Transcriptomic and targeted metabolomic analyses provide insights into the flavonoids biosynthesis in the flowers of Lonicera macranthoides

Abstract

Background: Flavonoids are one of the bioactive ingredients of Lonicera macranthoides (L. macranthoides), however, their biosynthesis in the flower is still unclear. In this study, combined transcriptomic and targeted metabolomic analyses were performed to clarify the flavonoids biosynthesis during flowering of L. macranthoides.

Results: In the three sample groups, GB_vs_WB, GB_vs_WF and GB_vs_GF, there were 25, 22 and 18 differentially expressed genes (DEGs) in flavonoids biosynthetic pathway respectively. A total of 339 flavonoids were detected and quantified at four developmental stages of flower in L. macranthoides. In the three sample groups, 113, 155 and 163 differentially accumulated flavonoids (DAFs) were detected respectively. Among the DAFs, most apigenin derivatives in flavones and most kaempferol derivatives in flavonols were up-regulated. Correlation analysis between DEGs and DAFs showed that the down-regulated expressions of the CHS, DFR, C4H, F3'H, CCoAOMT_32 and the up-regulated expressions of the two HCTs resulted in down-regulated levels of dihydroquercetin, epigallocatechin and up-regulated level of kaempferol-3-O-(6''-O-acetyl)-glucoside, cosmosiin and apigenin-4'-O-glucoside. The down-regulated expressions of F3H and FLS decreased the contents of 7 metabolites, including naringenin chalcone, proanthocyanidin B2, B3, B4, C1, limocitrin-3,7-di-O-glucoside and limocitrin-3-O-sophoroside.

Conclusion: The findings are helpful for genetic improvement of varieties in L.macranthoides.

Keywords: Lonicera macranthoides; Anthocyanin; Flavone; Flavonoid biosynthesis; Flavonol; Flower; Targeted metabolome; Transcriptome.

Fig. 1

Venn Diagram and the up- and down-regulated differentially expressed genes in the three sample groups. GB, green flower bud; WB, white flower bud; WF, white flower; GF, golden flower

Fig. 2

KEGG pathway classification of differentially expressed genes in the three sample groups of L. macranthoides.(A) In GB_vs_WB. (B) In GB_vs_WF. (C) In GB_vs_GF.

Fig. 3

qPCR validation of RNA-seq data using 9 related genes in L. macranthoides. 18 S rRNA expression values were used as the internal reference. Values are the means ± SD of three biological replicates. GB, green flower bud; WB, white flower bud; WF, white flower; GF, golden flower

Fig. 4

The total flavonoids detected in the samples

Fig. 5

The differentially accumulated flavonoids in the three sample groups. (A) Venn diagram depicting the shared and specific flavonoids. (B) The up- and down-regulated flavonoids. GB, green flower bud; WB, white flower bud; WF, white flower; GF, golden flower

Fig. 6

Clustering heat map of the flavonoids detected in the total samples. Each sample is visualized in a single column, and each metabolite is represented in a single row. Red indicates high abundance of metabolites, while green indicates low abundance. GB, green flower bud; WB, white flower bud; WF, white flower; GF, golden flower

Fig. 7

The pathway associated with flavonoid biosynthesis in L. macranthoides and heat maps of differentially expressed genes (DEGs) and differentially accumulated flavonoids in three sample groups. The red font represents DEG. ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; CCoAOMT, caffeoyl-CoA O-methyl-transferase; CHI, chalcone isomerase; CHS, chalcone synthase; C4H, cinnamate-4-hydroxylase; C3’H, p-coumarate 3’-hydroxylases; DFR, dihydroflavonol reductase; F3’5’H, flavonoid 3’,5’- hydroxylase; F3’H, flavonoid 3’-hydroxylase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; FNS, flavone synthase; HCT, hydroxycinnamoyl CoA shikimatelquinate hydroxycinnamoyltransferase; LAR, leucocyanidin reductase; PAL, phenylalanine ammonialyase; 4CL, 4-coumaric acid coenzyme a ligase

Fig. 8

Connection network between genes and flavonoids. K3OG, Kaempferol-3-O-(6’’-O-acetyl)- glucoside; LOG, luteolin-7-O-(6’’-malonyl)-glucoside; K3OGG, Kaempferol-3-O-(6’’-acetyl)glucosyl- (1→3)-galactoside. The size of the triangle and the circle represents the number of genes or metabolites associated with them, respectively

Fig. 9

The four developmental stages of flower in L. macranthoides. From left to right, green flower bud (GB), white flower bud (WB), white flower (WF) and golden flower (GF).