transformed with pET‐30a vector. (d) Anthocyanin composition profile in DPP calli carrying 35S:DcSAT1 or pCAMBIA 1301 vector (control).

Figure 6

Regulation of DcUCGXT1 and DcSAT1 by DcMYB113. (a) Relative transcript levels of DcUCGXT1 and DcSAT1 in different root tissues of PPHZ, DPP, CPP, KRD, two 35S:DcMYB113 and three Pro_PPHZ:DcMYB113 transgenic KRD lines. Data are means of three biological replicates ± SDs. (b) Validation of interaction of DcMYB113 with the DcUCGXT1 and DcSAT1 promoter fragments by yeast one‐hybrid assays. (c) Transient expression analyses confirming transactivation of DcUCGXT1 and DcSAT1 by DcMYB113. (d) Transactivation activity of DcMYB113 on DcUCGXT1 and DcSAT1 expressed as ratio of luciferase (LUC) to Renilla (REN) activity. Data are means of six replicate reactions ± SDs.

Figure 7

Weighted gene co‐expression network analysis of genes expressed in root tissues of PPHZ, CPP, KRD, as well as two 35S:DcMYB113 and three Pro_PPHZ:DcMYB113 transgenic KRD lines. (a) Hierarchical cluster tree showing 39 co‐expression modules with different colours in lower panel. Each gene is represented as a leaf in the tree. (b) Module‐trait (total anthocyanin content) correlations and P‐values (in parentheses). Colour in right panel shows correlation from –1 (blue) to 1 (red). In left panel, 39 modules are represented by different colours. (c) Genes identified in ‘pink’ module are clustered in four groups. Colour indicates node connectivity (degree) value lower than 60 (yellow), or from 60 (white) to 90 (purple). (d) Cytoscape representation of co‐expressed genes with edge weight ≥0.10 in ‘pink’ module. Colour shows node connectivity (degree) value from 60 (white) to 90 (purple).