Identification and differential expression analysis of anthocyanin biosynthetic genes in leaf color variants of ornamental kale

Abstract

Background: Anthocyanins perform diverse biological functions in plants and are beneficial to human health. Leaf color is the most important trait of ornamental kale and the characteristics of changes in leaf color make it an ideal material to elucidate genetic mechanisms of anthocyanins accumulation in Brassica oleracea. To elucidate the anthocyanin distribution, metabolic profiles and differentially expressed anthocyanin biosynthetic genes between different colored accessions can pave the way for understanding the genetic regulatory mechanisms of anthocyanin biosynthesis and accumulation in ornamental kale.

Results: In this study, anthocyanin distributions in red- and white-leaved ornamental kale accessions were determined. Thirty-four anthocyanins were detected in the red-leaved accession. The complete set of anthocyanin biosynthetic genes in the B. oleracea reference genome was identified and differential expression analysis based on RNA-seq was conducted. Eighty-one anthocyanin biosynthetic genes were identified in the B. oleracea reference genome. The expression patterns and differential expressions of these genes in different leaf types indicated that late biosynthetic genes (BoDFR1, BoANS1 and 2, and BoUGT79B1.1), positive regulatory genes (BoTTG1, BoTT8, and Bol012528), a negative regulatory gene (BoMYBL2.1), and transport genes (BoTT19.1 and BoTT19.2) may play roles in anthocyanin accumulation in ornamental kale. A genetic regulatory network of anthocyanin accumulation in ornamental kale was constructed.

Conclusions: The distribution of pigments and anthocyanin profiles explained the leaf color phenotypes of ornamental kales. The identification of key genes and construction of genetic regulatory network in anthocyanin accumulation in ornamental kale elucidated the genetic basis of leaf color variants. These findings enhance the understanding of the genetic mechanisms and regulatory network of anthocyanin accumulation in B. oleracea, and provide a theoretical basis for breeding new cultivars of Brassica vegetables with enhanced ornamental and nutritional value.

Keywords: Anthocyanin accumulation; Anthocyanin biosynthetic genes; Brassica oleracea; Color variants; Differentially expressed genes; Ornamental kale; Regulatory network.

Fig. 1

Individual plants, new and mature leaves, and anatomical distribution of leaf pigments in ornamental kale DH lines ‘05-DH-65’ and ‘06-DH-71’. (a) ‘05-DH-65’ plant; (b) ‘06-DH-71’ plant; (c) ‘05-DH-65’ new (central) leaf; (d) ‘05-DH-65’ mature (outer) leaf; (e) ‘06-DH-71’ new (central) leaf; (f) ‘06-DH-71’ mature (outer) leaf; (g) ‘05-DH-65’ new leaf lamina, transverse section; (h) ‘05-DH-65’ mature leaf lamina, transverse section; (i) ‘06-DH-71’ new leaf lamina, transverse section; (j) ‘06-DH-71’ mature leaf lamina, transverse section; (k) ‘05-DH-65’ new leaf vein, transverse section; (l) ‘05-DH-65’ mature leaf vein, transverse section; (m) ‘06-DH-71’ new leaf vein, transverse section; (n) ‘06-DH-71’ mature leaf vein, transverse section; (o) ‘05-DH-65’ new leaf petiole, longitudinal section; (p) ‘05-DH-65’ mature leaf petiole, longitudinal section; (q) ‘06-DH-71’ new leaf petiole, longitudinal section; (r) ‘06-DH-71’ mature leaf petiole, longitudinal section

Fig. 2

a Liquid chromatography chromatograms (520 nm) of anthocyanin extracts from new leaves of the ornamental kale DH line ‘05-DH-65’. Horizontal axis shows retention time (min); vertical axis shows the strength of the chromatographic peak response intensity. The compound name is provided for each peak. b Total anthocyanins content in new and mature leaves of the DH lines ‘05-DH-65’ and ‘06-DH-71’

Fig. 3

Distribution of anthocyanin biosynthetic genes (ABGs) on the pseudo-chromosomes of B. oleracea reference genome versions Cap02–12 (a) and TO1000 (b). The blue bars represent the nine chromosomes (C01 to C09) of B. oleracea. The relative positions of BoABGs are marked on the pseudo-chromosomes of genome versions Cap02–12 (a) and TO1000 (b). Gene annotations and names are provided on the left and right sides of the bars, respectively. The scale indicates the physical distance of the chromosomes

Fig. 4

The anthocyanin biosynthetic pathway and expression levels of structural and positive regulatory genes in B. oleracea. The pathway can be divided into two sections: the phenylpropanoid and the flavonoid pathways. Two types of genes are involved in the flavonoid pathway: early biosynthetic genes (EBGS) and late biosynthetic genes (LBGs). Red type indicates biosynthetic enzymes. The ellipses “M”, “B”, and “W” represent the MYB, bHLH, and WD40 proteins involved in the positive regulation of anthocyanin synthesis. The expression levels of coding genes is indicated by blue and red shading, which represents low to high expression levels, respectively. The color scale corresponds with the mean-centered log2-transformed FPKM values, which are identical to those in Fig. 4

Fig. 5

The significant differential expression ABGs in new and mature leaves between red accession ‘05-DH-65’ and white accession ‘06-DH-71’ by EdgeR and DEseq analysis. The red and green colors indicated significant up- and down-regulation of genes in new and mature leaves of ‘05-DH-65’, and the gray ones showed on significant expression difference

Fig. 6

Regulatory network of anthocyanins accumulation in red- and white-leaved ornamental kales. In red leaves, BoTTG1, BoTT8, and Bol012528 form the MBW complex to activate BoDFR1, BoANS1 and 2, BoUGT79B1.1, and BoUT78D2 to promote anthocyanins biosynthesis on the cytosolic surface of the ER. In white and green leaves, the negative regulator BoMYBL2.1 is highly expressed to repress the LBGs. The transporter genes BoTT19.1 and 19.2 are highly expressed in red leaves to transport anthocyanins from the ER to the vacuole, thereby promoting the red leaf phenotype. High expression levels of BoTT19.1 and 19.2 may also feedback to activate LBGs by transporting greater amounts of anthocyanins