Comparative Metabolomic Analysis Reveals Distinct Flavonoid Biosynthesis Regulation for Leaf Color Development of Cymbidium sinense 'Red Sun'

Abstract

The colorful leaf is an important ornamental character of Cymbidium sinense (C. sinense), especially the red leaf, which has always been attracted by breeders and consumers. However, little is documented on the formation mechanism of the red leaf of C. sinense. In this study, the changing patterns of flavonoid-related metabolites, corresponding enzyme activities and genes expression in the leaves of C. sinense 'Red Sun' from red to yellow and finally to green was investigated. A total of 196 flavonoid-related metabolites including 11 anthocyanins metabolites were identified using UPLC-MS/MS-based approach. In the process of leaf color change, 42 metabolites were identified as having significantly different contents and the content of 28 differential metabolites turned to zero. In anthocyanin biosynthetic pathway, content of all 15 identified metabolites showed downregulation trend in the process of leaf color change. Among the 15 metabolites, the contents of Naringenin chalcone, Pelargonidin O-acetylhexoside and Anthocyanin 3-O-beta-d-glucoside decreased to zero in the green leaf stage. The changing pattern of enzyme activity of 10 enzymes involved in the anthocyanin biosynthetic pathway showed different trends from red leaves that have turned yellow and finally green, while the expression of genes encoding these enzymes was all down-regulated in the process of leaf color change. The results of this study revealed the types of flavonoid-related metabolites and the comprehensive analysis of metabolites content, enzyme activities and genes expression providing a new reference for breeders to improve the leaf color of C. sinense 'Red Sun'.

Keywords: Cymbidium sinense; differential metabolites; enzyme activity; gene expression; leaf color; metabolomic analysis.

Figure 1

Phenotypes of Cymbidium sinense ‘Red Sun’ red leaves (RL) (a), yellow leaves (YL) (b) and green leaves (GL) (c).

Figure 2

Chlorophyll (a), carotenoids content (b), total flavonoid (c) and total anthocyanin (d) content of different color leaves of Cymbidium sinense ‘Red Sun’. Bars represent the mean of three biological replicates ±SE. Lowercase letters indicate significant differences at p < 0.05.

Figure 3

Hierarchical clustering analysis of all metabolites detected in this study. The abscissa indicates three biological replicates of red leaves (RL1, RL2, and RL3), yellow leaves (YL1, YL2, and YL3) and green leaves (GL1, GL2, and GL3), and the ordinate indicates the metabolites detected in this study. The red segments indicate a relatively high content of metabolites, while the blue segments indicate a relatively low content of metabolites. The relative metabolite contents represented by color segments at the corresponding locations are listed in Table S1.

Figure 4

(a) Volcano plot of differential metabolites for RL vs. GL, RL vs. YL and YL vs. GL. The colors of the scatter points in Figure 4a indicate the final screening results: red indicates metabolites that were significantly up-regulated; green indicates metabolites that were significantly down-regulated; grey indicates metabolites with no significant difference. (b) Venn diagram analysis of differential metabolites. RL, red leaves; YL, yellow leaves; GL, green leaves.

Figure 5

Differences in the content of 28 metabolites in the process of leaf color change. Y-scale represent the integral value of chromatographic peak area.

Figure 6

The changing patterns of enzymes activity and intermediate products contents related to anthocyanin synthesis in the process of Cymbidium sinense ‘Red Sun’ leaf color change. Red and blue shading in the lower right corner indicates the relatively high-or low content, respectively. PAL, Phenylalanine ammonia lyase, C4H, cinnamate 4-hydroxylase, 4CL, 4-coumarate CoA ligase, CHS, chalcone synthase, CHI, chalcone isomerase, F3H, flavone 3-hydroxylase, F3′H, flavonoid 3′-hydroxylase, F3′5′H, flavonoid 3′,5′-hydroxylase, DFR, dihydroflavonol reductase, ANS, anthocyanidin synthase. Units on y-scale of enzymes activity is U/g.

Figure 7

Expression pattern of genes coding enzymes related to anthocyanin synthesis in the process of Cymbidium sinense ‘Red Sun’ leaf color change.