Unraveling the Mechanism of Purple Leaf Formation in Brassica napus by Integrated Metabolome and Transcriptome Analyses

Abstract

Brassica napus as both oilseed and vegetable, is widely cultivated in China. The purple leaf of B. napus is rich in anthocyanins and can provide valuable nutrients. Although several high-anthocyanin cultivars have been reported, the molecular mechanism underlying anthocyanin biosynthesis in B. napus remains lesser-known. Therefore, in this study, we conducted integrative metabolome and transcriptome analyses in three B. napus cultivars with different leaf colors. Overall, 39 flavonoids were identified (including 35 anthocyanins), and 22 anthocyanins were differentially accumulated in the leaves, contributing to the different leaf colors. Cyanidin-3,5,3'-O-triglucoside was confirmed as the main contributor of the purple leaf phenotype. Meanwhile, other anthocyanins may play important roles in deepening the color of B. napus leaves. A total of 5,069 differentially expressed genes (DEGs) and 32 overlapping DEGs were identified by RNA-sequencing; hence, the correlation between anthocyanin content and DEG expression levels was explored. Two structural genes (DFR and ANS), three GSTs (homologous to TT19), and 68 differentially expressed transcription factors (TFs), especially MYB-related TFs and WRKY44, were identified in three B. napus varieties characterized by different leaf color, thereby indicating that these genes may contribute to anthocyanin biosynthesis, transport, or accumulation in B. napus leaves. The findings of study provide important insights that may contribute to gaining a better understanding of the transcriptional regulation of anthocyanin metabolism in B. napus.

Keywords: Brassica napus; anthocyanin; metabolome; purple leaves; transcriptome.

FIGURE 1

Leaf morphology and total anthocyanin content in three B. napus varieties. (A) Upper epidermis of green leaf type (GLT), reddish-green leaf type (RGLT), and purple leaf type (PLT). Scale Bars = 2 cm. (B) Lower epidermis of GLT, RGLT, and PLT. (C) Transverse section of GLT, RGLT, and PLT. Scale Bars = 500 μm. (D) Total anthocyanin contents in GLT, RGLT, and PLT. The different capital letters indicate significant differences at P ≤ 0.01.

FIGURE 2

Heatmap of anthocyanin metabolites in the three different colored leaves of B. napus. PLT, purple leaf type; GLT, green leaf type; RGLT, reddish-green leaf type. GLT, green leaf type; RGLT, reddish-green leaf type; PLT, purple leaf type.

FIGURE 3

Metabolome analysis of differentially accumulated metabolites (DAMs). (A) Venn diagram analysis among GLT vs. PLT, GLT vs. RGLT, and RGLT vs. PLT. (B) Cluster heat map of DAMs among GLT, RGLT, and PLT. (C) The relative content of overlapping DAMs (four anthocyanins) in the leaves of the three B. napus cultivars.

FIGURE 4

Volcano map and Venn diagram analysis of differentially expressed genes (DEGs) in the different colored leaves of B. napus. Red dots indicate upregulated DEGs, green dots indicate downregulated DEGs, and blue dots indicate genes that were not differentially expressed. (A–C) Volcano map of DEGs among GLT vs. PLT, GLT vs. RGLT, and RGLT vs. PLT. (D) Venn diagram analysis of DEGs among GLT vs. PLT, GLT vs. RGLT, and RGLT vs. PLT.

FIGURE 5

GO and KEGG analysis and gene expression heatmap of the 32 overlapping DEGs in the different colored leaves of B. napus. (A) GO analysis of the 32 overlapping DEGs. (B) KEGG enrichment analysis of the 32 overlapping DEGs. (C) The gene expression heatmap of the 32 overlapping DEGs.

FIGURE 6

Distinct expression patterns of transcription factors (TFs). (A) Number of differentially expressed TFs in the GLT, RGLT, and PLT groups. (B) Overview of the enriched TF family. The histograms show the number of genes belonging to each TF family or in each B. napus variety. (C) Heatmaps represent the expression fold change (Log₂FC) of TFs among GLT, RGLT, and PLT groups.

FIGURE 7

Candidate structural genes related to the anthocyanidin biosynthesis pathway. Heatmap shows the expression levels of GLT, RGLT, and PLT from left to right, respectively. The color change of the heatmap from green to red indicates the expression levels ranging from low to high. △, □, and ○ indicate the differential expressed levels in GLT vs. PLT, GLT vs. RGLT, and RGLT vs. PLT groups, respectively.

FIGURE 8

Correlation network of 30 metabolites and 14 key genes involved in flavonoid and anthocyanin biosynthesis in B. napus leaves. Blue circle and pink diamond represent genes and metabolites, respectively. The degree represents the number of genes or metabolites. Relation represents the correlations with the coefficient value ≥ 0.80 (positive) or ≤ –0.80 (negative). Cya-3-O-xyl, cyanidin-3-O-xyloside; Cya-3-O-(coumaryl)-glu, cyanidin-3-O-(coumaryl)-glucoside; Cya-3-O-5-O-(6-O-coumaryl)-diglu, cyanidin-3-O-5-O-(6-O-coumaryl)-diglucoside; Cya-3-O-sam, cyanidin-3-O-sambubioside; Cya-3-sam-5-glu, cyanidin-3-O-sambubioside-5-O-glucoside; Cya-3-(6-caf-sop)-5-glu, cyanidin-3-(6-caffeylsophoroside)-5-glucoside; Cya-3-O-xyl, cyanidin-3-O-xyloside; Cya-3-O-sop, Cyanidin-3-O-sophoroside; Cya-triglu, cyanidin-3,5,3’-O-triglucoside; Del-3-rut-5-glu, delphinidin-3-rutinoside-5-glucoside; Kaempferol-3-O-rut, kaempferol-3-O-rutinoside; Mal-3-O-(6-O-malonyl)-glu, malvidin-3-O-(6-O-malonyl-beta-D-glucoside); Pel-3-O-sop-5-O-(malonyl)-glu, pelargonidin-3-O-sophoroside-5-O-(malonyl)-glucoside; Pel-3-O-gal, pelargonidin-3-O-galactoside; Pel-3-(6-caf-sop)-5-glu, pelargonidin-3-(6-caffeylsophoroside)-5-glucoside; Pel-3-O-sop, pelargonidin-3-O-sophoroside; Pel-3-O-5-O-(6-O-coumaryl)-diglu, pelargonidin-3-O-5-O-(6-O-coumaryl)-diglucoside; Pel-triglu, pelargonidin-3,5,3’-O-triglucoside; Pel-3-O-rut-5-O-glu, pelargonidin-3-rutinoside-5-glucoside; Peo-3,5-O-diglu, peonidin-3,5-O-diglucoside; Peo-3-O-ara, peonidin-3-O-arabinoside; Peo-3-(caf-glucosyl-glu)-5-glu, peonidin-3-(caffeoyl-glucosyl-glucoside)-5-glucoside; Pet-3-O-(coumaryl)-glu, petunidin-3-O-(coumaryl)-glucoside; Naringenin-7-O-glu, naringenin-7-O-glucoside; Peo-3-O-sop, peonidin-3-O-sophoroside; Peo-triglu, peonidin-3,5,3’-O-triglucoside; Peo-3-O-glu, peonidin-3-O-glucoside; Pel-3-O-glu, pelargonidin-3-O-glucoside; Peo-3-O-(6-O-p-coumaryl)-glu, peonidin-3-O-(6-O-p-coumaryl)-glucoside.

FIGURE 9

The qRT-PCR analysis of the expression patterns for genes involved in B. napus anthocyanin biosynthesis. Green, red, and purple represent the expression levels of genes in GLT, RGLT, and PLT, respectively.