Flavonoid Synthesis-Related Genes Determine the Color of Flower Petals in Brassica napus L

Abstract

The color of rapeseed (Brassica napus L.) petal is usually yellow but can be milky-white to orange or pink. Thus, the petal color is a popular target in rapeseed breeding programs. In his study, metabolites and RNA were extracted from the yellow (Y), yellow/purple (YP), light purple (LP), and purple (P) rapeseed petals. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), RNA-Seq, and quantitative real-time (qRT-PCR) analyses were performed to analyze the expression correlation of differential metabolites and differential genes. A total of 223 metabolites were identified in the petals of the three purple and yellow rapeseed varieties by UPLC-MS/MS. A total of 20511 differentially expressed genes (DEGs) between P, LP, YP, versus Y plant petals were detected. This study focused on the co-regulation of 4898 differential genes in the three comparison groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and quantitative RT-PCR analysis showed that the expression of BnaA10g23330D (BnF3'H) affects the synthesis of downstream peonidin and delphinidin and is a key gene regulating the purple color of petals in B. napus. L. The gene may play a key role in regulating rapeseed flower color; however, further studies are needed to verify this. These results deepen our understanding of the molecular mechanisms underlying petal color and provide the theoretical and practical basis for flower breeding targeting petal color.

Keywords: Brassica napus; petal color; transcriptome sequencing; ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).

Figure 1

The phenotypes of the four differently-colored flowers of Brassica napus (A) The wild phenotype (B) Rapeseed petals. (a) Yellow petals, (b) yellow/purple petals, (c) purple petals, (d) light purple petals.

Figure 2

The physiological indicators. (A) Anthocyanin extracts in the different petals during the full flowering period. (B) Carotenoid extracts in the petals during the full flowering period. Data are means ± SD obtained from three biological replicates. Bars with different letters are significantly different at p < 0.05, according to Tukey’s test.

Figure 3

Overall qualitative and quantitative analyses of metabolomics data. (A) TIC overlap map of QC samples by MS detection. Abscissa represents the retention time (min) of metabolite detection. Ordinate represents the intensity of ion current (cps: count per second). (B) Pearson’s correlation coefficients among Y, LP, YP, and P. (C) PCA of Y, LP, YP, and P. X-axis represents the first principal component. Y-axis represents the second principal component.

Figure 4

Analysis of differential metabolites. (A) The upregulated and downregulated differential metabolites in different petal groups. (B) Venn diagram of upregulated differential metabolites in different petal groups. (C) Heatmap of the differential metabolites. Red bars indicate highly accumulated metabolites, and blue indicates less accumulated metabolites.

Figure 5

(A) Differential metabolites bar graph. Horizontal coordinates are log2FC of differential metabolites and vertical coordinates are differential metabolites. Red represents upregulated differential metabolites, and green represents downregulated differential metabolites. From left to right, Y vs. LP, Y vs. YP, Y vs. P. (B) Differential metabolites clustering heat map. From left to right, Y vs. LP, Y vs. YP, Y vs. P. (C) K means analysis. (D) KEGG analysis of differential metabolites. The horizontal coordinate indicates the Rich factor of each pathway, the vertical coordinate is the name of the pathway, and the color of the dot is the p-value, the redder it is, the more significant the analysis. The size of the dots represents the number of differential metabolites. From left to right, Y vs. LP, Y vs. YP, Y vs. P.

Figure 6

The DEGs between differently colored petals. (A) The upregulated and downregulated DEGs in different petal color groups. (B) Venn diagram of the upregulated DEGs between different petals color groups. Red bars indicate highly expressed genes, and green indicates less expressed genes.

Figure 7

GO analysis of the DEGs. (A) GO functional classification of DEGs between Y and LP. (B) GO functional classification of DEGs between Y and YP. (C) GO functional classification of DEGs between Y and P. Red arrows indicate the DEGs associated with metabolic processes.

Figure 8

(A) Canonical correlation analysis (CCA). From left to right, Y vs. LP, Y vs. YP, Y vs. P. (B) Left, transcriptome loading plot; right, metabolome loading plot.

Figure 9

KEGG pathway analysis of flavonoid biosynthesis pathway. From top to bottom, Y vs. LP, Y vs. YP, Y vs. P.

Figure 9

KEGG pathway analysis of flavonoid biosynthesis pathway. From top to bottom, Y vs. LP, Y vs. YP, Y vs. P.

Figure 10

The flavonoid biosynthetic pathway. Heat maps of the DEGs between LP, YP, P, and Y. The light purple and purple boxes indicate significantly upregulated/downregulated metabolites in the flavonoid metabolic pathway. The F3’H gene (BnaA10g23330D) circled in red is the key differential gene in the flavonoid pathway that regulates purple flower traits.

Figure 11

qRT-PCR analysis of selected DEGs in the rapeseed petal subtypes, identified by transcriptome analysis. Note: Y represents a yellow petal line; LP represents a light purple petal line; YP represents a yellow/purple petal line; P represents a purple petal line. BnaA10g23330D: BnF3ʹH; BnaCnng18670D: BnVSR; BnaA08g27660D and BnaC08g00430D: BnHCT; BnaC08g33510D: BnFLS; BnaC09g47360D: BnF3H; BnaC08g08350D, BnaC09g30320D and BnaA10g07160D: BnUFGT. Fold Change: Take Y as the reference point. Internal reference gene: β-actin. Data are means ± SD obtained from three biological replicates. Different letters above the error bars indicate significant differences at p < 0.05, according to Tukey’s test.