Orchids are important ornamental plants characterised by their fantastic floral morphology and high levels of corolla-colour polymorphism and variability, which have played an important role in pollination and have excited biologists since Darwin (Gigord et al., 2001; Aguiar et al., 2020; Basist et al., 2021). As an important member of orchids, Cymbidium species are probably the earliest orchid, having been cultivated since before the time of Confucius (551–479 BC) for traditional use in worship and ornamentation (Chen and Tang, 1982; Du Puy et al., 2007). Currently, members of Cymbidium are still very prevalent and dominate the world floriculture markets (Singh et al., 2019). Although the mechanism of genes encoding enzymes for flower coloration in orchids has been studied in some species (Mudalige-Jayawickrama et al., 2005; Han et al., 2006; Hieber et al., 2006; Chen et al., 2011; Hsiao et al., 2011; Liu et al., 2012; Yu et al., 2018), as one of the longest historic floricultural plants, Cymbidium kanran attracts wide attention. However, its coloration mechanism remains unclear.

Anthocyanins, as a series of secondary metabolites, are water-soluble pigments that contribute colours from orange, pink, red, magenta, purple, blue, cyan to “black” (Hatier and Gould, 2007; Tanaka et al., 2008; Petroni and Tonelli, 2011; Moreau et al., 2012; Li et al., 2014). These pigments are an ideal model for genetics, molecular biology, and cell biology (Passeri et al., 2016). To date, hundreds of anthocyanins have been extensively studied and characterised, which were originally based on six common types of anthocyanidins (chromophores of anthocyanins), namely, petunidin, pelargonidin, delphinidin, cyanidin, malvidin, and peonidin. Accordingly, the synthetic route of anthocyanins and important related genes are well-studied and characterised in some model and non-model plants. Anthocyanins are derived from flavonoid biosynthetic pathways, and their biosynthesis has been divided into three stages (Grotewold, 2006). The first stage is phenylalanine and phenylpropanoid metabolism, which is shared among other secondary metabolisms. The second stage, flavonoid metabolism, is the crux of anthocyanin biosynthesis. Chalcone synthase (CHS) is the first key enzyme to produce chalcones, the precursor for all classes of flavonoids. Subsequently, an enzymatic reaction of chalcones to naringenins is catalysed by chalcone isomerase (CHI), which is followed by a conversion to dihydrokaempferol (DHK) by flavanone 3-hydroxylase (F3H). The last step involves two participants, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3', 5'hydroxylase (F3' 5'H), corresponding to the respective products dihydroquercetin (DHQ) and dihydromyricetin (DHM). The final stage is anthocyanin metabolism, including two rate-limiting steps catalysed by dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS). Three kinds of products, delphinidin, pelargonidin, and cyanidin, undergo several modifications, such as glycosylation or methylation, by UDP-glucoside: flavonoid 3-O-glucosyltransferase (3GT) and anthocyanin methyltransferase (AMT).

Transgenic technology, expression, and suppression of the level of the anthocyanin synthetic gene have been verified as feasible to study the accumulation mechanism of anthocyanin in many plants. With the expression of UFGT, the skin colours of the Vitis and Litchi were turned red or violet from green (Ramazzotti et al., 2008; Zhao et al., 2012). Overexpression of SmANS from Salvia miltiorrhiza enhanced anthocyanin accumulation in the petals of S. miltiorrhiza f. alba and resulted in purple-red colour from white colour (Li et al., 2019). Using RNA interference (RNAi) technology to suppress CHS, ANS, and F3'5'H, the petals of gentian plants exhibited pure white to pale-blue and magenta flowers accordingly (Nakatsuka et al., 2008). Consequently, using transgenic approaches to introduce exogenous anthocyanin genes into the targeted plant can obtain the desired coloration traits to produce new flower colour (Meyer et al., 1987; Katsumoto et al., 2007).

Cymbidium kanran is an ideal species to study coloration mechanisms because of numerous variations in floral coloration and abundant traditional varieties. In this study, we used purple-red flowers (with many purple-red pigments) and white flowers (greenish-white without purple-red pigments) as research materials (Figure 1). Through a combination of transcriptome sequencing, chemical, and molecular analysis, the mechanism of anthocyanin deficiency in white was preliminarily revealed. This dataset provided a novel resource to study and to understand the molecular mechanisms of coloration in C. kanran.

The main pigments targeted for orchid flowers are anthocyanins that contribute to a variety of colours, such as red, purple, pink, and blue, while studies have shown that the main types of anthocyanidin aglycones in orchids are cyanidin, peonidin, and delphinidin (Liu et al., 2012; Vignolini et al., 2012; Wang et al., 2014; Liang et al., 2020; Zhang et al., 2020). In Oncidium Gower Ramsey, the red portion of lips contained a mixture of cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, and peonidin-3-O-glucoside compounds (Liu et al., 2012). Flowers of Phalaenopsis with red-purple, purple, purple-violet, and violet to violet-blue colour were due to highly accumulated cyanidin-based anthocyanins, but no true-blue colour and no delphinidin was detected (Liang et al., 2020). In some cultivars of Cymbidium hybrids, only cyanidin-, malvidin-, and peonidin-type anthocyanins were identified (Woltering and Somhorst, 1990; Johnson et al., 1999). In C. kanran, the flavonoid composition exhibited a sharp shift between purple-red and white flowers. Three types (cyanidin, peonidin, and malvidin) of anthocyanidins were also detected in purple-red flowers but were absent in white flowers. While the transcript level of the colour-related gene showed that CkC4H-3, CkCHS-1, CkDFR, and CkANS were highly expressed corresponding to purple-red floral tissues compared with white type floral tissues, but CkFLS had a higher level of expression in the white one, and a similar result was also achieved in Pleione limprichtii (Zhang et al., 2020). Dihydroflavonol reductase is a key enzyme in the biosynthesis of anthocyanins and can catalyze the reduction of three colourless dihydroflavonols, DHQ, DHK, and DHM, to leucoanthocyanidins. Because of the similar structure of the three substrates, it is easy to understand that DFRs from many species can utilise all three substrates (Heller et al., 1985; Meyer et al., 1987; Stich et al., 1992; Helariutta et al., 1993; Tanaka et al., 1995; Livak and Schmittgen, 2001; Ma et al., 2008; Chen et al., 2013). dihydroflavonol reductases from some species specifically convert one or two types of dihydroflavonols. In Petunia and Cymbidium, pelargonidin-based brick red/orange flowers do not exist in nature because DFRs cloned from these two species cannot reduce DHK efficiently (Gerats et al., 1982; Forkmann and Ruhnau, 1987; Johnson et al., 1999), suggesting that DFRs have a striking substrate specificity (Springob et al., 2003). In Fragaria species, two DFRs were isolated, and one enzyme variant strongly preferred DHK as a substrate, whereas the other had a higher specificity for DHQ than for DHM (Miosic et al., 2014). Similar results were also reported in Ginkgo biloba and Freesia hybrida (Cheng et al., 2013; Li et al., 2017). In C. kanran, relative quantification showed that cyanidin, peonidin, and malvidin were detected, and the cyanidin content was far greater than that of peonidin and malvidin, indicating that the DFR isolated from C. kanran could catalyze DHQ conversion to leucocyanidin. However, more in vitro enzyme assays are needed to verify the substrate specificity of DFR with DHM and DHK.

Orchid flowers are striking for their specific patterns of colours in the perianths, and the regulation and formation of pigmentation is determined by specific cells of the different floral organs (Yu and Goh, 2001). In the case of C. kanran, the distributions of anthocyanin production depended on the tissue specificity. The pigmentation in the petals showed anthocyanins concentrated in the outer layer of subepidermal cells to the xylem (Figure 2A), but in the lip, the red anthocyanins only accumulated in epidermal cells (Figure 2C). Orchid flowers have three petals, and one of the petals is morphologically different in structure and is known as the lip or labellum. However, the differences between the petals and lip were represented not only in shape but also in tissue and genes. The epidermal cells in the lip displayed different forms, in which the adaxial side cells were large and bubble-like, but the abaxial side cells were similar to the petal epidermal cells, which were small and flat. However, anthocyanins accumulated in the region from subepidermal cells to the xylem in the petal (Figure 2A), while in the lip, anthocyanins accumulated only in the epidermal cells of the adaxial surface (Figure 2C). Accordingly, pigments in the adaxial side of lips displayed bright-coloured pigmentation compared with those in petals (Figures 1J,K, 2A,C). Similar results were also reported in the leaves of Tipularia discolour orchid, the same anthocyanins in different anatomical locations led to a broad range of colours (Hughes et al., 2020). In addition, differentially expressed genes in transcriptome analysis of C. kanran supported that the molecular changes also existed between the lip and petals. In petals, CkCHS-1 was highly expressed, but CkCHS-2 was inversely expressed in the lip. In the Petunia hybrid, 12 genes encode CHS from a gene family, which were detected specifically in the margin of the corolla throughout all stages of flower development. Among them, CHS-A and CHS-J were expressed predominantly. These results indicated that CHS expression is regulated by different mechanisms in marginal picotee petals (Saito et al., 2006). According to a previous study, MADS-box genes might collectively regulate the genes involved in colour differentiation in petals and sepals during Cattleya orchid flower development, which may result in the spatiotemporal expression of anthocyanin biosynthesis pathway structural genes to determine colour differentiation (Li et al., 2020).

Knowledge of the molecular genetic basis of the flavonoid biosynthetic pathway allows us to improve the colour quality of plants. The simplest strategy to modify flower colour is to change the amounts of endogenous anthocyanins by regulating the essential enzymes required for flavonoid biosynthesis. For example, transgenic gentian plants with a suppressed ANS showed pale-blue-coloured flowers, while those with a suppressed CHS exhibited a more serious phenotype, i.e., pale-blue to pure white flowers (Nakatsuka et al., 2008). The manipulation of DFR and FLS, which are competing steps for flux towards anthocyanins and flavonols, led to an increase or decrease in anthocyanin content in the flower (Davies and Schwinn, 2010). In addition, the approach of regulating endogenous modification genes has also been utilised in genetic engineering. For example, F3'5'H suppression by RNAi induced a flower colour change from purple to red in cyclamen (Boase et al., 2010). Similar transformations were also displayed in both F3'5'H and A5/3'AT (anthocyanin 5,3'-aromaticacyltransferase)-suppressed transgenic gentian plants (Nishihara and Nakatsuka, 2010). In C. kanran, the validation of the transient expression of key genes involved in colour formation showed that only triple genes, CkCHS-1, CkDFR, and CkANS could accumulate red spots in both the petal and lip tissue, indicating that the regulatory mechanism of coloration is complicated. To date, studies on some genetically defined genotypes or mutants revealing blocks of anthocyanin pathway gene expression have already been verified in many plants. In Forsythia, it was shown to be blocked in anthers at both the DFR and ANS levels but at the ANS level, only in petals (Rosati et al., 1999). However, the absence of anthocyanins in the skins of white berry varieties is due to a block only of UFGT expression in Vitis (Boss et al., 1996). Additionally, a point mutation of the CHS gene could result in the formation of the white flower of Matthiola incana (Hemleben et al., 2004). Another study in Oncidium also revealed that the failure of anthocyanin accumulation may be due to the inactivation of OgCHS caused by epigenetic methylation of the 5'-upstream promoter regions (Liu et al., 2012). However, the silencing of three different CHS genes of the purple-colour flower of Dianthus chinensis via VIGS could obtain white or pale purple flower (Liu et al., 2021), suggesting that several CHS genes may be involved in the formation of coloration. In C. kanran, it is noteworthy that CkCHS-2 was abundantly expressed in white flowers but expressed at low levels in purple-red flowers (Figures 5J,K), and CkCHS-1 was highly expressed in purple-red flowers but had lower expression levels in white flowers (Figures 5G,H), indicating the complicated regulation of CHSs in C. kanran. Therefore, further studies need to be performed on the regulatory mechanism of anthocyanin pathway-related genes in C. kanran.

Thus, we tentatively speculated about the pathways for purple-red and white C. kanran (Figure 9). In purple-red C. kanran, flavonoids are initially derived from phenylalanine and phenylpropanoid metabolism. Subsequently, CkCHS is generated to produce chalcones, and CkCHI catalyzes chalcones into naringenin. Furthermore, naringenin can be converted through CkF3H, CkF3'H, and CkF3'5'H to produce dihydroxyflavonols including DHK, DHQ, and DHM. Following the above reaction, CkDFR and CkANS further catalyze the divergent conversion of dihydroflavonols to produce colourless cyanidin and delphinidin. In the end, CkUFGT and other GTs catalyze the glucosylation of anthocyanidins (peonidin, malvidin, and cyanidin). As for white C. kanran, the main divergence of flavonoid metabolism is anthocyanin metabolism, the GTs cannot be regulated and fulfilled to catalyze the glucosylation of anthocyanidins. White-type flowers of Cymbidium are uncommon in nature, and traditional division propagation requires a long period, which leads to a low reproductive rate and high price. In this study, the molecular mechanism of anthocyanidin deficiency in white-type flowers establishes a foundation for genetic engineering to generate white-type C. kanran. The simplest strategy is to suppress the expression levels of CkCHS-1, CkDFR, and CkANS.

Concurrently, the regulatory mechanism of genes involved in the anthocyanin metabolic pathway has also been carried out in orchids. In Oncidium, OgMYB1 directly activated the expression of OgCHI and OgDFR to regulate anthocyanin accumulation in the lip (Chiou and Yeh, 2008). In Phalaenopsis, three MYB transcription factors determine full-red pigmentation, red spots, and venation patterns (Hsu et al., 2015). A recent study in C. hybrids also indicated that CyMYB1 regulates temporal- and temperature-dependent anthocyanin accumulation in petals (Nakatsuka et al., 2019). In C. kanran, we also isolated several differentially expressed MYB genes by Biochemical and Biophysical Research Communications transcriptome analysis and attempted to reveal the molecular mechanism of MYB genes regulating pigmentation patterning, which may provide genetic resources for C. kanran molecular breeding in the future.

The white and purple-red phenotype C. kanran flowers had provided a unique experimental system to examine the mechanism for flower anthocyanin pigmentation. Compared with white flowers, purple-red flowers are comprised of three types of anthocyanidin compounds, namely, peonidin, malvidin, and cyanidin, which were shown by HPLC analysis. A total of 102 candidate genes involved in the flower coloration of C. kanran were identified using transcriptome analysis. Gene expression profiles showed that four of these genes were positively correlated with anthocyanidin biosynthesis by qRT-PCR. Transient expression of CkCHS-1, CkDFR, and CkANS by particle bombardment results in the accumulation of anthocyanins in white flowers. These results indicated that CkCHS-1, CkDFR, and CkANS are the key genes involved in floral pigment accumulation in C. kanran. This study also provided a platform for C. kanran functional genomic research.

The datasets presented in this study can be found in online repositories. The name of the repository and accession number can be found below: National Centre for Biotechnology Information (NCBI) BioProject, https://www.ncbi.nlm.nih.gov/bioproject/, PRJNA744371.

ZZ, XW, and ZL designed the experiments. ZZ, XW, YY, and ZY performed all experiments. ZZ, XW, and ZW analysed the data. ZZ and XW wrote the manuscript. ZL revised the manuscript. All the authors have read and agreed to the published version of the manuscript.

This research was funded by the Natural Science Foundation of Zhejiang Province (grant nos. LY20C160005 and LY19C150003), National Key R & D Program of China (grant nos. 2018YFD1000401 and 2019YFD1000400), and Wenzhou Agricultural New Variety Breeding Cooperative Group Project (grant no. 2019ZX004-3).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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