Flavonoid biomarkers and co-expression gene networks characterized in ten Hemerocallis citrina accessions via multi-omics

Abstract

Hemerocallis citrina Baroni (H. citrina), a traditional medicinal and edible plant, is rich in bioactive flavonoids. This study investigated flavonoid metabolites in H. citrina flowers from ten accessions using UPLC-MS/MS-based metabolomics. A total of 642 flavonoids and 14 tannins were identified in the flowers. Among these, 375 flavonoids were classified as flavonols and flavone, representing for 39.33% and 17.53%, respectively. The identification of 309 differential flavonoid metabolites was achieved through analyses of nine pairwise comparison groups. These differential flavonoids in flowers of H. citrina could be distinctly categorized into two groups, clearly separating the 7 edible accessions from the 3 non-edible accessions. Significant variations in 14 flavonoid markers were observed among 10 H. citrina accessions originating from different regions, as determined by hierarchical clustering and ROC analyses. Transcriptomic analyses revealed that the majority of differentially expressed genes were enriched in the flavonoid metabolism pathway among these accessions. Integrated transcriptomic and metabolic analyses identified 14 differential flavonoid metabolites (DFMs) and 47 differentially expressed genes (DEGs) associated with flavonoid biosynthesis through Pearson's correlation analysis and WGCNA analyses. qRT-PCR validation of 13 DEGs confirmed the consistency of transcriptomic data. A flavonoid-gene correlation network indicated that the 14 DFMs might be directly regulated by 17 DEGs, comprising 13 flavnoid metabolism-related genes and 4 transcript factors. These findings provide biological and chemical insights into flavonoid metabolic difference across H. citrina origins, offering a theoretical basis for food and medical applications, and enabling clear differentiation between edible and non-edible H. citrina flowers in the market.

Keywords: Hemerocallis citrina; Correlation network; Differentially expressed genes; Flavonoid metabolism; Flowers.

Fig. 1

Characterization of flavonoid metabolites in flowers from 10 H. citrina accessions. (A): Types of flavonoid metabolites; (B): Principal component analysis (PCA); (C): Differential flavonoid metabolites from pairwise comparative analysis; D: Hierarchical cluster analysis of flavonoid metabolites.

Fig. 2

The statistic analysis of representive marker flavonoid metabolites in floral samples from ten H. citrina accessions. A-N: 14 representive marker flavonoid metabolites identified in floral samples from ten H. citrina accessions; O: the heatmap of Differential flavonoid metabolites by hierarchical clustering analysis. Note: the error bars representing the variability of all sample data on the graph were calculated using statistical methods (mean ± SD).

Fig. 3

The analysis of differentially expressed genes in floral samples from ten H. citrina accessions. (A): The number of DEGs identified across 9 pairwise comparison combination from floral samples of ten H. citrina accessions; (B): Go enrichment analysis of DEGs; C: KEGG enrichment analysis of DEGs; D: Hierarchical clustering analysis of DEGS across floral samples from ten H. citrina accessions.

Fig. 4

Differential gene expression profiles of ten H. citrina accessions.Note: the error bars representing the variability of all sample data on the graph were calculated using statistical methods (mean ± SD); Different lowercase letters above the bars indicate significant differences (p < 0.05).

Fig. 5

Interaction regulatory network of differential metabolites and differentially expressed genes.

Fig. 6

Hierarchical cluster analysis of differential flavonoid metabolites and differentially expressed genes. (A): Cluster tree containing 30% DFMs; (B): Cluster tree containing 45% DFMs; (C): Cluster tree containing 100% DFMs; (D): Cluster tree containing 15% DEGs; (E): Cluster tree containing 30% DEGs F: Cluster tree containing 100% DEGs