Metabolite analysis reveals flavonoids accumulation during flower development in Rhododendron pulchrum sweet (Ericaceae)

Abstract

The azalea (Rhododendron simsii Planch.) is an important ornamental woody plant with various medicinal properties due to its phytochemical compositions and components. However little information on the metabolite variation during flower development in Rhododendron has been provided. In our study, a comparative analysis of the flavonoid profile was performed in Rhododendron pulchrum sweet at three stages of flower development, bud (stage 1), partially open flower (stage 2), and full bloom (stage 3). A total of 199 flavonoids, including flavone, flavonol, flavone C-glycosides, flavanone, anthocyanin, and isoflavone were identified. In hierarchical clustering analysis (HCA) and principal component analysis (PCA), the accumulation of flavonoids displayed a clear development stage variation. During flower development, 78 differential accumulated metabolites (DAMs) were identified, and most were enriched to higher levels at the full bloom stage. A total of 11 DAMs including flavone (chrysin, chrysoeriol O-glucuronic acid, and chrysoeriol O-hexosyl-O-pentoside), isoflavone (biochanin A), and flavonol (3,7-di-O-methyl quercetin and isorhamnetin) were significantly altered at three stages. In particular, 3,7-di-O-methyl quercetin was the top increased metabolite during flower development. Furthermore, integrative analyses of metabolomic and transcriptomic were conducted, revealing that the contents of isoflavone, biochanin A, glycitin, and prunetin were correlated with the expression of 2-hydroxyisoflavanone dehydratase (HIDH), which provide insight into the regulatory mechanism that controls isoflavone biosynthesis in R. pulchrum. This study will provide a new reference for increasing desired metabolites effectively by more accurate or appropriate genetic engineering strategies.

Keywords: Flavonoids; Flavonol; Flower development; Isoflavone; Rhododendron pulchrum sweet.

Figure 1. Three flowering stages of R. pulchrum.

(A) Stage 1: bud; (B) stage 2: pre-flowering; (C) stage 3: fully open flower.

Figure 2. HCA and PCA of flavonoids in R. pulchrum during flower development.

(A) Heatmap of flavonoids detected in the total samples. Red indicates high abundance, and green indicates low abundance. (B) Score plot of PCA in different flower development stages. Each point represents a sample from three stages and mixed samples. Stages 1, 2, and 3 represent the bud, pre-flowering, and fully open flower stages, respectively. The mix represents quality control (QC) sample, from the mixture of all samples.

Figure 3. Analysis of differential accumulation of metabolites (DAMs) during flower development.

(A) Numbers of upregulated or downregulated DAMs in the three comparisons. (B) Heat maps of 78 DAMs identified during flower development. Red indicates high abundance, and green indicates low abundance. All DAMs were divided into three clusters (1,2,3) according to their change trend. Ace, acetyl; Ade, adenosine; Api, apigenin; Aca, Acacetin; Chr, chrysoeriol; Cya, Cyanidin; GR, glucopyranoside; Dim, dimethyl; Eri, Eriodictyol; Fer, feruloyl; GE, b-guaiacylglyceryl ether; Glu, glucoside; Gly, glycerin; Gua, guanosine; Gluc, Glucuronic acid; Gen, Genistein; Hex, hexoside; hexosyl; Kae, Kaempferol; Ino, Inosine; Iso, Isorhamnetin; Isos, Isosakuranetin; Lut, luteolin; mal, malonyl; met, methyl; Nar, Naringenin; neo, neohesperidoside; Pen, pentoside;rut, rutinoside; oct, octadecatetraenoic acid; que, quercetin; Rob, robinoside; rha, rhamnoside; Ros, Rosinidin;Phe, phenylformic acid; Pen, pentosyl; Pel, Pelargonidin; Que, Quercetin; Sac, saccharopine; Sin, sinapoyl; SE, syringyl alcohol ether; syr, syringic acid; SA, saccharic acid; Tri,Tricin.

Figure 4. Differential accumulation of metabolites (DAMs) in R. pulchrum during flower development.

(A) Venn diagram of DAMs shared by two comparisons or all three comparisons. (B) The 11 DAMs shared among the three comparisons. (C) five DAMs specifically exhibited a significant difference in accumulation between the pre-flowering (stage 2) and the fully open flower stage (stage 3). (D) The eight DAMs specifically exhibited a significant difference in accumulation between the bud (stage 1) and the pre-flowering stage (stage 2). The dentification of DAMs among different flowering stages was determined by PLSDA with the VIP values >1 and ANOVA (p ≤ 0.05). The data in B, C, D are means ± SD from two biological replicates. Api, apigenin; Chr, chrysoeriol; Fer, feruloyl; Gluc, glucuronic acid; Hex, hexosyl; Kae, kaempferol; Met, methyl; Pen, pentoside; Que, quercetin; Rut, rutinoside; Ros, rosinidin; Tri, tricin.

Figure 5. Top 20 upregulated and downregulated DAMs in the two comparisons.

(A) Bud vs partially open flower stage, (B) partially open flower vs fully open flower stage. Api, apigenin; Chr, chrysoeriol; Cya, cyanidin; Fer, feruloyl; Glu, glucoside; Gluc, glucuronic acid; Hex, hexosyl; Kaem, kaempferol; Lut, luteolin; Mal, malonyl; Met, methyl; Nar, naringenin; Pen, pentoside; Que, quercetin; Rut, rutinoside; Ros, rosinidin; Robi, robinobioside.

Figure 6. Expression pattern of unigenes and accumulation profiles of DAMs in flavonoids biosynthesis pathway in R. pulchrum during flower development (three stages: bud, partially open flower, and fully open flower stage).

The color scale from blue (low) to orange (high) represents the FPKM values. The unigene names are indicated at the side of each step. The color scale from green (low) to red (high) represents the abundance of the metabolites. Unigene names were abbreviated as follows: trans-cinnamate 4-monooxygenase (CYP73A), shikimate O-hydroxycinnamoyl transferase (HCT), 2-hydroxyisoflavanone synthase (CYP93C), naringenin 3-dioxygenase (F3H), isoflavone 7-O-glucosyltransferase (IF7GT), flavonoid 3′,5′-hydroxylase (CYP75B), flavonol synthase (FLS), flavonoid 3′,5′-hydroxylase (CYP75A), flavonoid O-methyltransferase (AOMT).