Genome-Wide Identification and Expression Analysis of Terpene Synthase Genes in Cymbidium faberi

Terpene synthases (TPSs) are essential for forming terpenes, which play numerous functional roles in attracting pollinators, defending plants, and moderating the interaction between plants. TPSs have been reported in some orchids, but genome-wide identification of terpenes in Cymbidium faberi is still lacking. In this study, 32 putative TPS genes were classified in C. faberi and divided into three subfamilies (TPS-a, TPS-b, and TPS-e/f). Motif and gene structure analysis revealed that most CfTPS genes had the conserved aspartate-rich DDxxD motif. TPS genes in the TPS-a and TPS-b subfamilies had variations in the RRX8W motif. Most cis-elements of CfTPS genes were found in the phytohormone responsiveness category, and MYC contained most of the numbers associated with MeJA responsiveness. The Ka/Ks ratios of 12/13 CfTPS gene pairs were less than one, indicated that most CfTPS genes have undergone negative selection. The tissue-specific expression patterns showed that 28 genes were expressed in at least one tissue in C. faberi, and TPS genes were most highly expressed in flowers, followed by leaves and pseudobulbs. In addition, four CfTPS genes were selected for the real-time reverse transcription quantitative PCR (RT-qPCR) experiment. The results revealed that CfTPS12, CfTPS18, CfTPS23, and CfTPS28 were mainly expressed in the full flowering stage. CfTPS18 could convert GPP to β-myrcene, geraniol, and α-pinene in vitro. These findings of CfTPS genes of C. faberi may provide valuable information for further studies on TPSs in orchids.

Most full-length TPSs contain two conserved domains defined in PFAM: PF01397 (N-terminal) containing a conserved RRX 8 W motif and PF03936 (C-terminal) containing a DDxxD motif and NSE/DTE motif (El-Gebali et al., 2019;Jiang et al., 2019). In addition, TPSs can be divided into seven major categories: TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g, and TPS-h (Chen et al., 2011). TPS-a, TPS-b, and TPS-g categories are angiospermspecific. The TPS-a category can encode sesquiterpene synthases of these three categories and can be further classified into monocot-specific TPS-a-1 and dicot-specific TPS-a-2 groups. In recent reports, all characterized TPSs in TPS-b with RRX 8 W motifs are monoterpene synthases. The TPS-g category can encode monoterpene synthases without the RRX 8 W motif. In addition, TPS-d is a gymnosperm-specific category that encodes monoterpenes, sesquiterpenes, and diterpenes. TPS-e/f can encode kaurene or copalyl diphosphate synthases that function in gibberellic acid synthesis in vascular plants. TPS-c may represent the ancestral category, and TPS-h only appears in Selaginella moellendorffii in recent reports (Chen et al., 2011;Li et al., 2012).
Cymbidium faberi is a plant of Orchidaceae with a long history of cultivation due to its characteristic flower fragrance and beautiful flower shape (Ramya et al., 2018). There are over 100 compounds in the C. faberi floral scent, and some terpenes have been identified in the blooming C. faberi flowers by headspace vapors (Omata et al., 1990). This study first analyzed the classification, phylogenetics, and expression patterns of TPS genes in C. faberi. Our results will provide valuable information for further studies on C. faberi and other orchids.

Plant Materials
The wild C. faberi collected in this study were cultivated in the greenhouse at Forest Orchid Garden in Fujian Agriculture and Forestry University (Fuzhou, Fujian Province, China) under natural light and temperatures. The temperature was about 20-25°C. Flowers, leaves, and pseudobulbs of C. faberi were sampled at the flowering stage. The buds about 1 cm before anthesis, semi-open flowers about 3 cm, and fully open flowers were also used in this study. All samples of C. faberi were frozen in liquid nitrogen for storage at −80°C until use.

Identification of CfTPS Genes in the C. faberi Protein Database
Two domains -PF01397 representing the TPS N-terminal domain and PF03936 representing the TPS C-terminal domain from PFAM 1 -were used as queries to search the C. faberi protein database (El-Gebali et al., 2019). The C. faberi genome data will be published separately. An HMM search (built-in Tbtools) was used in this study with an e-value cut at 10 −3 . To avoid missing potential TPS genes, TPS sequences from A. thaliana in the TAIR database 2 were also used to screen the C. faberi protein database using BLASTP (built-in Tbtools; Chen et al., 2018). The candidate TPS genes were checked manually by Pfam to verify putative full-length TPS genes, and TPS genes lacking either PF03936 or PF01397 were excluded. The grand average of hydrophobicity (GRAVY), molecular weight (Mw), isoelectric points (pI), aliphatic index (AI), and instability index (II) of the TPS proteins were predicted by the ExPASy database (Artimo et al., 2012). 3 Subcellular localization was predicted by Plant-mPloc (Chou and Shen, 2010), 4 AtSubP (Kaundal et al., 2010), 5 and Ploc-mPlant (Cheng et al., 2017). 6 Terzyme 7 and BLATP 8 were used to predict gene function (Priya et al., 2018).

Motifs and Gene Structure Analysis
Conserved motifs in the C. faberi TPS sequences were employed and analyzed by MEME software 9 with default parameters (Bailey et al., 2009). We identified 20 motifs in this study. The exon-intron structure of the sequences was determined using GSDS software (Hu et al., 2015). 10

Phylogenetic Analysis of TPS Genes
The transcriptomes of TPS sequences from P. equestris and A. shenzhenica were downloaded from their genome databases. TPS sequences from S. moellendorffii were downloaded from NCBI. 11 TPS sequences from Picea abies were downloaded from UniProt, 12 and TPS protein sequences from A. thaliana and Oryza sativa were downloaded from Phytozome. 13 All these sequences were aligned with MAFFT (Rozewicki et al., 2019). The maximum likelihood (ML) method was used for the phylogenetic tree, which was constructed with RAxML on the CIPRES Science Gateway web server (RAxML-HPC2 on XSEDE; Miller et al., 2011). Bootstrap values were 1,000 replicates with the JTT model. The most appropriate protein evolution model for the alignment was predicted by ProTest (Darriba et al., 2011). The generated tree was redrawn and annotated by EVOLVIEW (He et al., 2016). 14 The sequences of the CfTPS proteins used in this study are listed in Supplementary Table S7.

Promoter Element Analysis of TPS Genes
Tbtools software extracted the promoter sequences and 2,000 bp regions upstream of 32 CfTPS genes . Afterward, the online software PlantCARE 15 was used to identify

Calculation of Ka and Ks Ratios
Gene pairs with similar genetic relationships were selected based on the phylogenetic tree. DNAMAN software was used to select the gene pairs with a consistency greater than 60%. Tbtools software was then used to calculate Ka (non-synonymous rate), Ks (synonymous substitution), and Ka/Ks (evolutionary constraint) values. Divergence time (T) was calculated by using the formula T = Ks/ (2 × 9.1 × 10 −9 ) × 10 −6 million years ago (Mya; Zhang et al., 2018). In general, Ka/Ks < 1.0 represents purifying or negative selection, Ka/Ks = 1.0 represents neutral selection, and Ka/Ks > 1.0 represents positive selection (Zhang et al., 2006).

Transcriptome Data and GO Classification Analysis
An RNA sequencing transcriptome database of leaves, pseudobulbs, petals, sepals, labellums, and gynostemium was established to study the expression patterns of CfTPS genes. Fragments per kilobase of transcript per million mapped reads (FPKM) values of CfTPS genes were used to evaluate translation abundance. DESeq was used to conduct gene differential expression analysis, and gene ontology (GO) classification analysis was performed based on the differentially expressed gene analysis. The heatmaps of CfTPS expression patterns were drawn by Tbtools software, and the color in the heatmap was expressed as the log2-transformed expression levels of each CfTPS gene . Synthesis SuperMix for qPCR was also used to remove genomic DNA. The real-time reverse transcription quantitative PCR (RT-qPCR) primers of CfTPS were designed by Primer Premier 5 software and can be found in Supplementary Table S12.

Extraction of RNA and RT-qPCR Analysis
Primer specificity was confirmed using Primer-BLAST on the NCBI website. 16 PerfectStart™ Green qPCR SuperMix (TransGen Biotech, Beijing, China) was used for RT-qPCR analysis. The C. faberi reference gene GAPDH (GenBank Accession: JX560732; Tian et al., 2020) was used as the internal control and quantified by the 2 -△△CT method (Livak and Schmittgen, 2001). There were three biological replicates in the RT-qPCR analysis.

CfTPS18 Enzyme Assays
The ORF of CfPS18 was synthesized and ligated into pET28a vector. Then, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) pLysS cells (Transgen, China). Primers are given in Supplementary Table S12

GC-MS Analysis
The volatiles were exposed to SPME fiber with 50/30 μm DVB/ CAR/PDMS (divinylbenzene/carboxen/polydimethylsiloxane; Supelco Co., Bellefonte, PA, United States). The extract was analyzed using a gas chromatograph (Agilent 6,890 N) and mass spectrometer (Agilent 5975B, Santa Clara, CA, United States) outfitted with a silica capillary column (DB-5MS; 60 m × 0.25 mm × 0.25 μm). The temperature program was as follows: 55°C for 2 min, 3°C min −1 up to 80°C, 5°C min −1 up to 180°C, 10°C min −1 up to 230°C, and finally, 20°C min −1 to 250°C. The ion source temperature was 230°C, and the electron energy was 70 eV. The GC-MS interface zone temperature was 250°C, and the scan range was 50-500 m/z. Reactions only added with GPP were used as the blank control. There were three biological replicates for the experiment. The retention time was compared with the NIST Mass Spectral Library.

TPS Gene Identification and Protein Features in C. faberi
To retrieve the TPS genes in C. faberi, two domains, PF01397 and PF03936 in the PFAM, were used to search the C. faberi 16 https://blast.ncbi.nlm.nih.gov/Blast.cgi protein database (El-Gebali et al., 2019). BLASTP (built-in Tbtools) was also used to screen the C. faberi protein database . After removing artefacts, 32 TPS genes were obtained. The deduced proteins of these genes were in the range of 115 for CfTPS1 and CfTPS2 to 902 amino acids for CfTPS12 and had predicted molecular weights ( (Chou and Shen, 2010;Kaundal et al., 2010;Cheng et al., 2017). The results showed that 16 CfTPS proteins were marked in the chloroplast, and 16 CfTPS proteins were marked in chloroplast or cytoplasm, indicating that these three predictors produce different results and need to be further analyzed. We also annotated 32 CfTPS genes using BLASTP 17 and Terzyme software (Supplementary Table S4; Priya et al., 2018). The results showed that 11 CfTPSs were annotated sesquiterpene synthases, 15 CfTPSs were annotated monoterpene synthases, and six CfTPSs were annotated diterpene synthases. The secondary structure predicted by the SOPMA program revealed that the average of α-helices, random coils, extended strands, and β-turns comprised 70.86, 21.37, 4.35, and 3.63% of the structure, respectively (Geourjon and Deléage, 1995). The results showed that α-helices were predominant in all CfTPS proteins ( Table 1).

Motif and Gene Structure Analysis
To understand the intron-exon structure of CfTPS genes, we analyzed TPS gene structure with GSDS software (Hu et al., 2015). The exons in CfTPS genes ranged in numbers from 2 to 15, and the results showed that most of the genes in the same category contained a similar intron-exon structure.
To further analyze the motifs of the CfTPS genes, we identified 20 motifs using MEME software (Bailey et al., 2009). The numbers of C. faberi TPS motifs ranged in length from 4 to 16. CfTPS3 and CfTPS23 contained the most motifs, with 16, while CfTPS1, CfTPS2 had only four motifs. Motif 3 can be found in all CfTPS proteins except CfTPS9 and CfTPS16. Motif 4 was also the most common CfTPS protein (28/32). Twenty-five CfTPS genes contained the RRX 8 W motif (motif 1), and 30 CfTPS genes contained the DDxxD motif (motif 2; Figure 2C). Accordingly, different clusters have different forms of motifs. The same cluster's CfTPS proteins generally contained similar motifs. These results of intron-exon structure 17 http://blast.ncbi.nlm.nih.gov  (Chou and Shen, 2010;Kaundal et al., 2010;Cheng et al., 2017). 9 Gene function predicted by Terzyme website. C10, C15, and C20 represent monoterpene, sesquiterpene, and diterpene, respectively. Row data is listed in Supplementary Tables S1-S5. and motif analysis verified the closeness of the phylogenetic tree in C. faberi (Figure 2).

Phylogenetic Analysis of CfTPS Genes
A phylogenetic tree was constructed to analyze the evolutionary patterns of the CfTPS genes. Thirty-two CfTPS genes were used, and TPS protein sequences from six species (A. shenzhenica, P. equetris, O. sativa, A. thaliana, P. abies, and S. moellendorfii) were used. The maximum likelihood (ML) method was used for the phylogenetic tree, which was constructed with RAxML on the CIPRES Science Gateway web server (RAxML-HPC2 on XSEDE; Miller et al., 2011). Bootstrap values were 1,000 replicates with the JTT model. The phylogenetic tree indicated that TPS proteins belonged to seven categories. This classification result was consistent with a recent study: TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g, and TPS-h (Chen et al., 2011). Thirty-two CfTPS proteins belonged to three categories according to the phylogenetic tree (Figure 3): TPS-a, TPS-b, and TPS-e/f. Of these three categories, the TPS-a and TPS-b clades contained the most members and were the most expanded categories, with 13 and 15 genes, respectively, and were consistent with other plant species, such as A. thaliana, C. sinensis, V. planifolia, D. catenatum, P. equestris, and D. officinale (Aubourg et al., 2002;Yu et al., 2020;Zhou et al., 2020;Huang et al., 2021). The remaining four TPS genes belonged to the TPS-e/f subfamily. We aligned the multiple sequences using MAFFT to analyze the TPS RRX 8 W motif in the N-terminus, DDxxD, and NSE/ DTE motifs in the C-terminus (Rozewicki et al., 2019). The alignment showed that almost all the CfTPS proteins in the TPS-a and TPS-b subfamilies had the highly conserved aspartaterich motif DDxxD, except CfTPS9 and CfTPS16 in the TPS-a subfamily and CfTPS17, CfTPS27, and CfTPS28 in the TPS-b subfamily (Jiang et al., 2019). TPS genes in TPS-a and TPS-b had variations in the RRX 8 W and RxR motif. However, in the TPS-a and TPS-b categories, the RRX 8 W motif was not found in nine CfTPS genes. The RRX 8 W motif was absent in the TPS-e/f subfamily, and the NSE/DTE motif was found in 16 CfTPS genes (Figure 4). Among them, DDxxD and NSE/ DTE are important in the metal-dependent ionization of the prenyl diphosphate substrate, and the RRX 8 W motif is essential in the cyclization of monoterpene synthase (Bohlmann et al., 1998;Chen et al., 2011;Jiang et al., 2019). In addition, the members in the TPS-a subfamily detected in both dicot and monocot plants encode only sesquiterpenes (Jiang et al., 2019). The secondary "R" in this family is not conserved (Martin et al., 2010). TPS-b encodes monoterpenes containing the conserved RRX 8 W motif (Chen et al., 2011;Jiang et al., 2019).

Promoter Analysis of CfTPS Genes
To retrieve the potential function of CfTPS genes, we obtained a 2,000-bp region upstream of the 32 CfTPS genes and analyzed them using the online software Plantcare (Lescot et al., 2002). In total, we found 784 cis-acting regulatory elements in the promoter areas of CfTPS genes. We classified them into three categories according to the function of these elements: plant growth and development, stress responsiveness, and phytohormone responsiveness. The plant growth and dependent category (166/784) contained nine cis-acting regulatory elements and consists of AAGAA motifs, As-1 elements, A1-rich elements, etc. Most of them had As-1 elements (66/166), which are related to shoot expression. The stress responsiveness category (216/874) contained ARE, DRE, LTR, ST-RE, ABRE, etc.  Miller et al., 2011). Bootstrap values were 1,000 replicates with the JTT model. The intron-exon structure was drawn by the GSDS website (Hu et al., 2015). Yellow boxes, blue boxes, and black lines exhibit exons, introns, and upstream or downstream-untranslated regions. (B) Phylogenetic tree and conserved motifs of CfTPS genes. Conserved motifs were determined by MEME software with default parameters (Kaundal et al., 2010). (C) Sequence logo of motif 1 (RRX 8 W) and motif 2 (DDxxD). Motif 1 shows the N-terminal motif RRX 8 W, and motif 2 shows the C-terminal motif DDxxD motif. Conserved motifs are available in Supplementary Table S6. STRE (48/216) is an essential element in the promoter that is related to stress. Interestingly, most of the cis-elements (401/784) were in the phytohormone responsiveness category, which contained CGTCA motifs, EREs, MYC motifs, TGAGG motifs, etc. Among them, most cis-elements were MYC (137/401), which is associated with MeJA responsiveness (Dombrecht et al., 2007). The results indicated that the CfTPS gene expression patterns might be regulated by MeJA treatment (Figure 5).

Ka/Ks Analysis in C. faberi
The Ka/Ks ratio can show positive selection (Ka/Ks > 1), negative or purifying selection (Ka/Ks < 1), and neutral selection (Ka/Ks = 1) during the evolution (Zhang et al., 2006). In this study, 13 gene pairs with similar genetic relationships were selected for Ka/Ks calculation. The results showed that the Ka/Ks ratios of 12/13 CfTPS genes were less than one, indicated that most CfTPS genes underwent negative selection (

Expression Patterns in Different C. faberi Organs and GO Classification of TPS Genes
An RNA sequencing transcriptome database of leaves, pseudobulbs, petals, sepals, labellums, and gynostemium was established to study the expression patterns of CfTPS genes. Eighteen genes were expressed in both leaves and flowers, and 20 genes were expressed in the labellums. Twenty-five genes were found in sepals and 24 in gynostemium. Four genes were not found to be expressed in any of the tissues.
FIGURE 3 | Phylogenetic tree of CfTPS genes based on the TPS protein sequences of seven plant species. The ML method was used for the phylogenetic tree, which was constructed with RAxML on the CIPRES Science Gateway web server (RAxML-HPC2 on XSEDE; Miller et al., 2011). The bootstrap values were 1,000 replicates with the JTT model. The generated tree was redrawn and annotated by the EVOLVIEW website (He et al., 2016). The TPS family was classified into seven categories: TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g, and TPS-h (Chen et al., 2011). TPS protein sequences in Cymbidium faberi are available in Supplementary Table S7. CfTPS12, CfTPS15, and CfTPS23 exhibited a high level of expression in leaves and pseudobulbs. Notably, CfTPS3, CfTPS12, CfTPS15, CfTPS17, CfTPS18, CfTPS28, and CfTPS31 displayed high transcript abundance in floral organs, suggesting that these TPS genes might be related to flower scent in C. faberi (Figure 6). Gene ontology classification analysis was performed based on the differentially expressed gene analysis. According to the classification results, molecular function contained most genes, and genes were mostly enriched in lyase activity, magnesium binding, and terpene synthase activity (Figure 7).

Expression Patterns of CfTPS in Flowers at Three Floral Developmental Stages
Real-time reverse transcription quantitative PCR was performed to investigate the expression patterns of CfTPS genes in flowers at three floral developmental stages. Four putative functional genes, CfTPS12, CfTPS18, CfTPS23, and CfTPS28, which are highly expressed in floral organs, were used. They were expressed differently among the three floral stages. Interestingly, these genes were mainly expressed in the full flowering stage (Figure 8).
Notably, CfTPS18 had the highest transcript levels during the floral developmental stages, consistent with the expression patterns displayed in Figure 5. Gene annotation indicated that CfTPS12, CfTPS18, CfTPS23, and CfTPS28 likely encoded monoterpene synthases and clustered in TPS-b subfamily ( Table 1).

Functional Characterization of CfTPS18 in E. coli
To investigate the enzyme activity, the full-length ORF sequence of CfTPS18 was cloned to vector pET28a and ectopically expressed in E. coli. Recombinant CfTPS18 enzyme was induced with 0.1 mM isopropyl-β-d-galactopyranoside and purified with Ni-NTA agarose (Clontech). SDS-PAGE analysis of CfTPS18 recombinant protein can be found in Supplementary Figure S1. After using GPP as substrate in the reactions, the products were analyzed by GC-MS. The result indicated that CfTPS18 could convert GPP into β-myrcene, geraniol, and α-pinene which were validated by the NIST Mass Spectral Library (Figure 9). The blank control with only GPP added to the reactions could not produce

DISCUSSION
Orchids are among the largest angiosperm families in angiosperms and demonstrate a diversity of epiphytic and terrestrial growth forms (Hsiao et al., 2011). Cymbidium faberi is one of the longest cultivated orchids planted in China and has high ornamental value due to its characteristic flower scent and beautiful flower shape (Omata et al., 1990;Hsiao et al., 2011;Sun et al., 2016). Terpenoids play an essential role in floral scent and can attract pollinating insects and defend against environmental stresses (Wagner and Elmadfa, 2003;Dudareva et al., 2006;Sun et al., 2016). Plants have enzymes called TPSs, which encode the synthesis of monoterpene (C10), sesquiterpene (C15), or diterpenes (C20) from DMAPP, GPP, and GGPP, respectively (Pichersky et al., 2006). Terpenoids are dominant in the flower scent of orchids.
In this study, we systematically retrieved and classified TPS genes in C. faberi.
We identified 32 CfTPS genes in the C. faberi genome according to the TPS N-terminal and C-terminal domains ( Table 1). We classified them into three categories: TPS-a, TPS-b, and TPS-e/f. TPS-b was the most expanded category, which was consistent with patterns in D. officinale, V. planifolia, and D. catenatum, and C. faberi have more genes in TPS-b than P. equestris Huang et al., 2021). However, it was not consistent with A. thaliana, O. sativa, and S. bicolor, which have a dominant subfamily TPS-a (Aubourg et al., 2002;Chen et al., 2011). These results suggest that orchids have more TPS genes in TPS-b than other angiosperm dicot species and are related to emit floral scent to attract pollinators (Huang et al., 2021). According to the phylogenetic tree, most of the TPS-b genes are present in dicots. TPS-a genes can be further split into monocot-specific TPS-a-1 and dicot-specific TPS-a-2 clades. The TPS gene family is a medium-sized family, and the numbers of TPS genes range from approximately 20-100 (Chen et al., 2011). For instance, 14 SmTPS, 23 GmTPS, 32 AtTPS, 34 DoTPS, 23 CsTPS, and 69 VvTPS were found in S. moellendorfii, G. max, A. thaliana, D. officinale, C. sinensis, and V. vinifera, respectively (Li et al., 2012;Yu et al., 2020;Zhou et al., 2020). In addition, TPS occupied 0.26 genes/M in A. thaliana (125 M), 0.14 genes/M in V. vinifera (487 M), 0.13 genes/M in S. moellendorfii (106 M), 0.02 genes/M in D. officinale (1.35 G), 0.02 genes/M in G. max (1.011 G), and 0.01 genes/M in C. faberi (3.77 G; Jaillon et al., 2007;Schmutz et al., 2010;Poczai et al., 2014;Zhang et al., 2016). These results indicate that the TPS family has undergone expansion throughout the evolutionary history of land plants, and different species may show a difference in the expansion mechanism (Chen et al., 2011;Jiang et al., 2019).
We also annotated 32 CfTPS genes, and the results showed that all TPSs in the TPS-a clade encode sesquiterpenes, and all TPSs in the TPS-b clade encode monoterpenes ( Table 1). All the CfTPS in the TPS-e/f clade were annotated as diterpene synthases. This is consistent with a recent study in which most TPS genes in the TPS-a subfamily were determined to be sesquiterpene synthases. TPS-e/f encoded monoterpene, sesquiterpene, and diterpene in a recent study (Chen et al., 2011). Sesquiterpenes, diterpenes, and monoterpenes are important to emit floral scents to attract pollinators and defend against environmental stress (Huang et al., 2021).
Each full-length TPS had two conserved domains, including the N-terminal domain containing the RRX 8 W motif and the C-terminal domain containing two highly conserved aspartate-rich motifs: DDxxD and NSE/DTE (Starks et al., 1997;Jiang et al., 2019). DDxxD and NSE/DTE are significant in the metal-dependent ionization of the prenyl diphosphate substrate, and the RRX 8 W motif is essential in the cyclization of monoterpene synthase (Bohlmann et al., 1998;Chen et al., 2011;Jiang et al., 2019). The secondary "R" in the TPS-a subfamily is not conserved (Martin et al., 2010). TPS-b contains the conserved RRX 8 W motif, which is related to monoterpene formation. TPS-c does not include the DDxxD motif (Chen et al., 2011;Jiang et al., 2019). Motif RxR was also conserved in TPS-a and TPS-b subfamilies. In this study, multiple sequence alignments showed that 28/32 CfTPS had highly conserved DDxxD motifs, 16/32 CfTPS had RRX 8 W motifs, 16/32 CfTPS had NSE/DTE motifs, and 9/32 CfTPS had RxR motifs. The RRX 8 W motif was not found in 11 CfTPSs in the TPS-a and TPS-b clades, and it was absent in the TPS-e/f subfamily. The results indicated that motif loss might have appeared during family evolution in C. faberi and that different subfamilies have different motif features.   Supplementary Table S9.
Different elements were observed in promoter areas of CfTPS genes. Most of the cis-elements were in the phytohormone responsiveness group, and the number of MYCs associated with MeJA responsiveness contained most of this group. The results indicated that the expression patterns of CfTPS may be regulated by MeJA treatment and may respond to multiple environmental stresses (Chaiprasongsuk et al., 2018). In recent studies, MeJA was shown to regulate TPS gene expression in D. officinale and C. sinensis Zhao et al., 2020;Zhou et al., 2020). However, this needs to be further investigated in C. faberi. The Ka/Ks ratio analysis indicated that the TPS gene family in C. faberi mainly underwent negative selection, making it more stable during the evolution. GO annotation analysis of CfTPS genes indicated that molecular function contained most genes, and genes were mostly enriched in lyase activity, magnesium binding, and terpene synthase activity. Expression pattern analysis indicated that CfTPS  Supplementary Table S10. genes were mainly expressed in the floral organs of C. faberi, indicating that they were related to floral scent. We selected four CfTPS genes with the highest transcript levels in floral organs for qRT-PCR analysis at three flowering stages. The results showed that they all belonged to the TPS-b clade and were mainly expressed in the full flowering stage. According to our annotation, four genes were all annotated as monoterpene synthases, which may play essential roles in floral scent and attracting pollinators in C. faberi. Enzymatic assays suggested that CfTPS18 could convert GPP to β-myrcene, geraniol, and α-pinene. In recent reports, DoTPS10 in D. officinale can convert GPP to linalool in vitro . DoGES1 in D. officinale can catalyze geraniol in vitro and in vivo . Linalool and geraniol belong to monoterpenes which play essential roles in floral scents. TPS that catalyze terpenes can also be found in V. vinifera, M. domestica, and Litsea cubeba (Martin et al., 2010;Nieuwenhuizen et al., 2013;Chen et al., 2020). To better understand terpenes production and function in C. faberi, more studies of expression profiles should be developed.

CONCLUSION
In this study, 32 CfTPS genes were identified from the genomes of C. faberi. We analyzed their conserved motifs, exon-intron structure, phylogeny, Ka/Ks ratios, and cis-acting regulatory elements. We also analyzed the expression patterns of CfTPS genes in leaves, pseudobulbs, petals, sepals, labellums, and gynostemium in wild C. faberi. Four putatively functional genes highly expressed in floral organs were used to analyze the expression patterns at three flowering stages. Enzymatic assays FIGURE 9 | Enzymatic assays after incubating recombinant CfTPS18. Reactions only performed with GPP were used as the blank control. The retention time was compared with the NIST Mass Spectral Library.  Supplementary Table S11, and the RT-qPCR primers of CfTPS are listed in Supplementary Table S12.
indicated that CfTPS18 could convert GPP to β-myrcene, geraniol, and α-pinene. The results will provide valuable information for further studies on floral scents in C. faberi and other orchids.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.