An integrated analysis of metabolomic and transcriptomic profiles reveals flavonoid metabolic differences between Anoectochilus roxburghii and Anoectochilus formosanus.

• The difference between Ar and Af leaf samples was first characterized applying transcriptome and metabolome analysis. • Correlation analysis between metabolites and metabolic enzyme gene expression revealed flavonoid metabolic differences between Ar and Af. • This study screened and obtained key genes potentially involved in the differentially accumulation of flavonoids between Ar leaves and Af leaves. • This study provides an in-depth understanding of differentially flavonoid accumulation between Ar leaves and Af leaves. Anoectochilus roxburghii (Wall.) Lindl (Ar) and Anoectochilus formosanus Hayata (Af) are two species of valuable Chinese herbal medicines that belong to the genus Anoectochilus in Orchidaceae and have often been applied as the Chinese herbal medicine "Jin Xian Lian". However, these species exhibit obvious differences in leaf color, and the difference in their medicinal value remains unclear. In this study, the metabolomes of leaf samples of Ar and Af were analyzed by high-performance liquid chromatography-mass spectrometry (LC–MS) in the positive and negative ion modes. The analysis of the leaves of both species in the positive ion mode identified 4013 metabolites enriched in 106 metabolic pathways, and the analysis in the negative ion mode found 1530 metabolites enriched in 98 metabolic pathways. A differential metabolic profiling analysis of the results obtained in positive ion mode found that 816 differentially accumulated metabolites were enriched in 80 metabolic pathways, whereas 297 differentially accumulated metabolites enriched in 55 metabolic pathways were obtained from the analysis in the negative ion mode. The number of flavonoids was higher than those of alkaloids and terpenoids. A quantitative analysis of the differentially accumulated metabolites showed that the most highly upregulated metabolites (Af/Ar) among the top 20 differentially accumulated metabolites in Af were mainly related to amino acid-related metabolic pathways, whereas the most highly downregulated metabolites (Af/Ar) in Af were mainly associated with flavonoid-related metabolic pathways. In addition, the transcriptome sequencing of Ar and Af leaf samples yielded 40.12 Gb of transcriptomic data. The analysis of differentially expressed genes (DEGs) classified 555 metabolism-related DEGs into 23 metabolism-related KEGG pathways, and the top DEGs were involved in the phenylpropanoid biosynthetic pathways, which included up to 138 DEGs associated with the flavonoid metabolism pathway. In addition, significant differences in the flavonoid-related KEGG pathways were found. A comprehensive analysis of the metabolomes and transcriptomes showed that the genes annotated as chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS), caffeoyl-CoA O -methyltransferase (CCOAOMT) and anthocyanidin synthase (ANS) were downregulated in Af leaves compared with Ar leaves, which might account for the downregulation of flavonoid-related metabolites in Af. In summary, both the metabolomic and transcriptomic analyses showed significant differences in flavonoid metabolism between the Ar and Af leaves, and the mechanisms underlying the molecular regulation of these differences were preliminarily analyzed. [ABSTRACT FROM AUTHOR]