Metabolomics Analysis Reveals the Accumulation Patterns of Flavonoids and Volatile Compounds in Camellia oleifera Petals with Different Color

Abstract

To systematically and comprehensively investigate the metabolic characteristics of coloring substances and floral aroma substances in Camellia oleifera petals with different colors, ultrahigh-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) and headspace solid phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) metabolomics methods were applied to determine the metabolic profiles of white, candy-pink and dark-red petals. The results revealed that 270 volatile organic compounds were detected, mainly terpenoids, heterocyclic, esters, hydrocarbons, aldehydes, and alcohols, in which phenylethyl alcohol, lilac alcohol, and butanoic acid, 1-methylhexyl ester, hotrienol, alpha-terpineol and 7-Octen-4-ol, 2-methyl-6-methylene-, (S)-, butanoic acid, 2-methyl-, 2-methylbutyl ester, 2,4-Octadienal, (E,E)- could act as the floral scent compounds. A total of 372 flavonoid compounds were identified, and luteolin, kaempferol, cyanidin and peonidin derivatives were considered as the main coloring substances for candy-pink and dark-red petal coloration. In conclusion, this study intuitively and quantitatively exhibited the variations in flower color and floral scent of C. oleifera petal with different colors caused by changes in variations of flavonoids and volatile organic compound composition, and provided useful data for improving the sensory quality and breeding of C. oleifera petals.

Keywords: Camellia oleifera petal; HS–SPME–GC–MS; UPLC–MS/MS; flavonoids; volatile organic compounds.

Figure 1

Differential petals chemotype in C. oleifera petals with different color, W = white petals, CP = candy-pink petal, DR = dark-red petal. (A): Correlation analysis of three C. oleifera petals based on identified VOCs and flavonoids; (B): Heatmap of the 270 VOCs identified in three C. oleifera petals with three biological replicates; (C): Heatmap of flavonoids identified in three C. oleifera petals with three biological replicates. The relative content values of all metabolites were denoted with a unique color, among which red color indicated a high accumulation level, and green color indicated a low accumulation level.

Figure 2

The 2D-PCA plot and OPLS-DA plot of the three C. oleifera petals based on the relative content of flavonoids and VOCs. (A): The 2D-PCA score plot of W, CP, and DR petals; (B): Score plots of the OPLS-DA model for W_vs_CP; (C): Score plots of the OPLS-DA model for CP_vs_DR; (D): Score plots of the OPLS-DA model for W_vs_DR. W = white petals; CP = candy-pink petal; DR = dark-red petal.

Figure 3

Venn diagram and pathway analysis of VOCs-DAMs in three C. oleifera petals. (A): The Venn diagram results among W_vs_CP, W_vs_DR, and CP_vs_DR comparisons. (B–D): KEGG pathway enrichment of the VOCs-DAMs of the W_vs_CP, W_vs_DR, and CP_vs_DR comparison, respectively. W = white petals; CP = candy-pink petal; DR = dark-red petal.

Figure 4

Violin plots of peak areas values of 12 crucial differential VOCs identified in W, CP, and DR petals. W = white petals; CP = candy-pink petal; DR = dark-red petal. The distribution and probability density of the 12 VOCs-DAMs were represented by a combination of box plots and density plots. The outer shapes represented the density of the peak area values distribution, the black rectangular box in the middle represents the quartile range, and the white circle in the middle represents the median.

Figure 5

(A–C): Volcano plots of DAMs in W_vs_CP, W_vs_DR, and CP_vs_DR comparisons; red indicated up-regulated differential metabolites, and blue indicated down-regulated differential metabolites; (D–F): KEGG pathway enrichment analysis of the DAMs in W_vs_CP, W_vs_DR, and CP_vs_DR comparisons.

Figure 6

Histogram of peak areas of 12 differentially accumulated metabolites identified in W, CP, and DR petals. W = white petals; CP = candy-pink petal; DR = dark-red petal. Duncan’s test was applied to evaluate the significant difference among petals with different color. ** indicated the significant difference at the p < 0.05 level; *** indicated the significant difference at p < 0.01 level.

Figure 7

Petals colors of three C. oleifera varieties: (A): “Camellia yuhsienensis” (White, W); (B): “Camellia semiserrata” (Candy-pink, CP); (C): “Camellia chekiangoleosa” (Dark-red, DR).