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Review

Evolution of Terpene Synthases in Orchidaceae

by
Li-Min Huang
1,
Hsin Huang
1,
Yu-Chen Chuang
1,
Wen-Huei Chen
1,2,
Chun-Neng Wang
3 and
Hong-Hwa Chen
1,2,*
1
Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan
2
Orchid Research and Development Center, National Cheng Kung University, Tainan 701, Taiwan
3
Department of Life Sciences, Institute of Ecology and Evolutionary Biology, National Taiwan University, Taipei 106, Taiwan
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(13), 6947; https://doi.org/10.3390/ijms22136947
Submission received: 2 April 2021 / Revised: 22 June 2021 / Accepted: 23 June 2021 / Published: 28 June 2021
(This article belongs to the Special Issue Orchid Biochemistry 2.0)
Browse Figures
Review Reports Versions Notes

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.

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.
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.
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.
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.

Author Contributions

L.-M.H. performed the phylogenetic analysis and motif prediction of TPSs; H.H. performed the gene arrangement analysis; Y.-C.C. performed the identification of orchid TPSs; W.-H.C. provided the suggestions for plant materials.; C.-N.W. provided discussion and composed the TPSs evolution; H.-H.C. conceived research plans and composed the article with assistances of all the authors, completed the writing, and served as the corresponding author for communication. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a grant from Ministry of Science and Technology, Taiwan (MOST 107-2313-B-006-003-MY3) to H.-H.C.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank the people that finished the whole genome sequence of the four orchid species, which allowed us to undertake this detail analysis.

Conflicts of Interest

No conflict of interest declared.

Appendix A

Table
Table A1. TPS genes used in phylogenetic analysis.
Table A1. TPS genes used in phylogenetic analysis.
SpeciesGene IDAccession Number of TPS Gene
Apostasia shenzhenica1Ash001768Ash001768
Ash001833Ash001833
Ash008718Ash008718
Ash008719Ash008719
Ash009730Ash009730
Ash010478Ash010478
Ash010480Ash010480
Ash012495Ash012495
Ash013718Ash013718
Vallina planifolia2KAG0449176KAG0449176
KAG0451042KAG0451042
KAG0451129KAG0451129
KAG0454496KAG0454496
KAG0454501KAG0454501
KAG0455064KAG0455064
KAG0455066KAG0455066
KAG0455553KAG0455553
KAG0455554KAG0455554
KAG0455713KAG0455713
KAG0455723KAG0455723
KAG0455730KAG0455730
KAG0456208KAG0456208
KAG0456209KAG0456209
KAG0456210KAG0456210
KAG0458420KAG0458420
KAG0458425KAG0458425
KAG0458429KAG0458429
KAG0460139KAG0460139
KAG0460140KAG0460140
KAG0460156KAG0460156
KAG0460160KAG0460160
KAG0496777KAG0496777
KAG0499157KAG0499157
KAG0501224KAG0501224
KAG0503399KAG0503399
KAG0503701KAG0503701
Dendrobium catenatum1Dca000690Dca000690
Dca000691Dca000691
Dca000692Dca000692
Dca000695Dca000695
Dca002950Dca002950
Dca002952Dca002952
Dca002953Dca002953
Dca003097Dca003097
Dca003101Dca003101
Dca004857Dca004857
Dca007288Dca007288
Dca007289Dca007289
Dca007806Dca007806
Dca010119Dca010119
Dca010463Dca010463
Dca010464Dca010464
Dca012868Dca012868
Dca012869Dca012869
Dca012871Dca012871
Dca013925Dca013925
Dca015828Dca015828
Dca016792Dca016792
Dca016793Dca016793
Dca017192Dca017192
Dca017693Dca017693
Dca018107Dca018107
Dca018109Dca018109
Dca019472Dca019472
Dca021138Dca021138
Dca021204Dca021204
Dca023162Dca023162
Dca023936Dca023936
Dca024570Dca024570
Dca024748Dca024748
Dca025036Dca025036
Phalaenopsis aphrodite3PATC043551PATC043551
PATC068781PATC068781
PATC127710PATC127710
PATC133907PATC133907
PATC137979PATC137979
PATC139978PATC139978
PATC141250PATC141250
PATC144727PATC144727
PATC150554PATC150554
PATC153230PATC153230
PATC155674PATC155674
PATC161091PATC161091
PATC175129PATC175129
PATC183449PATC183449
PATC187424PATC187424
PATC200022PATC200022
PATC208458PATC208458
Phalaenopsis equestris1Peq006275Peq006275
Peq006282Peq006282
Peq006283Peq006283
Peq006285Peq006285
Peq010211Peq010211
Peq011221Peq011221
Peq011664Peq011664
Peq011667Peq011667
Peq013045Peq013045
Peq013048Peq013048
Peq013713Peq013713
Peq020239Peq020239
Peq020483Peq020483
Peq021360Peq021360
Peq023325Peq023325
Phalaenopsis bellina4PbTPS01CL86.Contig1
PbTPS02CL214.Contig2
PbTPS03CL376.Contig6
PbTPS04CL376.Contig8
PbTPS05CL1323.Contig1
PbTPS06CL2295.Contig2
PbTPS07CL2800.Contig3
PbTPS08CL4514.Contig2
PbTPS09CL6288.Contig1
PbTPS10CL6288.Contig7
PbTPS11Unigene4722
Arabidopsis thaliana2AtTPS1At4g15870
AtTPS2At4g16730
AtTPS3At4g16740
AtTPS4At1g61120
AtTPS5At4g20230
AtTPS6At1g70080
AtTPS7At4g20200
AtTPS8At4g20210
AtTPS9At2g23230
AtTPS10At2g24210
AtTPS11At5g44630
AtTPS12At4g13280
AtTPS13At4g13300
AtTPS14At1g61680
AtTPS15At3g29190
AtTPS16At3g29110
AtTPS17At3g14490
AtTPS18At3g14520
AtTPS19At3g14540
AtTPS20At5g48110
AtTPS21At5g23960
AtTPS22At1g33750
AtTPS23At3g25830
AtTPS24At3g25810
AtTPS25At3g29410
AtTPS26At1g66020
AtTPS27At1g48820
AtTPS28At1g48800
AtTPS29At1g31950
AtTPS30At3g32030
AtTPS31At4g02780
AtTPS32At1g79460
Abies grandis2AAB70707AGU87910
AAB70907AF006193
AAB71085U87909
AAF61454AF139206
Selaginella moellendorffii2EFJ31965GL377573
EFJ37889GL377565
J9QS23_SmTPS9XM_002960304
|J9R388_SmTPS10XM_024672072
G9MAN7_SmTPS4XM_024672355.
G1DGI7_SmTPS7XM_024689660
EFJ12417GL377639
EFJ37773GL377565
EFJ33476GL377571
1 OrchidBase 4.0 (http://orchidbase.itps.ncku.edu.tw/est/home2012.aspx (accessed on 9 August 2020)). 2 NCBI database (https://www.ncbi.nlm.nih.gov/ (accessed on 9 August 2020). 3 Orchidstra 2.0 (http://orchidstra2.abrc.sinica.edu.tw/orchidstra2/index.php (accessed on 5 January 2021). 4 P. bellina trascriptome database (unpublished).

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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.
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.
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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.
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.
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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 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.
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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.
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.
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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.
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.
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Table
Table 1. The number of TPSs subfamilies in Orchidaceae and other plant species.
Table 1. The number of TPSs subfamilies in Orchidaceae and other plant species.
TPS Subfamily
Speciesabcde/fghTotalReference
Apostasia shenzhenica24001209This research
Vallina planifolia7120017027This research
Dendrobium catenatum13180040035This research
Phalaenopsis equestris470040015This research
Phalaenopsis aphrodite670040017This research
Phalaenopsis bellina170030011This research
Arabidopsis thaliana2261021032Aubourg et al. (2002) [6]
Solanum lycopersicum1282052029Falara et al. (2011) [10]
Oryza sativa1803092032Chen et al. (2014) [14]
Sorghum bicolor1521033024Paterson et al. (2009) [8]
Vitis vinifera301920117069Martin et al. (2010) [9]
Populus trichocarpa16142033038Irmisch et al., (2014) [13]
Selaginella moellendorffii003030814Li et al., (2012) [11]
Table
Table 2. The gene clusters of TPSs in the genome of Orchidaceae and Arabidopsis thaliana.
Table 2. The gene clusters of TPSs in the genome of Orchidaceae and Arabidopsis thaliana.
SpeciesNumber of ClustersNumber of ScaffoldsNumber of Clustered TPSsNumber of Total TPSsPercentage of Clustered TPSs (%)
Apostasia shenzhenica336966.7
Vallina planifolia75222781.5
Dendrobium catenatum87203557.1
Phalaenopsis equestris3381553.3
Arabidopsis thaliana [6]55133240.6
Table
Table 3. The gene density of TPSs in the genome of Orchidaceae and other plant species.
Table 3. The gene density of TPSs in the genome of Orchidaceae and other plant species.
SpeciesGenome Size (Mb)Cluster Length of TPSs (Kb)Total Length of TPSs (Kb)Cluster Density of TPSs (%)
Apostasia shenzhenica349265647.3
Vallina planifolia74459575878.6
Dendrobium catenatum110412524850.5
Phalaenopsis equestris10646215838.9
Arabidopsis thaliana1204310939.9
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Huang, L.-M.; Huang, H.; Chuang, Y.-C.; Chen, W.-H.; Wang, C.-N.; Chen, H.-H. Evolution of Terpene Synthases in Orchidaceae. Int. J. Mol. Sci. 2021, 22, 6947. https://doi.org/10.3390/ijms22136947

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Huang L-M, Huang H, Chuang Y-C, Chen W-H, Wang C-N, Chen H-H. Evolution of Terpene Synthases in Orchidaceae. International Journal of Molecular Sciences. 2021; 22(13):6947. https://doi.org/10.3390/ijms22136947

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Huang, Li-Min, Hsin Huang, Yu-Chen Chuang, Wen-Huei Chen, Chun-Neng Wang, and Hong-Hwa Chen. 2021. "Evolution of Terpene Synthases in Orchidaceae" International Journal of Molecular Sciences 22, no. 13: 6947. https://doi.org/10.3390/ijms22136947

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