Integrated Analyses of Metabolome and RNA-seq Data Revealing Flower Color Variation in Ornamental Rhododendron simsii Planchon

Abstract

Rhododendron simsii Planchon is an important ornamental species in the northern hemisphere. Flower color is an important objective of Rhododendron breeding programs. However, information on anthocyanin synthesis in R. simsii is limited. In this research, the regulatory mechanism of anthocyanin biosynthesis in R. simsii was performed through the integrated analysis of metabolome and RNA-seq. A total of 805 and 513 metabolites were screened by positive and negative ionization modes, respectively, In total, 79 flavonoids contained seven anthocyanidins, 42 flavanones, 10 flavans, 13 flavones, and seven flavonols. Methylated and glycosylated derivatives took up the most. Differentially accumulated metabolites were mainly involved in "flavone and flavonol biosynthesis", "cyanoamino acid metabolism", "pyrimidine metabolism", and "phenylalanine metabolism" pathways. For flavonoid biosynthesis, different expression of shikimate O-hydroxycinnamoyltransferase, caffeoyl-CoA O-methyltransferase, flavonoid 3'-monooxygenase, flavonol synthase, dihydroflavonol 4-reductase/flavanone 4-reductase, F3'5'H, chalcone synthase, leucoanthocyanidin reductase, and 5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase genes ultimately led to different accumulations of quercetin, myricetin, cyanidin, and eriodictyol. In flavone and flavonol biosynthesis pathway, differential expression of F3'5'H, flavonoid 3'-monooxygenase and flavonol-3-O-glucoside/galactoside glucosyltransferase genes led to the differential accumulation of quercetin, isovitexin, and laricitrin. This research will provide a biochemical basis for further modification of flower color and genetic breeding in R. simsii and related Rhododendron species.

Keywords: Rhododendron simsii Planchon; anthocyanins; flower color breeding; flower color variation; molecular mechanism.

Figure 1

Flower tissues of R. simsii varieties: (A) “red variety”; (B) “pink variety”; (C) “violet variety”.

Figure 2

Annotation of metabolites detected under positive ionization mode (A) and negative ionization mode (B) against HMDB database.

Figure 3

LIPID MAPS annotation of metabolites detected under positive ionization mode (A) and negative ionization mode (B).

Figure 4

Cluster analyses of variety-specific accumulation patterns.

Figure 5

Venn diagram of differentially accumulated metabolites detected under positive ionization mode (A) and negative ionization mode (B).

Figure 6

Cluster analyses of three R. simsii varieties based on metabolite profile.

Figure 7

Typical representatives of three R. simsii varieties: (A) “red variety” vs. “pink variety” under positive ionization mode; (B) “red variety” vs. “pink variety” under negative positive ionization mode; (C) “red variety” vs. “violet variety” under positive ionization mode; (D) “red variety” vs. “violet variety” under negative ionization mode; (E) “pink variety” vs. “violet variety” under positive ionization mode; (F) “pink variety” vs. “violet variety” under negative ionization mode.

Figure 8

Overview of flavonoid biosynthesis process in R. simsii flowers. Notes: Numbers refer to corresponding enzymes listed in Enzyme Commission hierarchy. The red, blue, and green codes represent upregulated, downregulated, and unstable differentially expressed genes, respectively.

Figure 9

Overview of flavone and flavonol biosynthesis process in R. simsii flowers. Notes: Numbers refer to corresponding enzymes listed in Enzyme Commission hierarchy. The red, blue, and green codes represent upregulated, downregulated, and unstable differentially expressed genes, respectively.