**1. Introduction**

As the largest family of monocotyledons, Orchidaceae has a long history of cultivation and contains abundant varieties. Among these varieties, *C. sinense* is a unique variety produced in China, which has been loved by consumers all around the world. The most important phenotypic feature of *C. sinense* is that it has a very rich variation in leaf color, which improves its ornamental and economic values. *C. sinense* contains a number of varieties based on the location and color of leaf variegation, including chimera art, claw art, crown art, crane art, middle penetration art, spot art and treasure art and so on [1]. At present, the leaf variegation varieties of *C. sinense* are basically yellow-green leaves, but there are few reports about red leaves. *C. sinense* 'Qihei' is the most common variety of green leaves. In the process of culture, the color of 'Qihei' leaf buds changes to red, and then the leaf color changes from red to yellow, finally to green, leaving only a little yellow at the tip of the leaf during the growth and development of leaf buds. The leaf variegation variety is named *C. sinense* 'Red Sun'. So far, the metabolic changes associated with the formation of red leaves in *C. sinense* 'Red Sun' are not known.

Compared with flower color, the influencing factors of leaf color are more complex. Generally, the greening of leaves is mainly due to the absolute proportion of chlorophyll, and the formation of yellow leaves is mostly due to the degradation of chlorophyll, leaving the color of carotenoids to dominate in leaves [1,2]. Anthocyanins are responsible for the red color of leaves, which is helpful for plants against various biotic and abiotic stresses [3,4]. In *Cymbidium* orchids, the activation of anthocyanin synthesis can be restored by introducing MYB and bHLH anthocyanin regulators simultaneously [5]. The synthesis pathway of anthocyanins begins with the precursor phenylalanine, resulting in the formation of dihydrokaempferol (DHK) by the catalysis of six enzymes. After the catalysis of four enzymes, DHK produces three kinds of steady-state anthocyanins, including pelargonidin, cyanidin and delphinidin, respectively [6–8]. At present, more than 600 anthocyanins found in nature are derived from these three substances and the accumulation of anthocyanins in plants has two main functions: one is to produce rich and colorful visual signals to promote pollination or seed transmission, and the other is to resist a series of biotic or abiotic stresses [9,10].

Plant metabolomics carries out a qualitative and quantitative analysis of small molecular metabolites in plants, so as to help researchers understand the synthesis and accumulation patterns of metabolites [11]. At present, the study of plant metabolites mainly involves the identification of metabolites, variety differentiation and auxiliary breeding and so on [12–15]. Flavonoids-targeted metabolomics refers to the analysis of small molecular metabolites of flavonoids (anthocyanins), which is often used to analyze the formation mechanism of plant color. Analysis of Flavonoids-targeted metabolomics in the white and purple flowers of *Phalaenopsis* identified 142 different flavonoid-related metabolites, of which the most important anthocyanin was the derivative of cyaniding [16]. A study on green and purple asparagus suggested that the difference in color was mainly caused by the contents of peonidin and cyanidin and their glycoside derivatives [17]. Metabolomic analysis the color difference of fig peel showed that the content of anthocyanin derivatives in purple peel was significantly higher than that in green fruit [18]. These results indicate that metabolomics is an important and effective method to analyze the formation mechanism of plant color.

In order to understand the key metabolites involved in the process of color change from red to green in the leaves of *C. sinense* 'Red Sun', flavonoids-targeted metabolomic analysis was used to analyze the change pattern of flavonoid-metabolites in the process of leaf color change. Physiology, enzyme kinetics and molecular biology experiments were carried out to explore the mechanism of leaf color difference. To the best of our knowledge, this study is the first effort to analyze the mechanism of the formation of *C. sinense* leaf variegation from the perspective of metabolites, and the results provide a new train of thought and basis for the study of *C. sinense* leaf variegation.
