Evolution of Terpene Synthases in Orchidaceae

Abstract

Terpenoids are the largest class of plant secondary metabolites and are one of the major emitted volatile compounds released to the atmosphere. They have functions of attracting pollinators or defense function, insecticidal properties, and are even used as pharmaceutical agents. Because of the importance of terpenoids, an increasing number of plants are required to investigate the function and evolution of terpene synthases (TPSs) that are the key enzymes in terpenoids biosynthesis. Orchidacea, containing more than 800 genera and 28,000 species, is one of the largest and most diverse families of flowering plants, and is widely distributed. Here, the diversification of the TPSs evolution in Orchidaceae is revealed. A characterization and phylogeny of TPSs from four different species with whole genome sequences is available. Phylogenetic analysis of orchid TPSs indicates these genes are divided into TPS-a, -b, -e/f, and g subfamilies, and their duplicated copies are increased in derived orchid species compared to that in the early divergence orchid, A. shenzhenica. The large increase of both TPS-a and TPS-b copies can probably be attributed to the pro-duction of different volatile compounds for attracting pollinators or generating chemical defenses in derived orchid lineages; while the duplications of TPS-g and TPS-e/f copies occurred in a species-dependent manner.

1. Introduction

Terpenoids are the largest group of natural metabolites in the plant kingdom, including more than 40,000 different compounds, and have multiple physiological and ecological roles. Terpene metabolites are not only essential for plant growth and development (e.g., gibberellin phytohormones), but also important intermediaries in the various interactions of plants with the environment [1]. For example, chlorophylls and carotenoids are photosynthetic pigments, while brassinosteroids, gibberellic acid, and abscisic acid are plant hormones [2,3]. Terpenoids can be classified based on the number of isoprene units, such as hemiterpene (C5), monoterpene (C10), sesquiterpene (C15), diterpene (C20), sesterterpene (25), triterpene (C30), sesquarterpene (C35), and tetraterpene (C40) (Gershenzon and Dudareva, 2007). The increased number of cyclizations, possibly from a precursor with five additional carbon atoms, gives structural diversity. Terpenoid structures are extremely variable and most of them are low molecular weight like monoterpene (C₁₀), sesquiterpene (C₁₅), and diterpene (C₂₀) [4]. The approximate number of monoterpenes is 1000 and more than 7000 sesquiterpenes [5].

Terepene synthases (TPSs) are key enzymes in terpenoids biosynthesis. To date, TPSs have been studied in several typical plant genomes, such as Arabidopsis thaliana (Arabidopsis, 32 TPSs) [6], Physcomitrella patens (earthmoss, 1 TPS) [7], Sorghum bicolor (Sorghum, 24 TPSs) [8], Vitis vinifera (grape, 69 TPSs) [9], Solanum lycopersicum (tomato, 29 TPSs) [10], Selaginella moellendorffii (spikemoss, 14 TPSs) [11], Glycine max (soybean, 23 TPSs) [12] Populus trichocarpa (poplar tree, 38 TPSs) [13], Oryza sativa (rice, 32 TPSs) [14], and Dendrobium officinale (Dendrobium orchid, 34 TPSs) [15]. According to the classification principle, TPSs can be generally classified into seven clades or subfamilies: TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, TPS-g, and TPS-h [16]. TPS-a, TPS-b, and TPS-g are angiosperm-specific subfamilies, while the TPS-e/f subfamily is present in angiosperms and gymnosperms. TPS-c exists in land plants. TPS-d is a gymnosperm-specific subfamily, and the TPS-h subfamily only appears in Selaginella moellendorffii [16].

The full length of plant TPSs has three conserved motifs on C- and N-terminal regions. The conserved motif of N-terminal domain is R(R)X₈W (R, arginine, W, tryptophan and X, alternative amino acid) and the C-terminal domain contains two highly conserved aspartate-rich motifs. One of them is the DDXXD motif, which is involved in the coordination of divalent ion(s), water molecules, and the stabilization of the active site [17,18,19]. The second motif in the C-terminal domain is the NSE/DTE motif. These two motifs flank the entrance of the active site and function in binding a trinuclear magnesium cluster [20,21]. Most terpene synthases belong to monoterpene synthase (MTPSs) [22], sesquiterpene synthase (STPSs), and diterpene synthase (DTPSs) [23]. They all share three conserved domains in the active site, including ‘DDXXD’, ‘DXDD’, and ‘EDXXD’. The ‘R(R)X₈W’ motif is also essential for monoterpene cyclization, while some MTPSs do not have it [16]. These circumstances can be seen in linalool synthase in rice (Oryza sativa L. cv. Nipponbare and Hinohikari) [24]; nerol synthase in soybean (Glycine max cv. ‘Bagao’), which has a signal peptide and is believed to be functional in plastid [25]; and FaNES1, the cytosolic terpene synthase identified in strawberry, which is able to use cytosolic GDP and FDP to produce linalool and nerolidiol [26].

TPSs in the same subfamilies are similar in sequence and have similar functions. Based on the protein sequence, angiosperm STPSs and DTPSs belong to TPS-a subfamily and monoterpene synthases belong to TPS-b subfamily. Subfamilies in TPS-c and e/f have enzyme activities of DTPSs; Gymnosperm-specific TPS-d subfamily owns the enzyme activities for MTPSs, STPSs, and DTPSs. TPS-g encodes MTPSs, STPSs, and DTPSs that produce mainly acyclic terpenoids. TPS-h is Selaginella moellendorffii-specific subfamily and putative encodes DTPSs [16,27]. Recently, large amounts of TPSs have been identified by using BLAST and thus used for functional characterization assay to further confirm the activity of TPSs. The functions of TPSs can be mono- or multi-functional, and the enzymes can be highly identical to each other. For instance, the DTPs of levopimaradiene/abietadiene synthase and isopimaradiene synthase showed 91% identity in Norway spruce [28]. Moreover, the functional bifurcation of these two enzymes were proved to be caused by only four amino acid residues [28]. Some TPSs are responsible for producing compounds that are related to plant growth and development, such as gibberellin biosynthesis [29], others are responsible in secondary metabolism like monoterpenes and sesquiterpenes for pollination and defense [30,31]. Molecules catalyzed by TPS are usually further modified by cytochromes p450 (CYPs) to generate diverse structures [32].

Orchids show extraordinary morphological, structural, and physiological characteristics unique in the plant kingdom [33]. Containing more than 800 genera and 28,000 species, the Orchidaceae, classified in class Liliopsida, order Asparagales, is one of the largest and most diverse families of flowering plants [33]. They are widely distributed wherever sun shines except Antarctica, and with a variety of life forms from terrestrial to epiphytic [34]. According to molecular phylogenetic studies, Orchidaceae comprises five subfamilies, including Apostasioideae, Cypripedioideae, Vanilloideae, Orchidaideae, and Epidendroideae [35]. Orchids emit various volatile organic compounds (VOCs) to attract their pollinators, and/or the enemy of herbivores for olfactory capture. The emitted VOCs are plant secondary metabolites, and the major natural products include terpenoids, phenylpropenoids, benzeniods, and fatty acid derivatives. The floral scent composed of the VOCs plays an important role in plants, such as pollinator attraction, defense, and plant-to-plant communication, especially in insect-pollinated plants [30,36].

Floral VOCs are characterized into several orchids, including α- and β-pinene for Cycnoches densiflorum and C. dianae [37]; phenylpropanoids in Bulbophyllum vinaceum [38]; α-pinene and e-carvone oxide for Catasetum integerrimum [39]; p-dimethoxybenzene for Cycnoches ventricosum and Mormodes lineata [39]; β-bisabolene and 1,8-cineole for Notylia barkeri [39]; e-ocimene and linalool for Gongora galeata [39]; monoterpenes in Orchis mascula and Orchis pauciflora [40]; (Z)-11-eicosen-1-ol in Dendrobium sinense [41]; terpenoid of (E)-4,8-dimethylnona-1,3,7-triene (DMNT) in Calanthe sylvatica [42] and Cyclopogon elatus [43]; (E)-β-ocimene and (E)-epoxyocimene for Catasetum cernuum and Gongora bufonia [44]; and farnesol, methyl epi-jasmonate, nerolidol, and farnesene in Cymbidium goeringii [45].

Phalaenopsis spp. is very popular worldwide for its spectacular flower morphology and colors. Most Phalaenopsis orchids are scentless but some do emit scent VOCs [46]. The scented species have been extensively used as breeding parents for the production of scented cultivars, such as P. amboinensis, P. bellina, P. javanica, P. lueddemanniana, P. schilleriana, P. stuartiana, P. venosa, and P. violace [47]. P. bellina and P. violacea are two scented orchids that are very popular in breeding scented cultivars. P. bellina emits mainly monoterpenoids, including citronellol, geraniol, linalool, myrcene, nerol, and ocimene [47,48], while P. violacea emits monoterpenoids accompanied with a phenylpropanoid, cinnamyl alcohol [46]. The VOCs of P. schilleriana contain monoterpenoids as well, including citronellol, nerol, and neryl acetate [49]. Because of the importance of terpenoids in plants, an increasing number of plants are required to investigate the function and evolution of TPSs.

In the present review, we summarized the recent progress in the understanding of the biosynthesis and biological function of terpenoids, and the latest advances in research on the evolution and functional diversification of TPSs in Orchidaceae. TPSs from different orchid species are reported to explore the evolutionary history and the evolution diversification of Orchidaceae TPSs.

2. Terpenoids and Their Biosynthesis in Plants

There are two compartmentalized terpenoid biosynthesis pathways, the mevalonic acid (MVA) pathway that occurs in the cytosol, and the methylerythritol phosphate (MEP) pathway that occurs in plastids to produce isopentenyl diphosphate (IPP) and its allylic isomer-dimethylallyl diphosphate (DMAPP) converted by isopentenyl diphosphate isomerase (IDI) (Figure 1) [50,51,52]. There are four major steps involved in the biosynthesis of terpenoid, beginning with isoprene unit (IPP) formation, which has five carbons. Second, IPP combines to DMAPP by geranyl diphosphate synthase (GDPS), geranylgeranyl diphosphate synthases (GGDPS) or farnesyl diphosphate (FDPS), and generates geranyl diphosphate (GDP), farnesyl diphosphate (FDP) or geranylgeranyl diphosphate (GGDP), respectively [1,27,53,54]. Third, the C₁₀-C₂₀ diphosphates go through cyclization and rearrangement to produce the basic carbon skeletons for terpenoids catalyzed by TPS [53]. The TPS family consists of enzymes that use GDP to form cyclic and acylic monoterpenes (C₁₀), FDP for sesquiterpene (C₁₅), and GGDP for diterpene (C₂₀) [16]. Moreover, FDP and GGDP can be dimerized to form the precursors of C₃₀ and C₄₀. The final step converts terpenes into different skeletons by oxidation, reduction, isomerization, conjugation, and other transformation [53]. TPSs are the key enzymes in terpenoid biosynthesis.

Figure 1. The MVA (left) and MEP (right) pathways responsible for IPP and DMAPP biosynthesis and monoterpene biosynthesis in plants. AACT, acetoacetyl-CoA thiolase; CMK, 4-(cytidine 5′ -diphospho)-2-C-methyl-d-erythritol kinase; DMAPP, dimethylallyl diphosphate; DXR, 1-deoxy-d-xylulose 5-phosphate reductoisomerase; DXS, 1-deoxyd- xylulose 5-phosphate synthase; FDP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; G3P, d-glyceraldehyde 3-phosphate; GDPS, geranyl diphosphate synthase; GDP, geranyl diphosphate; HDR, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; HMGS, 3-hydroxy-3-methylglutaryl- CoA synthase; IDI, isopentenyl diphosphate isomerase; IPP, isopentenyl diphosphate; MCT, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase; MDD, mevalonate diphosphate decarboxylase; MDS, 2-C-methyld-erythritol 2,4-cyclodiphosphate synthase; MVK, mevalonate kinase; MVAP, mevalonate 5-phosphate; MVAPP, mevalonate diphosphate; PMK, phosphomevalonate kinase; TPS, terpene synthase.

3. The Evolution of TPS Genes in Orchidaceae Species

We chose the whole genome sequences of four orchids, including A. shenzhenica [54] in Apostasioideae subfamily; Vanilla planifolia [55] in Vanilloideae subfamily; and D. catenatum [56] and P. equestris [57] in Epidendroideae subfamily. There were two justifications for this selection. First, these four orchids are distributed into three different subfamilies, and their whole genome sequences are available in NCBI (https://www.ncbi.nlm.nih.gov/ (accessed on 6 January 2021).) and OrchidBase database [58] (http://orchidbase.itps.ncku.edu.tw/est/home2012.aspx (accessed on 9 August 2020).). Second, A. ashenzhenica is the most original orchid, and P. equestris is the first whole genome sequenced orchid. V. planifolia produces vanillin and is important in the food industry, and D. catenatum is a medicinal orchid and produces important secondary metabolites for pharmaceutical purpose. We isolated the TPS genes of Orchidaceae through KAAS (http://www.genome.jp/tools/kaas/ (accessed on 21 February 2017).) annotation and BLASTp from the whole genome sequences of four orchids. Each full-length TPS is characterized by two conserved domains with Pfam [59] ID PF01397 (N-terminal) and PF03936 (C-terminal) [17]. A total of 9, 27, 35, and 15 TPS genes were identified from the whole genome sequences of A. shenzhenica, V. planifolia, D. catenatum, and P. equestris, respectively. In addition, P. aphrodite with white, scentless flowers and P. bellina scented flowers are native species. Their floral transcriptomes are available in Orchidstra and OrchidBase transcriptome database, respectively. 17 TPS genes in P. aphrodite and 11 TPS genes in P. bellina were identified from the transcriptome database. The TPS genes were denoted with numbers Ash-, KAG-, Dca-, Peq-, PATC-, and PbTPS- identified from A. shenzhenica, V. planifolia, D. catenatum, P. equestris, P. aphrodite, and P. bellina, respectively.

TPSs in P. equestris and D. officinale have been reported [15,60]. These TPSs are divided into four subfamilies (TPS-a, TPS-b, TPS-c, and TPS-e/f). So, we further investigated TPS evolution in Orchidaceae and provided insight into TPSs at the genome level. In this review, the encoded amino acid sequences of identified orchid TPS genes were aligned with those from Arabidopsis and Abies grandis, and those from Selaginella moellendorffii were used as outgroups (Appendix A Table A1). The phylogenetic tree was constructed using Neighbor-Joining method with Jones–Taylor–Thornton model and pairwise deletion with 1000 bootstrap replicates by using MEGA7 software. The orchid TPSs are grouped into TPS-a, -b, -e/f, and g subfamilies (Figure 2). Most of the orchid TPSs belong to TPS-a and TPS-b subfamilies (89/115, Table 1). In the TPS-a subfamily, copies from dicot and monocot species formed distinct subgroups, which is in accordance to previous studies [15,16]. However, compared to angiosperm dicot species, which have more TPSs in TPS-a subfamily, orchid (monocot) TPSs have more members in TPS-b subfamily than in TPS-a subfamily. Within TPS-b subfamily, these orchid TPSs form distinct clades separated from those of Arabidopsis (dicot) TPSs (Figure 2). Taken together, the persistence of dicot and monocot distinct clades within TPS-a and TPS-b implies that these TPSs have diverged since the ancestor of angiosperm. On the other hand, most of the duplicated orchid TPS-a and TPS-b copies were species-dependent (i.e., paralogs duplicated within each species). In particular, the number of duplicated orchid TPS-a and TPS-b copies increased in V. planifolia and D. catenatum (Figure 2). These data suggest that TPS-a and TPS-b copies evolved in a species-dependent manner and may have been positively selected to generate exceptionally more multiple copies. TPS-a and TPS-b are angiosperm-specific subfamilies that are responsible for sesquiterpene or diterpene and monoterpene synthases. These orchid volatile terpenes have critical roles in producing floral scents in order to be attractive to pollinators and to respond to environmental stresses [15]. It is therefore not surprising that TPS-a and TPS-b subfamilies have diverged greatly in orchid species.

Figure 2. Phylogenetic analysis of terpene synthases. TPSs in Orchidaceae, including A. shenzhenica; V. planifolia; D. catenatuml P. equestris Phalaenopsis aphrodite; P. bellina, Arabidopsis thaliana, and Abies grandis; and S. moellendorffii were used. Sequence analysis was performed using MEGA 7.0 to create a tree using the nearest neighbor-joining method. The coding sequence was used for analysis. The numbers at each node represent the bootstrap values. Various colors mean distinct subfamilies and special symbols represent different plant species, with solid circles, tangle, diamond, and triangle illustrating Orchidaceae, Arabidopsis thaliana, A. grandis, and S. moellendorffii, respectively.

Table 1. The number of TPSs subfamilies in Orchidaceae and other plant species.

	TPS Subfamily
Species	a	b	c	d	e/f	g	h	Total	Reference
Apostasia shenzhenica	2	4	0	0	1	2	0	9	This research
Vallina planifolia	7	12	0	0	1	7	0	27	This research
Dendrobium catenatum	13	18	0	0	4	0	0	35	This research
Phalaenopsis equestris	4	7	0	0	4	0	0	15	This research
Phalaenopsis aphrodite	6	7	0	0	4	0	0	17	This research
Phalaenopsis bellina	1	7	0	0	3	0	0	11	This research
Arabidopsis thaliana	22	6	1	0	2	1	0	32	Aubourg et al. (2002) [6]
Solanum lycopersicum	12	8	2	0	5	2	0	29	Falara et al. (2011) [10]
Oryza sativa	18	0	3	0	9	2	0	32	Chen et al. (2014) [14]
Sorghum bicolor	15	2	1	0	3	3	0	24	Paterson et al. (2009) [8]
Vitis vinifera	30	19	2	0	1	17	0	69	Martin et al. (2010) [9]
Populus trichocarpa	16	14	2	0	3	3	0	38	Irmisch et al., (2014) [13]
Selaginella moellendorffii	0	0	3	0	3	0	8	14	Li et al., (2012) [11]

Our phylogenetic analysis also reveals that the orchid TPS-e/f subfamily has increased copy numbers compared to that from A. thaliana (Table 1; Figure 2). Orchid TPS-g subfamily can only be found in A. shenzhenica and V. planifolia (Table 1; Figure 2), whereas those Epidendroideae TPS-g members have perhaps been lost during evolution. There are no orchid TPSs in TPS-c group that host copalyl diphosphate synthases (CPS) of angiosperm [61]. TPS-d and TPS-h are gymnosperm and Selaginella moellendorffii specific, respectively [16]. Our analysis showed that no orchid TPSs were grouped in these subfamilies, in accordance with previous conclusions by Chen et.al, and Trapp et.al. [16,62].

Motifs of identified orchid TPS proteins were predicted using MEME software (https://meme-suite.org/meme/tools/meme (accessed on 19 March 2021).) (Figure 3A), and five major functional conserved motifs of TPSs (R(R)X₈W, EDXXD, RXR, DDXXD, and NSE/DTE) were elucidated (Figure 3B). The TPS-a subfamily that encodes STPSs is mainly found in both dicot and monocot plants [9,11,16,63]. In this subfamily, STPSs contain the non-conserved secondary “R” (arginine) of motif R(R)X₈W that functions in the initiation of the isomerization cyclization reaction [64], or in stabilizing the protein through electrostatic interactions [65]. Compared with Arabidopsis, most orchid TPSs contain motif R(R)X₈W, except PATC144727, Peq011664, Dca017107, and PATC155674 in TPS-a subfamily (Figure 4A). In contrast, the angiosperm-specific TPS-b subfamily that encodes MTPSs contains the highly conserved R(R)X₈W motif. All TPSs in Arabidopsis TPS-b subfamily contain conserved R(R)X₈W motif, except AtTPS02 (Figure 4B). However, several members of orchid TPS-b subfamily have lost the conserved R(R)X₈W motif (Figure 4B). Motifs EDXXD, RXR, DDXXD, and NSE/DTE are highly conserved in TPS-a and -b subfamilies, while the conserved R(R)X₈W motif of orchid TPSs is divergent in TPS-b subfamily.

Figure 3. The amino acid sequences of the predicted motifs in TPS proteins. (A) Twenty-five classical motifs in TPS proteins were analyzed using the MEME tool. The width of each motif ranges from 6 to 50 amino acids. The font size represents the strength of conservation. (B) The amino acid sequences of five highly conserved motifs in TPS proteins.

Figure 4. Motif structures of TPS proteins. (A–D) are TPS-a, -b, -e/f, and -g subfamilies, respectively. Twenty-five classical motifs in TPS proteins were analyzed by using the MEME tool. The width of each motif ranged from 6 to 50 amino acids. Different color blocks represent distinct motifs. Star indicates TPSs of A. shenzhenica, and the red solid circle indicates the out group of Apostasia TPSs. The red and blue rectangle squares reveal orthologous and paralogous gene pairs, respectively.

DTPSs are evolved from kaurene synthase (KS) and CPS. MTPSs and STPSs are evolved from ancestral DTPS through duplication and then sub- or neo-functionalization during evolution [66]. A. shenzhenica has clear evidence of whole-genome duplication that is shared by all orchids [54]. Yet, the copies of TPS in A. shenzhenica are among the fewest and are worthwhile for further investigation. For Phalaenopsis orchids, paralogs of TPS genes could be identified from each species, implying the duplications were attributed to their common ancestor, and some persisted or lost in current species (Figure 4). For example, TPS-a copies of P. aphrodite, P. bellina, and P. equestris species can be found (some lost) in three parallel clades of the phylogenetic tree (PATC144727/Peq010211/PbTPS02, PATC137979/Peq021360, and PATC175129/Peq011667) (red tangle, Figure 4A). Similarly, TPS-b copies of P. aphrodite, P. bellina, and P. equestris can be repeatedly identified (some lost) in eight parallel clades, indicating the TPS-b gene copy duplications could be traced back to the common ancestor of Phalaenopsis species (PATC208458/Peq006283, PATC153230/PbTPS09, PATC150554/Peq006282, Peq006285/PbTPS07, Peq006275/PbTPS10, PATC127710/Peq013713, PATC068781/Peq013045 and PATC187424/Peq013048) (red tangle, Figure 4B).

Members of TPS-e/f subfamilies are mainly detected in angiosperm and conifers DTPSs of primary metabolism (i.e., gibberellin biosynthesis) [16,67]. Orchid TPS-e/f subfamilies comprise orthologous genes without R(R)X₈W (Figure 4C), which are consistent with Arabidopsis. The Ash009730 in TPS-e/f subfamily, predicted to be KS, was grouped with KAG0503701 and Dca000690 (red retangle with red star, Figure 4C). No TPSs were found in A. shenzhenica in TPS-f subclade. As copies of these orchid TPS-e/f subfamilies were duplicated within each species, the duplications seem to be species dependent.

TPS-g subfamily is closely related to the TPS-b but lacks the N-terminal “R(R)X₈W” motif and encodes MTPSs, STPSs, and DTPSs that produce mainly acyclic terpenoids [68,69]. A highly conserved arginine-rich RXR motif of sesquiterpene synthase reported that the motif is involved in producing a complex with the diphosphate group after the ionization of FPP in sesquiterpene biosynthesis [70]. TPS-g subfamily in Arabidopsis (AtTPS14) lacks both “R(R)X₈W” and “RXR” motifs. However, although TPSs of V. planifolia in TPS-g subfamily (those started with KAG in Figure 4D) lack the N-terminal “R(R)X₈W” motif, they still have the “RXR” motif (Figure 4D). This suggests that TPS-g subfamily of V. planifolia may have conserved enzyme activities that are capable of accepting a multi-substrate in terpene biosynthesis.

The pharmaceutical effective compounds in D. catenatum, a widely used Chinese herb, belong to terpenoid indole alkaloid (TIA) class [71], and many of them contain a terpene group. A sesquiterpene alkaloid-Dendrobine found in Dendrobium is believed to be responsible for its medical property [71]. Concomitantly, a significant increased number of TPS-a TPSs was detected in D. catenatumas as compared to that of other orchid species, which is responsible for sesquiterpene biosynthesis (Table 1). The increased number of TPS-b in Dendrobium may cause the floral fragrance in D. catenatum as well as the formation of TIA. P. bellina is a scented orchid with the main floral compounds of monoterpenes including linalool, geraniol, and their derivatives, which attract pollinators [48]. PbTPSs from the floral transcriptome database are majorly classified into the TPS-b subfamily (Table 1). Previously, the expression of both PbTPS5 and PbTPS10 were concomitant with the VOCs (monoterpene linalool and geraniol) emission in P. bellina [72]. This suggests that these genes may be involved in the biosynthesis of monoterpene in P. bellina. TPS-e/f enzymes have diverse functions, including linalool synthase, geranyllinalool synthase, and farnesene synthase in kiwifruit [73,74]. TPSs in the TPS-e/f subfamily are thought to be dicot-specific because so far no TPS-e/f activity has been reported in monocots. However, the number of TPS in TPS-e/f expands from 1 in Apostasia to 4 in Phalaenopsis (Table 1), suggesting that the duplication events of TPS- b and TPS-e/f have evolved in response to natural selection.

Together, our analyses suggest that orchid TPSs in each subfamily evolved from the early divergence orchid species, such as A. shenzhenica and/or V. planifolia. The large expansion of TPS copies in orchid groups such as V. planifolia, D. catenatum, and Phalaenopsis species might be due to high flexibility for adaptation and evolution through natural selection.

4. The Arrangement of TPS

The functional cluster phenomenon of TPS genes was detected in orchids. Orchid TPS gene clusters diverged with tandem or segmental duplications (Figure 5). Tandem duplication inferred that the duplication occurred in the same scaffold, such as Ash012495 grouped with Dca000691/Dca000692/Dca000697 cluster genes in TPS-b subfamily (Figure 4B and Figure 5C). TPS genes duplicated on different scaffolds is thought to be segmental duplication, e.x.: Ash008718/Ash008719 grouped with two cluster genes of V. planifolia (KAG0458420/KAG0458425/KAG0458429 and KAG0460140/KAG0460156/KAG0460160) in different scaffolds in the TPS-g subfamily (Figure 4D and Figure 5A,B). We identified that 6, 24, 20, and 8 TPSs in A. shenzhenica, V. planifolia, D. catenatum, and P. equestris, respectively, form clusters in the same genome scaffold (Table 2, Figure 5A–D). In addition, these clusters were present with TPSs of the same subfamily and therefore the enhancement of functions was predicted. In A. shenzhenica, V. planifolia, D. catenatum, and P. equestris, TPS genes have three, nine, eight, and three clusters, respectively (Table 2, Figure 5). Each cluster contains two TPS genes in A. shenzhenica, while more genes are present in the clusters of V. planifolia, D. catenatum, and P. equestris (Figure 4). TPS genes in the same cluster usually belong to the same subfamily except that V. planifolia has one large scaffold containing TPS genes of TPS-a, TPS-b, and TPS-e/f subfamilies, yet with huge distance between each subfamily cluster (44 Mb and 5 Mb, respectively). The percentages of clustered TPS genes were 66.7%, 81.5%, 57.1%, and 53.3% for A. shenzhenica, V. planifolia, D. catenatum, and P. equestris, respectively, while that was 40.6% in Arabidopsis thaliana (Table 2). The cluster density of orchid TPSs could infer the event of TPS gene duplication occurred during evolution. The genome sizes of A. shenzhenica, V. planifolia, D. catenatum, and P. equestris are 349 Mb, 7449 Mb, 1104 Mb, and 1064 Mb, respectively (Table 3). The cluster densities of TPSs in orchids were 47.3%, 78.6%, 50.5%, and 38.9% for A. shenzhenica, V. planifolia, D. catenatum, and P. equestris, respectively (Table 3). Interestingly, orchids have more clusters and higher TPS gene density as compared to that of Arabidopsis, with that of V. planifolia having the highest cluster gene density of TPS among the four orchids analyzed. Even though TPSs copies of derived orchids (D. catenatum and Phalaenopsis spp.) were increased compared with those in A. shenzhenica, the total number was not linked to the increased genome size.

Figure 5. Gene clusters in Orchidaceae genome. Clustered genes in the genomic scaffolds of A. shenzhenica (A), V. planifolia (B), D. catenatum (C), and P. equestris (D), respectively. The TPS genes located on the scaffolds are identified from the assembled whole genome sequences of A. shenzhenica, V. planifolia, D. catenatum, and P. equestris. The direction of arrows illustrates the forward translation of genes in the scaffolds. Various colors indicate the distinct TPS subfamilies. Blue, green, purple, and bisque colors represent TPS genes in TPS-a, -b, -e/f, and -g subfamilies, respectively. Break lines indicate the shrink length of genes.

Table 2. The gene clusters of TPSs in the genome of Orchidaceae and Arabidopsis thaliana.

Species	Number of Clusters	Number of Scaffolds	Number of Clustered TPSs	Number of Total TPSs	Percentage of Clustered TPSs (%)
Apostasia shenzhenica	3	3	6	9	66.7
Vallina planifolia	7	5	22	27	81.5
Dendrobium catenatum	8	7	20	35	57.1
Phalaenopsis equestris	3	3	8	15	53.3
Arabidopsis thaliana [6]	5	5	13	32	40.6

Table 3. The gene density of TPSs in the genome of Orchidaceae and other plant species.

Species	Genome Size (Mb)	Cluster Length of TPSs (Kb)	Total Length of TPSs (Kb)	Cluster Density of TPSs (%)
Apostasia shenzhenica	349	26	56	47.3
Vallina planifolia	744	595	758	78.6
Dendrobium catenatum	1104	125	248	50.5
Phalaenopsis equestris	1064	62	158	38.9
Arabidopsis thaliana	120	43	109	39.9

In plants, gene clusters were often observed for metabolic pathways, such as gene clusters found in oat and Arabidopsis related to triterpene biosynthesis pathway [75]. Local duplication of TPS gene families in plants has been described and often results in tandem repeats, as an important driver for the expansion [16,76]. The genes related in terpene synthesis are usually lined together, forming functional clusters in plants [77]. The functional clusters of TPS genes have already been reported in several plant species, such as Arabidopsis thaliana [6], Vitis vinifera [9], Solanum lycopersicum [77], Eucalpyus grandis [78], and rice [79,80]. Genomic clusters of TPS genes in E. grandis are up to 20 genes [78]. In several Solanum species, the gene duplications and divergence give rise to TPS gene clusters for terpene biosynthesis [77]. A dense cluster of 45 V. vinifera TPSs are present on chromosome 18 [9]. Arabidopsis TPS genes are reported with the phenomenon of several gene clusters [6]. In addition, a gene cluster with three TPS members, including Os08g07080, Os08g07100, and Os08g07120, is observed in Asian rice Oryza sativa and also appears in various rice species including O. glaberrima, O. rufipogon, O. nivara, O. barthii, and O. punctata. [80]. Both conserved and species-specific expression patterns of the clustered rice TPSs indicate the functions in insect-damaged plants [80]. The expression of these rice TPS genes and their catalytic activities for emission patterns of volatile terpenes is induced by insect damage and is largely consistent [80]. Interestingly, the evolution of TPSs with other biosynthesis-related genes was also found to form unexpected connection with time passed. For instance, the evolution of TPS/CYP pairs is different in monocot and dicot [81]. TPS/CYP pairs duplicate with ancestral TPS/CYP pairs as templates to be evolved in dicots, but the evolutionary mechanism of monocot shows that the genome rearrangement of TPS and CYP occurred independently [81]. In Solanum spp., TPS forms functional clusters with cis-prenyl transferase [77]. Both tandem and segmental duplications significantly contribute toward family expansion and expression divergence and play important roles in the survival of these expanded genes. A functional gene cluster is a group of closely-related genes lined together in a genome, and the study of gene clusters is important for the understanding of evolution within species.

Together, the orchid TPS genes formed genomic clusters, and the clusters increased in V. planifolia and D. catenatum. Combining the results from phylogenetic analysis and functional gene clusters, orchid TPSs may be expanded by tandem or segmental duplications. Interestingly, the genome duplication events occurred all the way along the evolution from Apostasioideae to Vanilloideae and Epidendroideae; the TPS clusters and copy numbers increased in orchid lineages, such as the early divergence A. shenzhenica. The large expansion of orchid TPS copies in V. planifolia, and D. catenatum species might have high flexibility in secondary biosynthesis through natural selection.

5. Conclusions

The basic evolution of TPS is from duplication and loss of TPS genes. In Orchidaceae, we discover that the duplication event of TPS occurred among all TPS subfamilies. TPs-a, TPS-b, and TPS-e/f subfamilies went through gene duplication, while TPS-g duplicated from Apostaceae to Vaniloideae, and then lost from Vaniloideae to Epidendroideae. The driving force of TPS evolution in each subfamily may be different. For example, in TPS-a and TPS-b, the necessity of generating volatile compounds for the interaction of orchids with their pollinators, producing chemical defenses and being responsive to environmental stress, may be the major reason for their rapid evolution. On the other hand, the duplications of TPS-g and TPS-e/f copies were mainly species dependent and the reason remains to be uncovered.