Integrative analysis of transcriptome and metabolome reveals flavonoid biosynthesis regulation in Rhododendron pulchrum petals

Abstract

Background: Color is the major ornamental feature of the Rhododendron genus, and it is related to the contents of flavonoid in petals. However, the regulatory mechanism of flavonoid biosynthesis in Rhododendron pulchrum remains unknown. The transcriptome and metabolome analysis of Rhododendron pulchrum with white, pink and purple color in this study aimed to reveal the mechanism of flavonoid biosynthesis and to provide insight for improving the petal color.

Results: Flavonoids and flavonols are the major components of flavonoid metabolites in R.pulchrum, such as laricitrin, apigenin, tricin, luteolin, isoorientin, isoscutellarein, diosmetin and their glycosides derivatives. With transcriptome and metabolome analysis, we found CHS, FLS, F3'H, F3'5'H, DFR, ANS, GT, FNS, IFR and FAOMT genes showed significantly differential expression in cultivar 'Zihe'. FNS and IFR were discovered to be associated with coloration in R.pulchrum for the first time. The FNS gene existed in the form of FNSI. The IFR gene and its related metabolites of medicarpin derivatives were highly expressed in purple petal. In cultivar 'Fenhe', up-regulation of F3'H and F3'5'H and down-regulation of 4CL, DFR, ANS, and GT were associated with pink coloration. With the transcription factor analysis, a subfamily of DREBs was found to be specifically enriched in pink petals. This suggested that the DREB family play an important role in pink coloration. In cultivars 'Baihe', flavonoid biosynthesis was inhibited by low expression of CHS, while pigment accumulation was inhibited by low expression of F3'5'H, DFR, and GT, which led to a white coloration.

Conclusions: By analyzing the transcriptome and metabolome of R.pulchrum, principal differential expression genes and metabolites of flavonoid biosynthesis pathway were identified. Many novel metabolites, genes, and transcription factors associated with coloration have been discovered. To reveal the mechanism of the coloration of different petals, a model of the flavonoid biosynthesis pathway of R.pulchrum was constructed. These results provide in depth information regarding the coloration of the petals and the flavonoid metabolism of R.pulcherum. The study of transcriptome and metabolome profiling gains insight for further genetic improvement in Rhododendron.

Keywords: Flavonoid biosynthesis; Metabolome; Rhododendron pulchrum; Transcriptome.

Fig. 1

OPLS-DA score plot of three materials and numbers of potential markers for each color petals. Note: a OPLS-DA score plot of ‘Baihe’ and ‘Fenhe’ petals; b OPLS-DA score plot of ‘Zihe’ and ‘Baihe’ petals; c OPLS-DA score plot of ‘Fenhe’ and ‘Zihe’ petals

Fig. 2

Flavonoid metabolites in R.pulchrum with different colors. Note: BMJ, cultivar ‘Baihe’; FMJ, cultivar ‘Fenhe’; ZMJ, cultivar ‘Zihe’. a Measurement of total flavonoids in R.pulchrum with different colors. b Flavonoid metabolites in ‘Baihe’ and ‘Fenhe’. c Flavonoid metabolites in ‘Zihe’ and ‘Baihe’. d Flavonoid metabolites in ‘Fenhe’ and ‘Zihe’

Fig. 3

Fold change of the flavonoid and anthocyanin metabolites in R.pulchrum with different colors. Note: BMJ, cultivar ‘Baihe’; FMJ, cultivar ‘Fenhe’; ZMJ, cultivar ‘Zihe’. a Flod change of the flavonoid metabolites. b Fold change of the anthocyanin metabolites

Fig. 4

Differently expressed genes in R.pulchrum with different colors. Note: a Statistic of differently expressed gene in cultivars 'Baihe'(BMJ),'Fenhe'(FMJ) and 'Zihe'(ZMJ). b The Venn diagram of differently expressed gene between three R.pulchrum cultivars

Fig.5

Transcript GO annotation classification statistics graph in three comparison groups. Note: BMJ, cultivar ‘Baihe’; FMJ, cultivar ‘Fenhe’; ZMJ, cultivar ‘Zihe’. a Transcript GO annotation classification between BMJ and FMJ; b Transcript GO annotation classification between ZMJ and BMJ; c Transcript GO annotation classification between FMJ and ZMJ

Fig. 6

KEGG pathway classification statistics graph in three comparison groups. Note: BMJ, cultivar ‘Baihe’; FMJ, cultivar ‘Fenhe’; ZMJ, cultivar ‘Zihe’. a KEGG pathway classification statistics between BMJ and FMJ; b KEGG pathway classification statistics between ZMJ and BMJ; c KEGG pathway classification statistics between FMJ and ZMJ

Fig. 7

Distribution of transcription factor types in three comparison groups. Note: BMJ, cultivar ‘Baihe’; FMJ, cultivar ‘Fenhe’; ZMJ, cultivar ‘Zihe’. a Distribution of transcription factor types in BMJ and FMJ group; b Distribution of transcription factor types in ZMJ and BMJ group; a Distribution of transcription factor types in FMJ and ZMJ group

Fig. 8

Biosynthesis pathway of flavonoid in three different color petals of R.pulchrum Sweet. Note: a Key structural genes and their expression level of involved in flavonoid biosynthesis pathway in R.pulchrum Sweet with different colors. b The expression levels of differential metabolites in flavonoid synthesis pathway

Fig. 9

A model for the flavonoid biosynthesis pathway in three different color petals of R.pulchrum Sweet. Note: a cultivar ‘Baihe’. b cultivar ‘Fenhe’. c cultivar ‘Zihe’. The red color indicates genes and metabolites with high expression, and the blue color indicates genes and metabolites with low expression

Fig. 10

WGCNA of genes in three different color petals of R.pulchrum Sweet. a Hierarchical clustering tree (cluster dendrogram) results showed 12 expression modules, labeled with different colors. b Module–sample association. Each row corresponds to a module, labeled with a color as in (a). The number of genes in each module is indicated on the left

Fig. 11

Coexpression network analysis in three different color petals of R.pulchrum Sweet. (a), (c), (e), (g), and (i) Heatmaps showing genes in cyan, midnightblue, mediumpurple3, lightcyan1, and grey60 modules that were significantly over-represented in BMJ, FMJ, and ZMJ, respectively. (b), (d), (f), (h), and (j) The correlation networks in the module corresponding to (a), (c), (e), (g), and (i), respectively. Candidate hub genes are shown in red

Fig. 12

Three different colors of Rhododendron pulchrum Sweet. Note: a. ‘Baihe’; b.‘Fenhe’; c.‘Zihe’