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Article

Bioinformatics Analysis and Expression Features of Terpene Synthase Family in Cymbidium ensifolium

by
Mengyao Wang
1,2,†,
Baojun Liu
1,†,
Jinjin Li
2,
Ningzhen Huang
1,
Yang Tian
2,
Liting Guo
2,
Caiyun Feng
2,
Ye Ai
2,* and
Chuanming Fu
1,*
1
Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin 541006, China
2
Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization, College of Landscape Architecture and Art, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2024, 10(10), 1015; https://doi.org/10.3390/horticulturae10101015
Submission received: 21 August 2024 / Revised: 17 September 2024 / Accepted: 23 September 2024 / Published: 24 September 2024
(This article belongs to the Special Issue Physiological and Molecular Biology of Ornamental Plants—2nd Edition)
Browse Figures
Versions Notes

Abstract

Terpene synthases (TPSs) are crucial for the diversification of terpenes, catalyzing the formation of a wide variety of terpenoid compounds. However, genome-wide systematic characterization of TPS genes in Cymbidium ensifolium has not been reported. Within the genomic database of C. ensifolium, we found 30 CeTPS genes for this investigation. CeTPS genes were irregularly distributed throughout the seven chromosomes and primarily expanded through tandem duplications. The CeTPS proteins were classified into three TPS subfamilies, including 17 TPS-b members, 8 TPS-a members, and 5 TPS-c members. Conserved motif analysis showed that most CeTPSs contained DDxxD and RRX8W motifs. Cis-element analysis of CeTPS gene promoters indicated regulation primarily by plant hormones and stress. Transcriptome analysis revealed that CeTPS1 and CeTPS18 had high expression in C. ensifolium flowers. qRT-PCR results showed that CeTPS1 and CeTPS18 were predominantly expressed during the flowering stage. Furthermore, CeTPS1 and CeTPS18 proteins were localized in the chloroplasts. These results lay the theoretical groundwork for future research on the functions of CeTPSs in terpenoid biosynthesis.

1. Introduction

Terpenoids constitute the most diverse and structurally complex class of compounds in plants [1,2]. They are essential to the attraction of pollinators to plants, protection from pathogens and predators, and exchange of information [3,4]. Moreover, terpenoids are utilized extensively due to their distinct flavor and scent in various types of industries, including cosmetics, food, perfume, and pharmaceuticals [5,6]. Terpenoids, composed of isoprene (C5) units, include monoterpenes (C10), sesquiterpenes (C15), and diterpenes (C20) [7]. Plants produce C5 isoprenoid precursors through two distinct pathways: the methylerythritol phosphate (MEP) pathway in the plastid and the mevalonate (MVA) pathway in the cytoplasm. The isomers dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) are produced via these two pathways [8]. Under the catalytic action of various isoprenyl diphosphate synthases (IPSs), different quantities of IPP and DMAPP synthesize the terpenoid precursors geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP) (Figure 1) [9]. The structural characteristics of terpene synthases (TPSs) are fundamental to the generation of terpene diversity [10]. TPSs are classified as monoterpene synthases, sesquiterpene synthases, and diterpene synthases, which synthesize the skeletons of monoterpene, sesquiterpene, and diterpene compounds using GPP, FPP, and GGPP as precursor substrates [11]. The terpene skeleton forms more diverse compounds in the modification of cytochrome P450 monooxygenases, glycosyltransferase, and acyltransferase [9,12,13].
Most TPS proteins exhibit the RRX8W motif near the N-terminus, as well as the DDxxD and NSE/DTE motifs near the C-terminus [14]. The TPS gene family can be divided into seven subfamilies, including angiosperm-specific subfamilies TPS-a, TPS-b, and TPS-g, the gymnosperm-specific subfamily TPS-d, and subfamilies TPS-c and TPS-e/f, which are shared by both angiosperms and gymnosperms [15]. The TPS-h subfamily is found exclusively in the Selaginella moellendorffii [16]. As the largest subfamily, TPS-a encodes sesquiterpene synthases in both monocot and dicot plant species. The TPS-b subfamily encodes monoterpene synthases with the RRX8W motif, while the TPS-g subfamily, which lacks this motif, encodes both monoterpene and sesquiterpene synthases. The “DxDD” motif, rather than the “DDxxD” sequence, distinguishes the TPS-c subfamily, which encodes diterpene synthase enzymes [15]. There are several TPS genes found in the genomes of Arabidopsis thaliana [17], Vitis vinifera [18], Rosaceae [19], Cinnamomum camphora [20], and Camellia sinensis [21].
Orchidaceae is one of the most diverse families among the angiosperms [22]. Orchid evolution has been significantly influenced by the interactions between pollinators and orchids [23]. Terpenoids are one of the main components of floral scent in orchids. Floral scents enhance horticultural aesthetics and improve fertilization rates by attracting pollinators [24,25]. Monoterpenes and sesquiterpenes are the primary volatile compounds in the floral scents of Phalaenopsis bellina and Cymbidium goeringii [26,27]. Currently, the genomes of multiple Orchidaceae plants contain the TPS gene families, including Apostasia shenzhenica, P. equestris [28], Dendrobium officinale [29], C. faberi [30], C. goeringii [31], and D. chrysotoxum [32]. Several TPS genes in Orchidaceae are involved in terpenoid biosynthesis. For instance, the DoTPS10 protein in D. officinale converts GPP directly to linalool [29]. Transient overexpression of DoGES1 in Nicotiana benthamiana leaves promotes the accumulation of geraniol [33]. The CfTPS18 protein in C. faberi can convert GPP into β-myrcene, geraniol, and α-pinene in Escherichia coli [30]. In P. bellina, TPS-b and TPS-e/f subfamily members are involved in monoterpene biosynthesis in flowers [34].
C. ensifolium is highly valued for its ornamental qualities, including fragrance, color, and flower form. Monoterpenes and sesquiterpenes are key components of the floral scent in C. ensifolium [35]. However, the molecular processes that drive terpenoid production in C. ensifolium remain unexplored. We identified 30 CeTPS gene family members and named them according to their chromosomal locations, then analyzed their physicochemical properties, phylogenetic relationships, gene structures, collinearity, and cis-regulatory elements. Expression patterns in various organs and flower developmental stages were examined using transcriptome sequencing data and quantitative real-time PCR (qRT-PCR) experiments, identifying genes potentially involved in terpenoid biosynthesis. Subcellular localization of key TPS proteins was also investigated. Our analysis provides a theoretical basis for future research on terpene metabolism and regulation in C. ensifolium.

2. Materials and Methods

2.1. Plant Materials

Plant materials for the study were sourced from the greenhouse at the Forest Orchid Garden, Fujian Agriculture and Forestry University. All the plant materials were cultivated under natural conditions, with temperatures ranging from 25 °C to 35 °C, humidity levels between 75% and 90%, and day length averaging 13 to 14 h. Samples of different organs (leaf, Le; root, Ro; pseudobulb, Ps; flower, Fl) of C. ensifolium were collected. Flowers from C. ensifolium were collected at three development stages (small bud, S1; large bud, S2; flowering, S3). Each sample was stored at −80 °C after cryopreserved in liquid nitrogen, ensuring their molecular integrity for accurate downstream analysis. Three replicates of each sample were obtained from diverse plant sources.

2.2. Identification and Physicochemical Properties of CeTPS

The NGDC database (https://ngdc.cncb.ac.cn/, accessed on 17 September 2023) provided the C. ensifolium genome sequencing and annotation data [35], while A. thaliana’s TPS gene family protein sequences were obtained from TAIR (https://www.arabidopsis.org/, accessed on 17 September 2023). Using the TBtools 1.132 BlastP program, the AtTPS protein sequences served as query sequences to extract homologous sequences from C. ensifolium [36]. We accessed the Pfam database (http://pfam.xfam.org/, accessed on 17 September 2023) to acquire Hidden Markov Models (HMMs) for TPS domains, PF01397 and PF03936, for accurate identification of TPS genes [37]. The Simple HMM Search tool in TBtools was used to identify protein sequences with two conserved structural domains, ensuring precise classification and analysis [36]. Duplicate protein sequences were eliminated after merging the results of the two searches. Candidate sequences were validated using SMART (http://smart.embl-heidelberg.de, accessed on 17 September 2023) and NCBI CDD (http://www.ncbi.nlm.nih.gov/cdd/, accessed on 17 September 2023) websites, and sequences lacking complete structural domains were removed to ensure accurate and reliable downstream analysis.
The molecular weight (MW), isoelectric point (pI), instability index (II), and grand average of hydropathicity (GRAVY) of CeTPS proteins were analyzed using the ExPASy online platform (https://www.expasy.org/, accessed on 19 September 2023) [38], providing detailed insights into their physicochemical properties. ProtComp 9.0 (http://linux1.softberry.com/berry.phtml?topic=protcomppl&group=programs&subgroup=proloc, accessed on 19 September 2023) was used to predict the subcellular localization of CeTPS proteins, providing insights into their cellular functions.

2.3. Chromosome Localization and Phylogenetic Analysis

Using the genome annotation data of C. ensifolium, Show Genes on Chromosome within TBtools was utilized to show the chromosomal positions of the CeTPSs in order to analyze their genomic distributions [36]. A phylogenetic tree was constructed using MEGA7.0 [39], based on TPS protein sequences from C. ensifolium, A. thaliana, Oryza sativa, A. shenzhenica, and P. equestris, to examine evolutionary relationships among TPS genes. ClustalW was utilized for multiple sequence alignment, and 1000 bootstrap repetitions of the Neighbor-Joining method were used to generate a phylogenetic tree [39]. We employed Evolview v2 (https://evolgenius.info/, accessed on 22 September 2023) for the enhancement and visualization of the phylogenetic tree [40].

2.4. Conserved Motifs, Gene Structure, and Synteny Analysis

MEME (http://meme-suite.org/tools/meme, accessed on 26 September 2023) was used to analyze conserved motifs in CeTPS protein sequences in order to find common sequence patterns and functional domains [41]. The analysis was conducted with a maximum of 20 predicted motifs, with the settings maintained at default. TBtools’ Gene Structure View was used to demonstrate the conserved motifs and structural details of the CeTPSs. One Step MCScanX in TBtools was used to investigate TPS gene duplications in C. ensifolium. To assess evolutionary selection pressures on TPS genes, we utilized TBtools’ Simple Ka/Ks Calculator to calculate the non-synonymous (Ka) and synonymous (Ks) substitution rates, as well as their ratio. We conducted an analysis of duplication events involving TPS genes across three distinct species: C. ensifolium, D. chrysotoxum, and C. goeringii. Multiple Synteny Plot in TBtools was used to visualize the duplication patterns [36].

2.5. Cis-Acting Regulatory Elements Analysis

Promoter sequences 2000 bp upstream of the CeTPS genes were obtained using TBtools to analyze regulatory elements controlling gene expression [36]. In order to investigate the regulation mechanisms of CeTPSs, PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 28 September 2023) was used for predicting the number and categories of cis-acting elements present in the promoter sequences [42]. Subsequently, the data were subjected to analysis and visualization utilizing Excel 2019 and TBtools [36].

2.6. Expression Patterns and qRT-PCR Analysis

This study initially identified potential CeTPSs involved in terpene biosynthesis using RNA-seq data from different organs. Samples of different organs (leaf, Le; root, Ro; pseudobulb, Ps; flower, Fl) of C. ensifolium were used for transcriptome analysis [35]. Calculations were performed to determine the Fragments per Kilobase per Million mapped reads (FPKM). Subsequently, we used the TBtools software to generate a comprehensive heatmap, illustrating the expression patterns of CeTPSs (FPKM values are in Supplementary Table S1) [36].
Three stages of flower development were examined for CeTPS gene expression patterns using qRT-PCR experiments. Total RNA was extracted from C. ensifolium flowers at three developmental stages using the Rapure Total RNA Plus Kit (Megan Biotechnology, Guangzhou, China). cDNA was synthesized using the Hifair® III 1st Strand cDNA Synthesis SuperMix kit (gDNA digester plus) from Yeasen Biotechnology (Shanghai, China). The qRT-PCR primers specific to CeTPSs were synthesized by Shanghai Sangon Biotech Company (Shanghai, China). Primer sequences used in this experiment are listed in Supplementary Table S2. qRT-PCR experiments were conducted on an ABI QuantStudio 6 Flex system (Applied Biosystems, Foster City, CA, USA). GAPDH was selected as the internal reference gene for accurate normalization of gene expression levels in the qRT-PCR experiments. To guarantee data reliability and robustness, the studies included three biological replicates. Using the 2−ΔΔCT formula, relative gene expression levels were calculated. The one-way ANOVA was conducted using SPSS 26.0 to assess gene expression differences across developmental stages, with results visualized using GraphPad Prism 9.5.

2.7. Subcellular Localization Analysis

We cloned the coding sequences (CDS) of CeTPS1 and CeTPS18 employing PCR amplification with primers detailed in Supplementary Table S3. These sequences were efficiently cloned into the pCAMBIA1302-35S-GFP vector. The 35S promoter-driven CeTPS-GFP vectors were successfully transformed into Agrobacterium tumefaciens strain GV3101 (Weidi Biotechnology, Shanghai, China). Agrobacterium strains carrying the CeTPSs were used to infect N. benthamiana leaves for transient expression experiments, with an empty vector as the control. After 48 h, CeTPS1 and CeTPS18 protein subcellular localization was observed utilizing the LSM880 confocal laser microscope (Carl Zeiss, Jena, Germany).

3. Results

3.1. Identification and Physicochemical Properties of CeTPS

An in-depth analysis of BlastP and HMMER search results was conducted. We identified 30 CeTPS genes, named CeTPS1 to CeTPS30, based on their respective chromosomal positions. A thorough analysis was conducted to investigate the physicochemical properties of the 30 CeTPS genes, with the results summarized in Table 1, providing insights into their roles and potential applications in C. ensifolium. The lengths of the CeTPS protein sequences exhibited considerable variation, ranging from a minimum of 194 amino acids (aa) (CeTPS17) to a maximum of 807 aa (CeTPS19), measuring 477 aa on average. The substantial variation in protein lengths suggests the potential presence of pseudogenes. The molecular weights (MW) of CeTPS proteins varied widely, from a minimum of 22.24 kDa (CeTPS17) to a maximum of 91.98 kDa (CeTPS19). This variation in MW reflects the different lengths and compositions of the amino acid sequences in these proteins. The average theoretical isoelectric point (pI) of CeTPS proteins was 5.76, with all pI values below 7, indicating their acidic nature. The instability index (II), indicating protein stability, varied widely among CeTPS proteins, from a stable 29.24 in CeTPS22 to an unstable 61.68 in CeTPS3. The grand average of hydropathicity (GRAVY) for all CeTPS proteins was below 0, indicating their hydrophilic nature. Furthermore, the chloroplasts and cytoplasm were the targets of the subcellular localization prediction results, as shown by the ProtComp 9.0 database.

3.2. Chromosome Localization and Phylogenetic Analysis of CeTPS

Chromosomal mapping revealed an uneven distribution of 28 CeTPS genes across the seven chromosomes of C. ensifolium, except for CeTPS29 and CeTPS30, which were not localized on the chromosomes (Figure 2). Chromosomes 8 and 16 in C. ensifolium showed a significant accumulation of CeTPSs, each containing up to eight genes due to the presence of two separate gene clusters. In contrast, chromosomes 2 and 12 each hold only one CeTPS gene. The clustering of most CeTPSs on chromosomes suggests they likely originated from tandem or segmental duplications. No correlation was found between the size of the chromosomes and the distribution of the CeTPSs. Based on the CeTPS and TPS protein sequences from other species, a phylogenetic tree was constructed using the Neighbor-Joining method (Figure 3). All TPS protein sequences were classified into five subfamilies: TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g. The CeTPS family was categorized into three subfamilies: TPS-b (17 members), TPS-a (8 members), and TPS-c (5 members) (Figure 3). TPS proteins from monocot and dicot species established separate subgroups in the TPS-a subfamily. This observation aligns with the findings of prior investigations [11]. In addition, CeTPS members were extremely similar to the TPS protein of A. shenzhenica and P. equestris from the Orchidaceae.

3.3. Analysis of CeTPS Conserved Motifs, Gene Structure, and Synteny

Utilizing the web program MEME, we identified 20 conserved motifs within the CeTPS protein sequences, providing valuable insights into their structural and functional conservation. The findings demonstrated that CeTPSs had between three and sixteen motifs (Figure 4B). The most conserved motifs were discovered in CeTPS20, CeTPS23, CeTPS25, and CeTPS28, whereas CeTPS3 and CeTPS17 only had three motifs. All CeTPS sequences contained Motif 2. Twenty-three CeTPS protein sequences contained the DDxxD (Motif 1) and RRX8W (Motif 3) motif, respectively. NSE/DTE motifs (Motif 8) were found in 18 CeTPS genes (Figure S1). In the TPS-a subfamily, all members except CeTPS12 contain the RRX8W motif. CeTPS27 of the TPS-b subfamily and all TPS-c subfamily members lack the conserved RRX8W motif, suggesting functional and structural differences within the TPS gene family in C. ensifolium. The RRX8W motif is essential for the cyclization process of monoterpenes [43]. The DDxxD and NSE/DTE motifs are essential for the enzymatic cleavage of prenyl diphosphate substrates, playing critical roles in substrate binding and catalysis in terpene synthase enzymes. These motifs facilitate the coordination of Mg2+ or Mn2+ ions at the C-terminus [4,14]. Results showed that members of the same subfamily had highly similar motif compositions, while distinct subfamilies were characterized by unique motifs, indicating conserved functions within subfamilies and functional divergence between them.
The CeTPSs displayed variability in exon count, spanning from two to fourteen exons (Figure 4C). The CeTPS19 gene had the most exons, while CeTPS3 and CeTPS17 had only two exons. Members of the same subfamily share comparable intron–exon structures. By analyzing the collinearity of the CeTPSs within C. ensifolium, we identified six pairs of tandem duplicates and one pair of segmental duplicates (Figure 2 and Figure 5). A Ka/Ks value of 1 indicates neutral selection, values greater than 1 signify positive selection, and values less than 1 suggest purifying selection [44]. The segmentally duplicated gene pair had a Ka/Ks ratio of 0.4476, while the tandem duplicates ranged from 0.5462 to 0.8025 (Table 2) These results, with all Ka/Ks values below 1, suggest that purifying selection has been applied to the CeTPS gene family, indicating that these genes are conserved and likely essential for maintaining specific functions in C. ensifolium. To explore the evolutionary relationships among Orchidaceae’s TPS family, we analyzed the collinearity of TPSs among C. ensifolium, D. chrysotoxum, and C. goeringii, revealing gene duplication events. CeTPSs had nine and eight pairs of homologous genes with C. goeringii and D. chrysotoxum, respectively (Figure 6).

3.4. Cis-Elements Analysis of CeTPS

Analysis of promoter sequences 2000 bp upstream of the CeTPSs identified 641 cis-acting elements, highlighting the complex regulatory mechanisms controlling gene expression and terpenoid biosynthesis in C. ensifolium. These cis-acting elements were categorized into three functional groups (Figure 7A,B). There were 135 cis-acting elements related to plant growth and development which were classified into 10 groups: As-1 element (33%), AAGAA-motif (21%), MRE (17%), AT-rich element (8%), CCAAT-box (5%), etc. In the CeTPS gene promoters, 8 categories were created from the identification of 155 cis-acting elements associated with plant stress response, including anaerobic induction (ARE, 43%), stress response (STRE, 18%), drought response (MBS, 15%), and wounding response (WUN-motif, 8%), etc. The phytohormone responsiveness category contained the most cis-acting elements (351/641), primarily responsive to MeJA (MYC, 31%; TGACG-motif, 13%; CGTCA-motif, 12%), ethylene (ERE, 23%), abscisic acid (ABRE, 5%), and auxin (TGA-element, 5%) (Figure 7C). Our findings revealed that MeJA serves as the primary regulator of CeTPS gene expression, corroborating previous research in plants [20,29].

3.5. Expression Patterns and qRT-PCR Analysis of CeTPS

Based on the transcriptome expression data of CeTPSs in various organs, we found that CeTPSs have tissue-specific expression characteristics (Figure 8A). Specifically, CeTPS14 and CeTPS16 showed comparatively high expression levels in the roots and leaves, while CeTPS23 and CeTPS28 showed significant expression levels in pseudobulbs. Additionally, CeTPS1 and CeTPS18 displayed high transcript abundance in flowers. We conducted qRT-PCR experiments to quantify the abundance of CeTPS1 and CeTPS18 transcripts at different developmental stages of the flower (Figure 8B). These genes showed the highest expression levels during blooming. It is hypothesized that these genes contribute to the biosynthesis of terpenoids in the flowers of C. ensifolium.

3.6. Subcellular Localization of CeTPS

To further validate the functions of the CeTPS1 and CeTPS18 proteins, we created expression vectors (35S: CeTPS1-GFP and 35S: CeTPS18-GFP) and transiently expressed them in N. benthamiana leaves. This method allowed us to track gene expression and visualize subcellular localization. Strong GFP fluorescence signals of CeTPS1-GFP and CeTPS18-GFP were detected in the cytoplasm, while non-overlapping punctate red spots were observed in the corresponding chlorophyll autofluorescence (Chl) images (Figure 9). The experimental results demonstrated that both CeTPS1 and CeTPS18 proteins localized in chloroplasts (Figure 9).

4. Discussion

Floral scent is crucial for the diverse evolution of orchids and is a key ornamental trait. Terpenes, which are primary components of orchid floral scents, are synthesized by TPS genes, whose roles in terpene production have been confirmed in various orchid species. C. ensifolium, a traditional Chinese orchid, has been cherished for its fresh and elegant fragrance [35]. However, comprehensive studies on the identification of the TPS gene family in C. ensifolium remain scarce, limiting our understanding of terpenoid biosynthesis and its potential applications. Therefore, analyzing and categorizing the TPS genes in C. ensifolium holds significant importance from a theoretical standpoint.
To accurately identify CeTPS genes, we combined HMMER search and BlastP results, followed by validation using CDD and SMART to exclude incomplete sequences, ultimately identifying 30 TPS gene family members in C. ensifolium. A comparative analysis of C. ensifolium TPS family members with other orchid species, including D. chrysotoxum (48) [32], D. officinale (34) [29], C. faberi (32) [30], and C. goeringii (40) [31], reveals that C. ensifolium has a medium-sized TPS gene family. Phylogenetic analysis of CeTPS proteins in C. ensifolium identified three unique subfamilies: TPS-a, TPS-b, and TPS-c (Figure 3), which differ from classifications in other Orchidaceae species, suggesting unique evolutionary adaptations [28,29,30,34]. Evidence suggests that C. ensifolium has lost TPS-e/f and TPS-g subfamily members over its evolutionary history. The CeTPS gene family comprised 8 TPS-a, 17 TPS-b, and 5 TPS-c subfamily members. Unlike the proportion of TPS-a subfamily in A. thaliana (68.75%), C. faberi (43.33%), and D. chrysotoxum (41.17%), C. ensifolium has only 26.67% of TPS-a subfamily members, and 56.67% of CeTPSs are distributed in the TPS-b subfamily. TPS genes in plants usually cluster together to generate functional gene clusters [45]. The TPS functional gene clusters exist in numerous plants, including A. thaliana [17], Solanum lycopersicum [45], Daucus carota [46], A. shenzhenica, V. planifolia, D. catenatum, and P. equestris [28]. The TPS gene clusters are highly conserved in plants, contributing to the stability and diversity of terpene biosynthesis pathways [45]. Chromosomal analysis of CeTPSs in C. ensifolium revealed that TPS-a, TPS-c, and TPS-b subfamily members were clustered on chromosomes Chr08, Chr09, and Chr16, respectively (Figure 2). Gene duplication is crucial for gene family expansion, diversification, and understanding evolutionary relationships among homologous genes. Gene duplications are classified into tandem, segmental, and genome-wide types. Tandem duplications place homologous genes on the same chromosome, while segmental duplications occur when duplicated genes are located on different chromosomes [47]. Seven duplication events, including six tandem and one segmental, were identified in the CeTPSs (Figure 2 and Figure 5), with Ka/Ks ratios ranging from 0.4476 to 0.8025 (Table 2), indicating purifying selection and suggesting evolutionary conservation. The findings indicate that tandem and segmental duplications have contributed to expanding the TPS gene family in C. ensifolium, driving genetic diversity and enhancing terpenoid biosynthesis essential for ecological interactions. Through further collinearity analysis of C. ensifolium, C. goeringii, and D. chrysotoxum, multiple TPS homologous genes were identified among these three species (Figure 6). The detection of these homologous genes highlights the evolutionary conservation among these orchid species and implies potential similarities in gene function or regulatory mechanisms. This knowledge lays a critical foundation for further exploration of the evolutionary processes and gene functions of the TPS gene in orchids.
Numerous cis-regulatory elements regulate phytohormone signaling and stress responses in C. ensifolium, including MeJA regulation, ERE regulation, anaerobic induction, drought response, etc. (Figure 7). The findings show that hormones and stress may modulate CeTPSs expression. Several environmental factors regulate the release of volatile terpenoids from plants, including light intensity, circadian clock, ambient temperature, and relative humidity [48]. MeJA generates geraniol by promoting the expression of the D. officinale TPS gene DoGES1 [33]. Some TPS genes in C. sinensis are suppressed or promoted in expression after treatments with MeJA and various stressors [21]. Genes responsible for MeJA biosynthesis in C. ensifolium are highly expressed in mature flower buds and fully bloomed flowers [35], implying that the MeJA signaling pathway may influence the expression of CeTPSs. Various transcription factors regulate the TPS gene in plants, playing a crucial role in modulating terpenoid biosynthesis by controlling gene expression in response to development and the environment. HcMYBs in Hedychium coronarium regulate HcTPS expression by binding to core MYB-binding sites [49]. Transient expression of the PbbHLH4 increases the content of monoterpenes in scentless phalaenopsis orchid flowers by 950-fold [50]. DobHLH4 interacts with DoJAZ1 and binds to the DoTPS10 promoter’s G-box, upregulating DoTPS10 expression, crucial for MeJA-mediated linalool accumulation in D. officinale [51]. Various transcription factors are thought to regulate the expression of CeTPS genes.
CeTPSs exhibit organ-specific expression patterns according to the transcriptome expression data. This observation suggests that members from different subfamilies may fulfill distinct roles in various plant organs, contributing to a diversity of biological processes. For example, CeTPS14 and CeTPS16 are predominantly expressed in roots and leaves, whereas CeTPS23 and CeTPS28 show significant expression in pseudobulbs (Figure 8A). This pattern indicates that these genes might be involved in stress responses or other physiological functions related to plant defense mechanisms. Furthermore, the elevated expression of CeTPS1 and CeTPS18 in flowers suggests their involvement in floral scent biosynthesis (Figure 8A). These findings provide essential theoretical foundations and valuable insights for future research on the specific roles of CeTPSs in plant growth, development, and their underlying molecular mechanisms across different organs. Floral organs primarily emit volatile organic compounds (VOCs). The release of floral scents follows a distinct pattern across the stages of flower development. Flowers emit more VOCs when they prepare for pollination [25]. We examined gene expression patterns in C. ensifolium at various stages of flower development, revealing key regulatory genes involved in scent production. Then we identified a peak in CeTPS transcript abundance, specifically during the flowering period. The present results align with earlier investigations [27]. Gene expression patterns indicated that CeTPS1 and CeTPS18 exhibited the highest expression levels during flowering (Figure 8B). CeTPS1 and CeTPS18 belong to the TPS-b subfamily. These genes are considered essential in the monoterpene biosynthesis pathways of C. ensifolium. These findings offer essential insights and establish a foundation for future research on floral scent biosynthesis and molecular breeding in orchids.

5. Conclusions

In this research, we identified 30 CeTPS genes within the C. ensifolium genome and performed an extensive analysis of their physicochemical properties. The CeTPS proteins were classified into three TPS subfamilies. Genes within the same subfamily shared remarkably comparable structures and conserved motifs, indicating functional conservation, whereas different subfamilies had distinct features, demonstrating functional differentiation. The genome of C. ensifolium contained seven pairs of duplicated genes with purifying selection, suggesting that the evolution of CeTPSs was conserved. Cis-element analysis of CeTPS gene promoters indicated regulation primarily by plant hormones and stress. Transcriptome analysis revealed the CeTPSs demonstrated organ-specific expression patterns. The qRT-PCR experiments further validated two functional genes that were highly expressed during flowering and analyzed their subcellular localization. These findings provide valuable insights and data, laying the groundwork for future research on floral scent biosynthesis mechanisms and molecular breeding in orchids.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10101015/s1, Figure S1: Motif logo of Motif1, Motif2, Motif3, and Motif8. Table S1: The FPKM values of CeTPSs of different organs of C. ensifolium; Table S2: Primers of qRT-PCR experiments; Table S3: Primers of subcellular localization.

Author Contributions

The experiments were devised and conceived by C.F. (Chuanming Fu) and Y.A.; B.L. conducted the bioinformatics analysis; the experiments and data analysis were carried out by M.W.; M.W. and B.L. drafted the manuscript; J.L., N.H. and Y.T. were responsible for the visualization and preparation of the figures; L.G. and C.F. (Caiyun Feng) prepared the plant materials. All authors contributed to revising the manuscript. M.W. and B.L. contributed equally to this work. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Guilin Innovation Platform and Talent Plan Project (No. 20210218-10), the Regional Fund of the National Natural Science Foundation of China (No. 32160096), the Basal Research Fund of Guangxi Academy of Sciences (No. CQZ-E-1910), and the Science and Technology Innovation Special Fund Project of Fujian Agricultural and Forestry University (No. KFb22057XA).

Data Availability Statement

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

Conflicts of Interest

The authors declare no conflict of interest.

References

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Figure 1. Pathways of terpenoid biosynthesis in plants. Two precursor substances of terpenoids are produced by the cytosolic mevalonate (MVA) and plastid methylerythritol phosphate (MEP) pathways. G3P: glyceraldehyde 3-phosphate; IPP: isopentenyl diphosphate; DMAPP: dimethylallyl diphosphate; FPPS: FPP synthase; GGPPS: GGPP synthase; GPPS: GPP synthase; FPP: farnesyl pyrophosphate; GGPP: geranylgeranyl pyrophosphate; GPP: geranyl pyrophosphate; STPS: sesquiterpene synthase; DTPS: diterpene synthase; MTPS: monoterpene synthase.
Figure 1. Pathways of terpenoid biosynthesis in plants. Two precursor substances of terpenoids are produced by the cytosolic mevalonate (MVA) and plastid methylerythritol phosphate (MEP) pathways. G3P: glyceraldehyde 3-phosphate; IPP: isopentenyl diphosphate; DMAPP: dimethylallyl diphosphate; FPPS: FPP synthase; GGPPS: GGPP synthase; GPPS: GPP synthase; FPP: farnesyl pyrophosphate; GGPP: geranylgeranyl pyrophosphate; GPP: geranyl pyrophosphate; STPS: sesquiterpene synthase; DTPS: diterpene synthase; MTPS: monoterpene synthase.
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Figure 2. Distribution of the CeTPSs on C. ensifolium chromosomes. The scale shows chromosomal distances in megabases (MB). The colored triangle icons indicate tandem repeat relationships between CeTPSs, and triangle icons of the same color CeTPSs have tandem repeats.
Figure 2. Distribution of the CeTPSs on C. ensifolium chromosomes. The scale shows chromosomal distances in megabases (MB). The colored triangle icons indicate tandem repeat relationships between CeTPSs, and triangle icons of the same color CeTPSs have tandem repeats.
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Figure 3. Phylogenetic analysis of TPS protein sequences from five plant species. Members of the TPS subfamilies from different species are marked with different colors and shapes. Green and cyan circles represent the TPS proteins from A. thaliana and O. sativa, respectively. Purple, blue, and yellow triangles represent the TPS proteins from C. ensifolium, A. shenzhenica, and P. equestris, respectively.
Figure 3. Phylogenetic analysis of TPS protein sequences from five plant species. Members of the TPS subfamilies from different species are marked with different colors and shapes. Green and cyan circles represent the TPS proteins from A. thaliana and O. sativa, respectively. Purple, blue, and yellow triangles represent the TPS proteins from C. ensifolium, A. shenzhenica, and P. equestris, respectively.
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Figure 4. Phylogenetic analysis, motif conservation, and gene structure of CeTPS. (A) Phylogenetic tree of CeTPS proteins. (B) Conserved motifs in CeTPS proteins. (C) Gene structure of CeTPSs.
Figure 4. Phylogenetic analysis, motif conservation, and gene structure of CeTPS. (A) Phylogenetic tree of CeTPS proteins. (B) Conserved motifs in CeTPS proteins. (C) Gene structure of CeTPSs.
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Figure 5. Collinear relationships of CeTPSs on chromosomes. The red line represents the CeTPSs with segmental duplications in C. ensifolium.
Figure 5. Collinear relationships of CeTPSs on chromosomes. The red line represents the CeTPSs with segmental duplications in C. ensifolium.
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Figure 6. Collinearity analysis of TPS genes in three orchid species. The red triangles indicate the positions of the CeTPSs on chromosomes. The red lines highlight the TPS genes with collinear relationships between three orchid species.
Figure 6. Collinearity analysis of TPS genes in three orchid species. The red triangles indicate the positions of the CeTPSs on chromosomes. The red lines highlight the TPS genes with collinear relationships between three orchid species.
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Figure 7. Promoter analysis of CeTPSs. (A) The count of distinct cis-elements identified in each CeTPS gene. (B) Total cis-regulatory elements related to plant growth and development, stress responsiveness, and phytohormone responsiveness. (C) The proportion of cis-elements related to plant growth and development, stress responsiveness, and phytohormone responsiveness.
Figure 7. Promoter analysis of CeTPSs. (A) The count of distinct cis-elements identified in each CeTPS gene. (B) Total cis-regulatory elements related to plant growth and development, stress responsiveness, and phytohormone responsiveness. (C) The proportion of cis-elements related to plant growth and development, stress responsiveness, and phytohormone responsiveness.
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Figure 8. Heatmap and qRT-PCR analysis of CeTPSs expression in C. ensifolium. (A) Expression patterns of CeTPSs in different organs (FPKM values) (Le: leaf; Ro: root; Ps: pseudobulb; Fl: flower). (B) Expression in flowers at three stages (S1: small bud; S2: large bud; S3: flowering). Bars display biological replicate mean values ± SE; significant statistical differences are shown by unique letters (p < 0.05, Duncan).
Figure 8. Heatmap and qRT-PCR analysis of CeTPSs expression in C. ensifolium. (A) Expression patterns of CeTPSs in different organs (FPKM values) (Le: leaf; Ro: root; Ps: pseudobulb; Fl: flower). (B) Expression in flowers at three stages (S1: small bud; S2: large bud; S3: flowering). Bars display biological replicate mean values ± SE; significant statistical differences are shown by unique letters (p < 0.05, Duncan).
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Figure 9. CeTPS proteins’ subcellular localization in N. benthamiana leaves (GFP: fluorescence image of green fluorescent protein; Chl: chlorophyll autofluorescence image; Bright: brightfield image; Merged: all channels combined).
Figure 9. CeTPS proteins’ subcellular localization in N. benthamiana leaves (GFP: fluorescence image of green fluorescent protein; Chl: chlorophyll autofluorescence image; Bright: brightfield image; Merged: all channels combined).
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Table 1. Physicochemical properties of the CeTPS proteins.
Table 1. Physicochemical properties of the CeTPS proteins.
NameIDAA (aa)Mw (kDa)pIIIGRAVYSubcellular Localization
CeTPS1JL01550741348.615.9757.61−0.500Chloroplast.
CeTPS2JL02712737844.716.0433.18−0.273Chloroplast. Cytoplasm.
CeTPS3JL02549921825.055.5961.68−0.405Chloroplast.
CeTPS4JL02628855263.555.3649.99−0.142Chloroplast.
CeTPS5JL02840054964.385.6252.65−0.194Chloroplast. Cytoplasm.
CeTPS6JL02850454964.605.6250.51−0.210Chloroplast. Cytoplasm.
CeTPS7JL02434440847.555.2848.98−0.106Chloroplast. Cytoplasm.
CeTPS8JL02623536442.915.9951.56−0.126Chloroplast. Cytoplasm.
CeTPS9JL02829442450.016.3852.50−0.258Chloroplast. Cytoplasm.
CeTPS10JL02470043651.155.4646.80−0.280Chloroplast. Cytoplasm.
CeTPS11JL02463349858.145.4647.35−0.231Chloroplast. Cytoplasm.
CeTPS12JL00815732937.996.5240.16−0.107Chloroplast. Cytoplasm.
CeTPS13JL02642770382.256.1538.04−0.383Chloroplast.
CeTPS14JL02733344653.016.1737.95−0.358Chloroplast.
CeTPS15JL02578944652.806.1637.71−0.345Chloroplast.
CeTPS16JL02247170382.206.1441.84−0.395Chloroplast.
CeTPS17JL02419419422.245.3642.68−0.185Chloroplast. Cytoplasm.
CeTPS18JL01249660270.075.9949.94−0.271Chloroplast.
CeTPS19JL00467780791.985.9548.28−0.286Chloroplast.
CeTPS20JL02780353563.065.6733.24−0.211Chloroplast. Cytoplasm.
CeTPS21JL00141028733.475.7332.94−0.175Chloroplast. Cytoplasm.
CeTPS22JL02656450859.716.2229.24−0.233Chloroplast. Cytoplasm.
CeTPS23JL02781752962.465.7433.84−0.208Chloroplast. Cytoplasm.
CeTPS24JL02831852862.475.3639.28−0.192Chloroplast. Cytoplasm.
CeTPS25JL02831953563.315.6033.94−0.227Chloroplast. Cytoplasm.
CeTPS26JL02763848557.505.4537.85−0.161Chloroplast. Cytoplasm.
CeTPS27JL02813332036.795.1431.87−0.372Chloroplast.
CeTPS28JL02705953563.115.8334.91−0.219Chloroplast. Cytoplasm.
CeTPS29JL02884450159.045.4637.25−0.153Chloroplast. Cytoplasm.
CeTPS30JL02898452862.545.3539.80−0.158Chloroplast. Cytoplasm.
Table 2. Ka/Ks analysis of CeTPS genes.
Table 2. Ka/Ks analysis of CeTPS genes.
Gene PairsKaKsKa/KsDuplication TypePurify Selection
CeTPS5CeTPS60.0179509090.0320748590.559656681TandemYes
CeTPS6CeTPS70.0299379440.0548066940.546246113TandemYes
CeTPS8CeTPS90.1460806620.227926560.6409111TandemYes
CeTPS17CeTPS180.4421144250.9877651760.447590618SegmentalYes
CeTPS23CeTPS240.02544540.0373893240.68055256TandemYes
CeTPS24CeTPS250.0276065160.0402781340.685397089TandemYes
CeTPS25CeTPS260.0026181840.0032626480.802472277TandemYes
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Wang, M.; Liu, B.; Li, J.; Huang, N.; Tian, Y.; Guo, L.; Feng, C.; Ai, Y.; Fu, C. Bioinformatics Analysis and Expression Features of Terpene Synthase Family in Cymbidium ensifolium. Horticulturae 2024, 10, 1015. https://doi.org/10.3390/horticulturae10101015

AMA Style

Wang M, Liu B, Li J, Huang N, Tian Y, Guo L, Feng C, Ai Y, Fu C. Bioinformatics Analysis and Expression Features of Terpene Synthase Family in Cymbidium ensifolium. Horticulturae. 2024; 10(10):1015. https://doi.org/10.3390/horticulturae10101015

Chicago/Turabian Style

Wang, Mengyao, Baojun Liu, Jinjin Li, Ningzhen Huang, Yang Tian, Liting Guo, Caiyun Feng, Ye Ai, and Chuanming Fu. 2024. "Bioinformatics Analysis and Expression Features of Terpene Synthase Family in Cymbidium ensifolium" Horticulturae 10, no. 10: 1015. https://doi.org/10.3390/horticulturae10101015

APA Style

Wang, M., Liu, B., Li, J., Huang, N., Tian, Y., Guo, L., Feng, C., Ai, Y., & Fu, C. (2024). Bioinformatics Analysis and Expression Features of Terpene Synthase Family in Cymbidium ensifolium. Horticulturae, 10(10), 1015. https://doi.org/10.3390/horticulturae10101015

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