Functions of TPS genes
The enzyme encoded by the P. patens PpCPS/KS gene catalyzes the formation of ent-kaurene and 16-hydroxy-ent-kaurene (Hayashi et al., 2006). ent-Kaurene is a precursor to ent-kaurenoic acid, which in P. patens serves the general physiological role in development that the gibberellins play in angiosperms (Hayashi et al., 2010). A specialized ecological role for PpCPS/KS is not presently known; however, the 16-hydroxy-ent-kaurene product of PpCPS/KS accumulates in P. patens with substantial concentrations of up to 0.5–1 mm and is also being released as a volatile compound (von Schwartzenberg et al., 2004). These levels of accumulation and release might be indicative of a role of 16-hydroxy-ent-kaurene in interactions with other organisms, but this hypothesis remains to be tested. No terpenes have yet been reported in S. moellendorffii, so possible ecological roles for TPS genes in this lycophyte species are not yet known either. In contrast, the ecological roles of terpenes in gymnosperms and angiosperms are many (Gershenzon and Dudareva, 2007), and much has been learned about the genes and enzymes that specify their production. In reviewing the information on the function of the TPS genes in the model systems discussed here, it is worth noting that the gymnosperm species studied are extremely long-lived trees with wind-pollinated cones, the two monocot angiosperm species, rice and sorghum, are grasses whose flowers are wind-pollinated, and among the three dicot angiosperm species, Arabidopsis is a small annual with scented flowers, poplar is a tree with wind-pollinated flowers, and grapevine is a cultivated perennial plant with scented flowers and highly aromatic fruits.
In gymnosperms, the largest number of TPS genes of specialized metabolism has been identified and functionally characterized in species of spruce (Picea) and in grand fir (Abies grandis), with a few individual TPS genes identified and characterized in other species such as loblolly pine (Pinus taeda), Douglas fir (Pseudotsuga menziesii), Ginkgo biloba and Taxus (Keeling and Bohlmann, 2006a; Keeling et al., 2011). The interest in diterpene synthases from Ginkgo and Taxus is due to their importance for the biosynthesis of medicinally useful metabolites, of which the anticancer drug taxol is perhaps the most prominent example of a high value terpenoid (Croteau et al., 2006). For the exploration of the TPS gene family in conifers (order Coniferales), and in particular members of the pine family (Pinacea), the major driving force has been to understand the biochemical, molecular, genomic and evolutionary underpinnings of the great chemical diversity of terpenoids in conifer defense against insects and pathogens, and to apply this knowledge for tree breeding and forest health protection. Conifers produce large quantities of oleoresin composed of complex mixtures of dozens of different acyclic and cyclic monoterpenes, sesquiterpenes, and diterpenes, the latter predominantly in the form of diterpene resin acids (Keeling and Bohlmann, 2006a,b). In addition, conifers emit volatile terpenoids in response to insect attack (Miller et al., 2005; Mumm and Hilker, 2006). Oleoresin terpenoid defenses act directly against invading insects and pathogens, as well as larger herbivores, while induced volatile emissions may function in indirect defense to attract the natural enemies of the attacking herbivores. The size of the TPS gene family and the diversity of biochemical functions of TPSs in any one conifer species mirror the complexity of oleoresin composition and volatile emissions. Published reports describe functions for 10 different TPS genes in Norway spruce (P. abies) (Fäldt et al., 2003a; Martin et al., 2004) and 11 different TPS genes in grand fir (Vogel et al., 1996; Bohlmann et al., 1997, 1998b, 1999; Steele et al., 1998). A recent large-scale transcriptome analysis identified 69 TPS genes in white spruce (P. glauca), 55 TPS genes in Sitka spruce (P. sitchensis), and 20 TPS genes in a hybrid white spruce (P. glauca × P. engelmannii), of which 21 have been functionally characterized as mono-, sesqui- and diterpene synthases of specialized metabolism (Keeling et al., 2011).
The majority of the conifer TPSs are multi-product enzymes, and it was in grand fir that the phenomenon of highly multi-product TPS enzymes was first highlighted with the discovery of the sequiterpene synthase genes encoding δ-selinene synthase (34 products) and γ-humulene synthase (52 products) (Steele et al., 1998). However, a few single-product TPSs have also been described in conifers. In the case of the Norway spruce ditepene synthases levopimaradiene/abietadiene synthase (PaTPS-LAS) and isopimaradiene synthase (PaTPS-Iso), multi-product and single-product enzymes exist as closely related (>90% identical) paralogous TPSs in the same species (Martin et al., 2004). Reciprocal site-directed mutagenesis revealed that the functional bifurcation of these two enzymes is due to only four amino acid residues (Keeling et al., 2008), thus providing an example of TPS gene duplication and neofunctionalization and the plasticity of TPS function. Recent studies confirmed that a critical mutation in the isopimaradiene synthase leading to change of product profiles in site-directed mutagenesis also occurs as a natural TPS variation in Sitka spruce (Keeling et al., 2011). This example is perhaps representative of the many events of TPS gene duplication, followed by sub- and neofunctionalization that have shaped the functional diversity of large families of active TPS genes of specialized metabolism in conifers. In addition to the more than 30 functionally characterized TPS genes of specialized metabolism functionally characterized in spruce, primary metabolism CPS and KS genes have also been characterized in white spruce and in Sitka spruce, where these genes appear to be actively expressed as single-copy genes (Keeling et al., 2010).
Expression of many of the conifer TPS genes involved in oleoresin defenses and volatile emissions is induced by herbivore or pathogen attack, and this effect can be mimicked to some extend by mechanical wounding of trees or treatment with methyl jasmonate (Miller et al., 2005; Zulak et al., 2009). Analysis of TPSs at the proteome and transcriptome levels in Norway spruce and Sitka spruce showed that methyl jasmonate-induced TPS transcription lead to increased protein abundance and enzyme activity and correlated with changes in the terpenoid metabolite profiles (Zulak et al., 2009; Hall et al., 2011). Spatially, the constitutive and induced expression of TPS genes and proteins in spruce is localized, at least in part, to the epithelial cells of resin ducts in stem tissues as determined by immunofluorescence localization (Zulak et al., 2010) and laser-capture microdissection (Abbott et al., 2010). In most conifers, constitutive and induced traumatic resin ducts are the primary site of massive accumulation of TPS products. Most of the products of mono- and sesquiterpene synthase activity accumulate without further modification. In contrast, most of the products of the conifer diterpene synthases are oxidized to diterpene resin acids by the activity of CYP720B cytochrome P450 enzymes, before being sequestered into resin ducts (Ro et al., 2005).
In Arabidopsis, 14 TPS genes have been functionally characterized, which include two diterpene synthase genes that encode CPS and KS respectively for the biosynthesis of gibberellins (Sun and Kamiya, 1994; Yamaguchi et al., 1998) and 12 for the biosynthesis of specialized metabolites. Because of its comprehensive molecular and genetic resources, Arabidopsis has become a particularly useful model for in depth studies on the spatial organization and temporal regulation of terpene metabolism in relation to the various biological functions of terpenes. Also, comparisons between ecotype- and genus-specific genomes allow investigating basic molecular mechanisms that control the intra- and inter-specific natural variation of terpene biosynthesis. Volatile terpenoids are emitted from several Arabidopsis tissues under constitutive and/or induced conditions (van Poecke et al., 2001; Aharoni et al., 2003; Chen et al., 2003; Fäldt et al., 2003b; Rohloff and Bones, 2005; Snoeren et al., 2010).
Most of the terpenoids detected so far in Arabidopsis are synthesized by approximately one-third of the enzymes of the Arabidopsis TPS family, with the enzymatic activities and products of the others yet to be determined. Microarray and RT-PCR analyses have indicated distinct or overlapping tissue-specificity of AtTPS gene expression, which has been investigated in greater detail for 10 genes (Figure 3). Flowers of the Arabidopsis ecotype Columbia (Col) emit a complex mixture of monoterpenes and over 20 sesquiterpene hydrocarbons with (E)-β-caryophyllene as the predominant compound (Chen et al., 2003; Tholl et al., 2005). Nearly all of the sesquiterpenes are produced by two flower-specific enzymes, the (E)-β-caryophyllene/α-humulene synthase (At5g23960, AtTPS21) and the multi-product sesquiterpene synthase AtTPS11 (At5g44630), and the other terpene compounds are synthesized by four additional enzymes (At4g16740, AtTPS03; At2g24210, AtTPS10; At1g61680, AtTPS14; At3g25810, AtTPS24) (Figure 3a). None of the floral terpenes is synthesized in flower petals, instead their formation occurs particularly in the stigma and sepals (AtTPS21), intrafloral nectaries and ovules (AtTPS11), and in pollen (AtTPS03) (Chen et al., 2003; Tholl et al., 2005; Huang et al., 2010). The terpenes that are emitted from these floral tissues at comparatively low rates can serve multiple functions: they may attract small pollinating insects at short range that contribute to cross-fertilization in natural populations. More recently, floral volatiles have been associated with defensive activities (Raguso, 2009) and Arabidopsis floral terpenes are likely to exhibit such functions to specifically protect floral organs such as the stigma against pathogen invasion. Differences in life histories and breeding systems influence floral terpene volatile emissions in the genus Arabidopsis. For example, in the highly scented flowers of the outcrossing perennial A. lyrata, formation of (E)-β-caryophyllene is substituted by the biosynthesis of benzenoid volatiles but instead has evolved as an insect-inducible trait in leaves catalyzed by a TPS21 orthologue (Abel et al., 2009).
Figure 3. Tissue- and cell type-specific expression patterns of Arabidopsis TPS genes in flowers (a), roots (b), and leaves (c) according to transcript and/or promoter-reporter gene analyses. Numbers indicate different TPS genes. Arrows and dots in (b) indicate root-specific expression. Expression profiles of two root-specific TPS genes with predicted function as sesquiterpene and diterpene synthases are shown. In (c), herbivore-induced AtTPSs are shown. 3, AtTPS03, α-farnesene synthase; 4, AtTPS04, (E,E)-geranyllinalool synthase; 8, AtTPS08, putative diterpene synthase; 11, AtTPS11, multi-sesquiterpene synthase; 12 and 13, AtTPS12, AtTPS13, (Z)-γ-bisabolene synthases; 21, AtTPS21, (E)-β-caryophyllene synthase; 22, AtTPS22, putative sesquiterpene synthase; 24, AtTPS24, multi-product monoterpene synthase; 23 and 27, AtTPS23, AtTPS27, 1,8-cineole synthases.
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Similar defensive functions as suggested for flower-specific TPS genes are predicted for 14 AtTPS genes with primary or exclusive expression in Arabidopsis roots. Four of these genes have been functionally characterized to encode monoterpene and sesquiterpene synthases (Chen et al., 2004; Ro et al., 2005) and diterpene synthase or sesquiterpene synthase activities have been predicted for others. According to high resolution transcriptome maps (Birnbaum et al., 2003; Brady et al., 2007) and promoter-reporter gene studies, many root-expressed genes exhibit cell type- and root zone-specific expression patterns (Figure 3b) that may emerge in defense against soil-borne, root-attacking organisms with different feeding or infection strategies. For example, promoter-GUS analyses demonstrated that the identical, duplicated genes AtTPS23 and AtTPS27 (At3g25820 and At3g25830), which are responsible for the formation of the monoterpene 1,8-cineole (Chen et al., 2004), are expressed in the vascular tissue of the root elongation zone and in epidermal cells and root hairs of older root-growth zones (Chen et al., 2004) (Figure 3b), where 1,8-cineole can directly be released into the rhizophere. Similarly, the two (Z)-γ-bisabolene synthase genes AtTPS12 (At4g13280) and AtTPS13 (At4g13300) are expressed in the stele of younger roots and in the cortex and endodermis of mature roots (Ro et al., 2006) (Figure 3b). Both genes have emerged by gene duplication and are found in pairs together with the co-expressed P450 genes CYP71A19 and CYP71A20 suggesting that these gene pairs form cell type-specific biosynthetic modules.
Leaves of the Arabidopsis ecotype Col release a mixture of volatiles containing the C16-homoterpene TMTT, the sesquiterpene (E,E)-α-farnesene, and the monoterpene β-myrcene in an induced response to herbivory, elicitor treatment, and the application of jasmonic acid or jasmonate mimics (van Poecke et al., 2001; Chen et al., 2003; Herde et al., 2008; Huang et al., 2010; Snoeren et al., 2010). AtTPS04 (At1g61120) catalyzes the formation of (E,E)-geranyllinalool as the first dedicated step in the biosynthesis of TMTT (Herde et al., 2008; Lee et al., 2010), while (E,E)-α-farnesene and β-myrcene are produced by the (E,E)-α-farnesene synthase AtTPS03 and presumably the β-myrcene/ocimene synthase AtTPS10, respectively (Bohlmann et al., 2000; Huang et al., 2010). Both AtTPS03 and AtTPS04 genes are induced locally at wound sites (Herde et al., 2008; Huang et al., 2010) (Figure 3c). The herbivore-induced volatile mixture was shown to attract parasitoids of herbivores (van Poecke et al., 2001; Loivamäki et al., 2008) and contributes to increased plant fitness (van Loon et al., 2000). Parasitoid attraction varies depending on the volatile blend emitted from different Arabidopsis ecotypes (Snoeren et al., 2010). For example, the ecotypes Col and Ws differ in their emission of (E)-β-ocimene, and this difference is due to a mutation that inactivates the (E)-β-ocimene-producing AtTPS02 in the Col ecotype (Huang et al., 2010).
Although the genome sequence for Populus was published in 2006, the functions of only three poplar TPS cDNAs have been characterized so far, including two encoding isoprene synthase and one encoding a sesquiterpene synthase. All of these genes are involved in the formation of volatile terpenes. Genes encoding isoprene synthase have been isolated from P. alba × tremula (Miller et al., 2001) and P. alba (Sasaki et al., 2005). The proteins encoded by these two isoprene synthase genes are 99% identical, suggesting that they are variants of the same gene. The release of volatile isoprene, produced by isoprene synthase, from Populus accounts for a large amount of the biogenic carbon found in the atmosphere. It is believed that isoprene emission has a role in the ecological response of Populus to abiotic stress (Sharkey and Yeh, 2001; Behnke et al., 2007). A full-length cDNA (PtdTPS1) was isolated from P. trichocarpa × deltoides and shown to encode a sesquiterpene synthase responsible for the synthesis of insect-induced (−)-germacrene D (Arimura et al., 2004). The herbivore-induced local and systemic mono- and sequiterpenoid emissions produced by poplar TPS genes may serve functions in multitrophic defence (Arimura et al., 2004) and in within-plant signaling (Frost et al., 2008).
In contrast to the poplar system, a substantial effort has been made to comprehensively analyze the genome organization and functions of the TPS gene family in grapevine (Martin et al., 2010). Terpenoids, in the form of free volatiles and as glycoside conjugates of monoterpene alcohols, are among the most important flavor compounds of grape berries and wine bouquet (Swiegers et al., 2005; Lund and Bohlmann, 2006). In addition, grapevine flowers produce a terpenoid-rich floral scent (Martin et al., 2009). In total, 43 VvTPS FLcDNAs from several grapevine varieties have been functionally characterized, representing mostly monoterpene synthases and sesquiterpene synthases (Lücker et al., 2004; Martin and Bohlmann, 2004; Martin et al., 2010). The majority of the functionally characterized VvTPS are multi-product enzymes. The products of these TPSs account for many of the major and minor terpenoids of berry and wine flavor, including a suite of monoterpene alcohols. A functionally characterized sesquiterpene synthase from Cabernet Sauvignon (VvValCS) and Gewürztraminer (VvValGw) produces (–)-valencene and (–)-7-epi-α-selinene as the main products (Lücker et al., 2004; Martin et al., 2009). This TPS gene is expressed in anthers and developing pollen grains and contributes to diurnal floral scent emission (Martin et al., 2009).
The rice TPS family has also been relatively well studied. Rice plants produce and emit a mixture of volatile terpenoids, including monoterpenes and sesquiterpenes, after insect herbivory (Yuan et al., 2008). By correlating terpenoid emission and expression of TPS genes analyzed by microarrays, three TPS genes were identified to be responsible for the production of the majority of insect-induced volatile terpenoids in rice. One of these genes encodes a monoterpene synthase making linalool as the single product, which is the most abundant insect-induced volatile from nipponbare rice plants (Yuan et al., 2008). The other two characterized TPS genes encode sesquiterpene synthases. Both of them make multiple sesquiterpenoids (Yuan et al., 2008).
Rice plants also produce a large number of labdane type diterpenoids. The known rice diterpenoids fall into five structurally related families: gibberellins, phytocassanes A–E, oryzalexins A–F, momilactones A and B, and oryzalexin S, which are derived from ent-kaurene, ent-cassa-12,15-diene, ent-sandaracopimaradiene, syn-pimara-7,15-diene and syn-stemar-13-ene, respectively (Peters, 2006; Toyomasu, 2008). Three rice TPSs have been shown to function as CPS, including OsCPS1 involved in gibberellin biosynthesis, OsCPS2 involved in the biosynthesis of non-gibberellin diterpenoids (Prisic et al., 2004) and OsCPSsyn producing syn-CPP for the biosynthesis of syn-labdane-related diterpenoids (Xu et al., 2004). A number of rice TPSs that use ent-CPP or syn-CPP as the direct substrate have also been characterized. These include OsKS1, which is a bona fide KS involved in gibberellin biosynthesis to produce ent-kaurene (Sakamoto et al., 2004), OsKSL4 (OsDTS2) for producing syn-pimaradiene (Wilderman et al., 2004), OsKS5 for producing ent-pimara-8(14),15-diene (Kanno et al., 2006), OsKS6 for producing ent-kaur-15-ene (ent-isokaurene) (Kanno et al., 2006; Xu et al., 2007), OsKSL7 (OsDTC1) for producing ent-cassadiene (Cho et al., 2004), OsKSL8 (OsDTC2) for producing syn-stemarene (Nemoto et al., 2004), OsKSL10 for producing ent-sandaracopimaradiene (Otomo et al., 2004a), and OsKSL11 for producing stemod-13(17)-ene (Morrone et al., 2006). These non-gibberellin diterpenoids function as phytoalexins to defend rice plants against microbial infection (Kato et al., 1994). Some of these compounds are produced by the rice plants then released into the environment to inhibit the germination and growth of neighboring plants, therefore acting as allelochemicals (Kato-Noguchi and Ino, 2003).
In contrast to rice, little is known about terpenes and TPS genes in sorghum. A recent study found that like rice sorghum plants emit volatile terpenoids, predominated by sesquiterpenes, upon insect herbivory. Colinearity analysis based on the synteny between the rice and sorghum genomes led to the identification of sorghum TPS genes, which are the orthologues of key rice TPS genes for producing insect-induced volatile terpenoids (X. Zhuang and F. Chen, unpublished results). Although not included in the analysis in this review, maize has served as another important monocot model for investigating terpenoid metabolism, especially in the context of plant-insect interactions. The majority of the TPS genes isolated from maize are involved in making herbivory-induced volatile terpenoids (Schnee et al., 2002, 2006; Köllner et al., 2008, 2009). Transgenic studies using some of these maize TPS genes convincingly showed that their products have a role in attracting parasitoids (Schnee et al., 2006) and entomopathogenic nematodes (Degenhardt et al., 2009b), the natural enemies of maize herbivores.