Metabolomic Study of Flavonoids in Camellia drupifera under Aluminum Stress by UPLC-MS/MS

Abstract

Aluminum (Al) affects the yield of forest trees in acidic soils. The oil tea plant (Camellia drupifera Lour.) has high Al tolerance, with abundant phenolic compounds in its leaves, especially flavonoid compounds. The role of these flavonoids in the Al resistance of oil tea plants is unclear. In this metabolomic study of C. drupifera under Al stress, ultra-pressure liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) was utilized to identify metabolites, while principal component analysis, cluster analysis, and orthogonal partial least squares discriminant analysis were applied to analyze the data on the flavonoid metabolites. The leaf morphology of C. drupifera revealed significant damage by excess aluminum ions under each treatment compared with the control group. Under Al stress at 2 mmol/L (GZ2) and 4 mmol/L (GZ4), the total flavonoid content in C. drupifera leaves reached 24.37 and 35.64 mg/g, respectively, which are significantly higher than the levels measured in the control group (CK) (p < 0.01). In addition, we identified 25 upregulated and 5 downregulated metabolites in the GZ2 vs. CK comparison and 31 upregulated and 7 downregulated flavonoid metabolites in GZ4 vs. CK. The results demonstrate that different levels of Al stress had a significant influence on the metabolite profile of C. drupifera. It was found that the abundance of the 24 differential flavonoid metabolites was gradually elevated with increasing concentrations of Al stress, including catechin, epicatechin, naringenin-7-glucoside, astilbin, taxifolin, miquelianin, quercitrin, and quercimeritrin. Moreover, the most significant increase in antioxidant activity (about 30%) was observed in C. drupifera precultured in leaf extracts containing 7.5 and 15 μg/mL of active flavonoids. The qRT-PCR results showed that the expression levels of key genes involved in the synthesis of flavonoids were consistent with the accumulation trends of flavonoids under different concentrations of Al. Therefore, our results demonstrate the key role of flavonoid compounds in the oil tea plant C. drupifera in response to Al stress, which suggests that flavonoid metabolites in C. drupifera, as well as other aluminum-tolerant plants, may help with detoxifying aluminum.

Keywords: Camellia drupifera; UPLC-MS/MS; aluminum stress; flavonoid metabolites.

Figure 1

Responses of C. drupifera to aluminum treatment. (a) Morphological characteristics and (b) total flavonoid content under different concentrations of aluminum. In the histogram, different letters indicate statistical significance (p < 0.05).

Figure 1

Responses of C. drupifera to aluminum treatment. (a) Morphological characteristics and (b) total flavonoid content under different concentrations of aluminum. In the histogram, different letters indicate statistical significance (p < 0.05).

Figure 2

Heatmap of 78 flavonoid metabolites. The data on the flavonoid metabolite contents were normalized for hierarchical cluster analysis. Each column represents one sample, while each metabolite is represented by one row. Red color in the scale bars indicates an increase in relative metabolite abundance, while green color in the scale bars indicates a decrease, with the magnitude of change according to the scale (log₂ (fold change)).

Figure 3

PCA analysis of the flavonoid metabolites in the leaves of C. drupifera under aluminum stress.

Figure 4

OPLS-DA model plots for (a) GZ2 vs. CK; (b) GZ4 vs. CK; (c) GZ4 vs. GZ2.

Figure 5

Volcano maps of differential metabolites. (a) GZ2 vs. CK; (b) GZ4 vs. CK; (c) GZ4 vs. GZ2.

Figure 6

Venn diagram showing the numbers of metabolites in GZ2 vs. CK and GZ4 vs. CK.

Figure 7

K-means cluster analysis showing the dynamic accumulation of differential metabolites under different concentrations of aluminum.

Figure 8

KEGG enrichment analysis of the differential metabolites in the comparisons of (a) GZ2 vs. CK and (b) GZ4 vs. CK.

Figure 8

KEGG enrichment analysis of the differential metabolites in the comparisons of (a) GZ2 vs. CK and (b) GZ4 vs. CK.

Figure 9

Effect of flavonoid pretreatment on antioxidant activity, expressed as the percentage of inhibition of DPPH radicals in C. drupifera leaves. * indicates statistical significance (p < 0.05).

Figure 10

Results of qRT-PCR analysis of the six genes (IFS, F3H, DFR, FLS, CHS1, and PAL) in the flavonoid biosynthetic pathway of C. drupifera under different concentrations of Al. Different letters indicate statistical significance (p < 0.05).