*2.5. Intermediates Content, Enzymes Activities and Genes Expression Associated with the Anthocyanin Biosynthetic Pathway*

In order to further analyze the regulation mechanism of anthocyanin in the process of *C. sinense* 'Red Sun' leaf color change, the detected intermediates of anthocyanin synthesis pathway and all the enzyme activities involved in anthocyanin synthesis were analyzed [19] (Figure 6). It can be observed that all 15 detected intermediates show a downward trend in the process of leaf color change. In total, 11 kinds of anthocyanins metabolites were detected, and the contents of eight of them (Cyanidin 3-rutinoside, Cyanidin 3-O-galactoside, Peonidin 3-O-glucoside chloride, Cyanidin 3-O-malonylhexoside, Cyanidin O-acetylhexoside, Anthocyanin 3-O-beta-d-glucoside, Pelargonidin 3-O-malonylhexoside and Pelargonidin O-acetylhexoside) changed significantly during the change of leaf color, especially the content of Pelargonidin O-acetylhexoside (molecular weight: 474.096 Da), a derivative of Pelargonidin, and Anthocyanin 3-O-beta-d-glucoside (molecular weight: 449.089 Da), a derivative of Cyanidin finally decreased to 0 in green leaves. After measuring the activity of all the enzymes involved in anthocyanin synthesis, it was found that the activity of henylalanine ammonia-lyase (PAL), trans-cinnamate 4-monooxygenase (C4H), 4-coumarate-CoA ligase (4CL) and flavonoid 3',5'-hydroxylase (F3 5 H) was up-regulated, the activity of chalcone synthase (CHS), chalcone isomerase (CHI), naringenin 3-dioxygenase (F3H) and flavonoid 3'-monooxygenase (F3 H) was down-regulated. The activity of DFR was down-regulated at first and then up-regulated, while the activity of ANS was up-regulated and then decreased. QRT-PCR analysis of the genes encoding these enzymes showed that all genes were significantly down-regulated (Figure 7). Comprehensive analysis of enzyme activity and the corresponding gene expression pattern showed that the changing trend of enzyme activity of CHS, CHI, F3H, F3'H was consistent with that of gene expression pattern, while that of the other six enzymes was different from that of gene expression pattern.

**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.

#### **3. Discussion**

The regulation of plant leaf color is a complex process. Most of the previous studies analyzed the mechanism of leaf color formation by methods of physiology, cytology and molecular biology [1], but the mechanism of leaf color regulation from the perspective of small molecular metabolites needs to be studied further. In this study, based on UPLC-MS/MS, the changes of metabolites in the process of leaf color change of *C. sinense* 'Red Sun' were qualitatively and quantitatively analyzed, and the unique pattern of flavonoid-related metabolites in the process of leaf color change was constructed for the first time. At the same time, the regulation mechanism of leaf color was further analyzed by enzyme kinetics and gene expression analysis.

As far as we know, this is the first time to analyze the types of flavonoid-related metabolites in the leaves of *C. sinense* 'Red Sun' by UPLC-MS/MS (flavonoids-targeted) method. A total of 196 flavonoid-related metabolites were detected. These substances belong to anthocyanin, chalcone, dihydroflavonoid, dihydroflavonol, flavanols, flavone, flavonoid, flavonoid carbonoside, flavonoids, flavonols and isoflavones. Based on wide target metabolomics analysis, only 6 and 15 differential flavonoid-related metabolites were detected in tea leaves and *Ginkgo biloba* leaves, respectively [20,21]. Based on phenolic-targeted secondary metabolites analysis in purple fig peel, only 15 differential flavonoid-related metabolites (including four anthocyanins metabolites) were detected [18]. In this study, 119 kinds of differential flavonoid-related metabolites were found (including 10 kinds of anthocyanins). The above results show that Flavonoids-targeted metabolites method can identify more kinds of flavonoid-related metabolites, and has more advantages in mining the types and contents of flavonoid-related metabolites, especially anthocyanins metabolites.

*Phalaenopsis* and the materials of this study belong to Orchidaceae. In the metabolomic analysis between petals of white and purple *Phalaenopsis*, 142 differential flavonoid-related metabolites, including 17 anthocyanins metabolites, were detected by flavonoids-targeted metabolomic analysis [16]. In accordance with the results of this research, among the 119-differential flavonoid-related metabolites detected, there were 8 differential anthocyanins. The differential metabolites of eight anthocyanins identified in the leaves of *C. sinense*'Red Sun' are Cyanidin 3-rutinoside (Keracyanin chloride), Cyanidin 3-O-galactoside, Peonidin 3-O-glucoside chloride, Cyanidin 3-O-malonylhexoside, Pelargonidin 3-O-malonylhexoside, Cyanidin O-acetylhexoside, Pelargonidin O-acetylhexoside and Anthocyanin 3-O-beta-d-glucoside. Compared with the 18 anthocyanin differential metabolites detected in the petals of *Phalaenopsis* [16], only Cyanidin 3-O-malonylhexoside and Cyanidin O-acetylhexoside are the same. Among the three main categories of pigmented glycosides, pelargonidin mainly shows orange/red, cyaniding mainly shows pink/magenta and delphinidin mainly shows purple/blue [22,23]. The metabolites with the highest proportion were cyanidin derivatives found in the petals of *Phalaenopsis* and leaves of *C. sinense* 'Red Sun'. Interestingly, during the process of *C. sinense* 'Red Sun' leaf color change, the two anthocyanins from existence to absence are Pelargonidin 3-O-malonylhexoside and Anthocyanin 3-O-beta-D-glucoside; the content of derivatives of six kinds of cyanidin and a kind of delphinidin was high in purple petals but zero in white petals of *Phalaenopsis* [16]. The above results are consistent with the phenotype of the corresponding materials.

As the upstream reaction of anthocyanin and other flavonoids, Phenylalanine was first converted to P-coumaroyl-CoA under the catalysis of PAL, C4H and 4CL [24]. The activities of PAL, C4H and 4CL were all up-regulated during the change of leaf color, while the coding genes expression of these three enzymes decreased significantly, which indicated that the activities of these three enzymes were also subject to post-transcriptional modification or post-translational modification [25]. Then P-coumaroyl-CoA was transformed into Naringenin under the action of CHS and CHI [24]. The activities of CHS and CHI were down-regulated in the process of leaf color change, which was consistent with the change pattern of gene expression. CHS is the first key enzyme in anthocyanin synthesis, its activity determines the formation of anthocyanin metabolic pathway, and the loss of its activity will lead to the loss of anthocyanin and other flavonoids [26,27]. From the results of qRT-PCR, the expression of CHS-3 in the red leaf stage was 813 times higher than that in the green leaf stage, indicating that CHS may play an important role in the process of leaf color change. Overall, similar correlations between gene expression and anthocyanin levels were also observed during the differential pigment deposition in crabapple cultivars with dark red, pink and white petal colors [28]. Next, Naringenin forms DHK under the catalysis of F3H. DHK then forms Dihydroquercetin (DHQ) and Dihydromyricetin (DHM) under the catalysis of F3 H and F3 5 H, respectively [29]. The enzyme activity of F3H and F3 H was down-regulated, while that of F3 5 H was up-regulated, and the coding gene expression of these three enzymes was significantly down-regulated. Both natural mutants and transgenic studies have proved that the competitions of three enzymes lead to different branching pathways at this critical point [30], and our results support this argument. DHK, DHQ and DHM formed unstable anthocyanins under the catalysis of DFR and ANS. The enzyme activity of DFR decreased at first and then increased, while the activity of ANS increased at first and then decreased, but the coding genes expression level of the two enzymes was significantly down-regulated. DFR from different plants has specific substrates biases for DHK, DHQ and DHM [6,31], and the downstream DFRs and ANSs is necessary for large sum of anthocyanin accumulation in *Phalaenopsis* [32,33]. These unstable anthocyanins eventually went through the action of UFGT to form stable anthocyanins [34]. During the period of color change, UFGT activity firstly decreased, and then increased, while the *UFGT* gene expression level was significantly down-regulated (Figure S5). Previous studies have found that overexpression of UFGT causes plants to show darker colors, such as crimson or purple, while overexpression of DFR or ANS only deepens the color to pink or lavender [34–36]. In this study, the results of qRT-PCR showed that the expression of ANS in red leaf stage was 833 times higher than that in green leaf stage, while the expression of UFGT in red leaf stage was 14 times higher than that

in green leaf stage. The question of whether ANS or UFGT had a greater effect on the leaf color of *Cymbidium* remains to be further verified.
