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

**Jie Gao** †**, Rui Ren** † **, Yonglu Wei, Jianpeng Jin, Sagheer Ahmad, Chuqiao Lu, Jieqiu Wu, Chuanyuan Zheng, Fengxi Yang \* and Genfa Zhu \***

Guangdong Key Laboratory of Ornamental Plant Germplasm Innovation and Utilization, Environmental Horticulture Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China; gaojie@gdaas.cn (J.G.); Renruinjau@163.com (R.R.); hjyylab@126.com (Y.W.); 13424050551@163.com (J.J.); sagheerhortii@gmail.com (S.A.); chuqiaolu18@163.com (C.L.); jiebaoqiqiqiu@163.com (J.W.); 15014323983@163.com (C.Z.)

**\*** Correspondence: yangfengxi@gdaas.cn (F.Y.); zhugenfa@gdaas.cn (G.Z.); Tel.: +86-020-8516-1014 (F.Y.)

† These authors contributed equally to this work.

Received: 5 February 2020; Accepted: 6 March 2020; Published: 9 March 2020

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