Combined Multi-Omics and Co-Expression Network Analyses Uncover the Pigment Accumulation Mechanism of Orange-Red Petals in Brassica napus L

Abstract

Rapeseed (Brassica napus L.) has been cultivated as an ornamental plant in recent years. However, the metabolic and regulatory processes involved in pigment accumulation in. B. napus flowers are poorly understood. To address this knowledge gap, we conducted a multi-omics analysis of the orange-red-flowered 'OrP' and the yellow-flowered 'ZS11' rapeseed cultivars. The total anthocyanin content of 'OrP' petals was 5.420-fold and 3.345-fold higher than 'ZS11' petals at the S2 and S4 developmental stages, respectively. The red coloration of 'OrP' flowers resulted primarily from the presence of anthocyanin pigment derivatives. The up-regulated differentially expressed genes (DEGs) of four stages in 'OrP' were found to be significantly enriched in phenylpropanoid, flavonoid, and anthocyanin metabolism-associated GO and KEGG terms. Weighted Gene Co-expression Network Analysis (WGCNA) revealed that 51 DEGs were linked to anthocyanin metabolism, including several structural genes such as BnaCHS, BnaF3H, BnaF3'H, BnaCHS, BnaDFR, BnaANS, BnaUGTs, and the transcription factor (TF) genes BnaHY5, BnaBBX22, BnaPIL1, BnaPAP2, BnaTT8, BnaTTG2, and BnaMYBL2. Furthermore, we found that three main factors affecting the relative content of anthocyanins in petals were likely responsible for the fading of 'OrP' petals, namely the significantly down-regulated expression of genes (BnaDFR, BnaANS, BnaPAP2, BnaTT8, and BnaTTG2) related to anthocyanin biosynthesis, the significantly up-regulated expression of genes (Bna.BGLUs, Bna.PRXs, and BnaMYBL2) related to anthocyanin degradation or the negative regulation of anthocyanin biosynthesis, and the rapidly increasing petals area.

Keywords: Brassica napus L.; WGCNA; anthocyanin; flower color; metabolome; transcriptome.

Figure 1

The morphological characteristics and pigment contents of the ‘OrP’ and ‘ZS11’ flowers at different developmental stages. (A,B) Morphological map of the orange-red-flowered rapeseed ‘OrP’ and the yellow-flowered rapeseed ‘ZS11’. (C) Morphological appearance of the petals of ‘OrP’ and ‘ZS11’ at different developmental periods. The stages are as follows: B1 to B8, the maximum length of the buds ranging from 1 to 8 mm; S0, petals just exposed from the sepals; S1, petals exposed from open buds before spreading; S2, petals half spread; S3, petals just fully spread; S4, petals fully spread to the maximum size. Scale bar: 5 mm. (D) The chromaticity values Δa (red-green color) of the petals in different stages for ‘OrP’ and ‘ZS11’. (E–G) The total anthocyanin, carotenoid, and chlorophyll contents of the petals in different stages for ‘OrP’ and ‘ZS11’. Error bars indicate the standard deviation of three independent replicates. Asterisks (*, **, or ***) denote significant differences at p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 2

The metabolome quality and differential metabolite analysis. (A) Principal component analysis, PC1 and PC2 are plotted on the x- and y-axes, respectively. (B) Correlation heat map showing that ‘OrP’ and ‘ZS11’ separated significantly. (C) Heatmap of the differentially accumulated metabolites, up- and down-regulated metabolites (red and blue color) of the values of log₂ (fold change).

Figure 3

The number of DEGs and GO and KEGG enrichment analyses in different compared combination groups of ‘OrP’ and ‘ZS11’. (A) The number of up- and down-regulated DEGs in different comparison groups. (B) Venn diagram of DEGs in different comparison groups. (C,D) GO enrichment analysis of the up- and down-regulated DEGs. The size of each point shows the number of genes in the term. BP, biological process; CC, cellular component; MF, molecular function. (E,F) KEGG enrichment analysis of the up- and down-regulated DEGs.

Figure 4

WGCNA of the anthocyanin contents and colorimetric values of the petals in ‘OrP’ and ‘ZS11’. (A) The hierarchical cluster tree showing the identified co-expression modules. (B) The correlations of the samples with WGCNA modules. Red and blue scale indicate positive and negative correlations.

Figure 5

The metabolic pathway analysis of structural genes involved in anthocyanin biosynthesis. Red to blue scale indicate up- and down-regulation. G1–G4: Different compared combination groups of ‘OrP’ and ‘ZS11’ during B5, B8, S1, and S3 stages, respectively. EBGs, early biosynthetic genes; LBGs, late biosynthetic genes; PAL, Phenylalanin ammonialyase; C4H, Cinnamate 4-hydroxy-lase; 4CL, 4-coumarate CoA ligase; ACC, Acetic acid-CoA carboxylase; CHS, chaleone syn-thase; CHI, Chaleone isomerase; F3H, Flavanone 3-hydroxylase; F3′H, Flavonoid 3′-hydroxylase; F3′5′H, Flavanone 3′5′-hydroxy-lase; FLS, Flavonol synthase; DFR, Dihydroflavonol 4-reductase; ANS, Anthocyanin synthase; LAR, Leucoantho cyanidin reductase; ANR, Anthocyanidin reductase; UFGT, UDPG-flavonoid-3-O-glycosyltranferase; MT, Methylferase; 3′-OMT, 3′-O-Methyltransferase; 5′-OMT, 5′-O-Methyltransferase.

Figure 6

The expression profile of the structural genes and transcript factor genes involved in anthocyanin biosynthesis. Clustering heat map showing the abundance of genes by log₂ of the FPKM values, where different colors from blue to red show gene expression level differences, with higher expression colored red and lower expression colored blue.

Figure 7

Working model of anthocyanin biosynthesis in ‘OrP’ and ‘ZS11’ petals. The arrows and blunt-ended lines indicate activation and repression, respectively. LBGs, late biosynthetic genes; ADEs, anthocyanin degradation enzymes.

Figure 8

Quantitative real-time PCR (qRT-PCR) validation of the structural genes and candidate transcript factors involved in anthocyanin biosynthesis. Error bars indicated the standard deviation of three independent replicates. Asterisks (**, ***) denoted significant differences at p < 0.01 and p < 0.001, respectively.