Many angiosperm plants, including basal dicots, eudicots, and monocots, emit (
E,
E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, which is derived from geranyllinalool, in response to biotic challenge. An Arabidopsis (
Arabidopsis thaliana) geranyllinalool synthase (
GLS) belonging to the e/f clade of the terpene synthase (
TPS) family and two Fabaceae
GLSs that belong to the
TPS-g clade have been reported, making it unclear which is the main route to geranyllinalool in plants. We characterized a tomato (
Solanum lycopersicum)
TPS-e/f gene, TPS46, encoding
GLS (
SlGLS) and its homolog (
NaGLS) from
Nicotiana attenuata. The
Km value of SlGLS for geranylgeranyl diphosphate was 18.7 µ
m, with a turnover rate value of 6.85 s
–1. In leaves and flowers of
N. attenuata, which constitutively synthesize 17-hydroxygeranyllinalool glycosides,
NaGLS is expressed constitutively, but the gene can be induced in leaves with methyl jasmonate. In tomato,
SlGLS is not expressed in any tissue under normal growth but is induced in leaves by alamethicin and methyl jasmonate treatments. SlGLS, NaGLS, AtGLSs, and several other
GLSs characterized only in vitro come from four different eudicot families and constitute a separate branch of the
TPS-e/f clade that diverged from kaurene synthases, also in the
TPS-e/f clade, before the gymnosperm-angiosperm split. The early divergence of this branch and the
GLS activity of genes in this branch in diverse eudicot families suggest that
GLS activity encoded by these genes predates the angiosperm-gymnosperm split. However, although a
TPS sequence belonging to this
GLS lineage was recently reported from a basal dicot, no representative sequences have yet been found in monocot or nonangiospermous plants.Geranyllinalool is an acyclic diterpene alcohol with a wide distribution in the plant kingdom; it has been identified as component of essential oils of distantly related plant species such as
Jasmin grandiflorum,
Michelia champaca, and
Homamelis virginiana (
Sandeep, 2009). Geranyllinalool is the precursor of 4,8,12-trimethyltrideca-1,3,7,11-tetraene (
TMTT), a volatile C
16-homoterpene emitted from the foliage of many angiosperm species including Arabidopsis (
Arabidopsis thaliana), tomato (
Solanum lycopersicum), maize (
Zea mays), fava bean (
Vicia faba), lima bean (
Phaseolus lunatus), alfalfa (
Medicago sativa), and
Eucalyptus spp. (
Van Poecke et al., 2001;
Ament et al., 2004;
Williams et al., 2005;
Hopke et al., 1994;
Leitner et al., 2010;
Webster et al., 2010). In addition, various hydroxygeranyllinalool glycosides have been isolated from many Solanaceous species such as
Capsicum annuum,
Lycium chinense, and at least 26
Nicotiana species (
Yahara et al., 1993;
Iorizzi et al., 2001;
Snook et al., 1997).The biosynthetic pathway leading to geranyllinalool, as for all other terpenoids, begins with the condensation of isopentenyl diphosphate and its allylic isomer, dimethylallyl diphosphate. Sequential condensation of one isopentenyl diphosphate molecule with three dimethylallyl diphosphate molecules produces geranylgeranyl diphosphate (GGPP), the C-20 intermediate of the diterpenoid pathway. Next, a terpene synthase (
TPS) catalyzes a two-step reaction in which carbocation formation of the C20 precursor is followed by an allylic rearrangement that results in the production of the tertiary alcohol geranyllinalool (
Herde et al., 2008).Although geranyllinalool and its derivatives,
TMTT and geranyllinalool glycosides, have been reported in a wide variety of plant species, a geranyllinalool synthase (
GLS) involved in
TMTT biosynthesis was only recently identified in Arabidopsis (
Herde et al., 2008). AtTPS04 belongs to the
TPS-e/f subfamily along with the previously identified
Clarkia spp. linalool synthases (
Chen et al., 2011). More recently, two
TPSs from
Vitis vinifera and one from the daisy
Grindelia hirsutula, also members of the
TPS-e/f subfamily, were found to exhibit
GLS activity in vitro (
Martin et al., 2010;
Zerbe et al., 2013). However, no in planta information has been presented for these, nor any evidence showing their involvement in
TMTT biosynthesis.The common characteristic of the
TPS-e/f
GLSs so far identified is that they lack a predicted plastidial transit peptide, and direct evidence for nonplastidial localization was obtained in Arabidopsis by observing the AtTPS04-GUS fusion protein in the cytosol and endoplasmic reticulum (
Herde et al., 2008). On the other hand, two
TPS-g subfamily proteins from the closely related Fabaceae species
Medicago truncatula and
Phaseolus lunata (MtTPS03 and PlTPS2, respectively) were shown to be plastidic and to catalyze the formation of geranyllinalool in vitro when GGPP was provided as a substrate and also when expressed in a heterologous plant species (
Arimura et al., 2008;
Brillada et al., 2013). However, the same enzymes also produced linalool and nerolidol when supplied with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), respectively (
Arimura et al., 2008;
Brillada et al., 2013). Given the present paucity of in vivo and in vitro studies of geranyllinalool biosynthesis in plants, it is not clear whether geranyllinalool in plants is typically produced via
TPS-g or
TPS-e/f type
TPSs, or both.The role of geranyllinalool itself in plant tissues is not well established. Often geranyllinalool coexists in floral or vegetative tissues with its homoterpene derivative
TMTT. The contribution of
TMTT to the floral scent of insect-pollinated species suggests a putative role in attraction of pollinators (
Tholl et al., 2011). On the other hand, in many angiosperm species, including tomato,
TMTT is a component of volatile blends released from vegetative tissues upon herbivore attack, sometimes in parallel with its constitutive emission from floral tissues (
Hopke et al., 1994;
Ament et al., 2004;
de Boer et al., 2004;
Kant et al., 2004;
Williams et al., 2005,
Herde et al., 2008). The latter case suggests that
TMTT might play a defensive role in both vegetative and floral tissues.
TMTT production from insect-infested plants is considered as an indirect defense mechanism because
TMTT attracts insect predators of the insect herbivores (
Brillada et al., 2013). Interestingly, production of
TMTT, and the homoterpene (
E)-4,8-dimethyl-1,3,7-nonatriene, from herbivore-attacked lima bean plants has been found to correlate with enhanced expression of defense genes in neighboring nonaffected control plants (
Arimura et al., 2000). In these cases, homoterpenes are believed to act as stress-responsive signals that enable intraspecies plant-to-plant communication.A plant defense role has also been suggested for 17-hydroxygeranyllinalool diterpene glycosides (
HGL-DTGs) present in leaves and flowers of
Nicotiana species, with higher concentrations measured in buds (
Heiling et al., 2010;
Jassbi et al., 2010). Several studies have found negative correlation between total leaf
HGL-DTG content and the mass of the larvae that feed on them (
Jassbi et al., 2008;
Dinh et al., 2013). Eleven
HGL-DTGs that differ in sugar moieties and number of malonylesters have been isolated from
Nicotiana attenuata. The sugar groups of these compounds are Glc and rhamnose and are conjugated to the hydroxygeranyllinalool skeleton via bonds at C3 and C17 hydroxylated carbons. Additional sugars may be added to these sugars on their hydroxyl groups at C2, C4, and C6, and manolyl esters are typically formed at the C6 hydroxyl group of the glucoses. The concentrations of these
HGL-DTGs are higher in young and reproductive tissues. While their total levels appear to be constant, the concentration of individual compounds change upon herbivore attack, with a proportionally greater increase in malonylated compounds. Unlike many other defense-related specialized metabolites, the
N. attenuata
HGL-DTGs are not found on the leaf surface or the trichomes, but, instead, they accumulate inside the leaves (
Heiling et al., 2010).Here, we show that in the Solanaceae species cultivated tomato and
N. attenuata, geranyllinalool is synthesized by
TPSs that belong to the
TPS-e/f subfamily and that the corresponding genes are related to Arabidopsis TPS04. The tomato and
N. attenuata enzymes were biochemically characterized, and the kinetic parameters were determined. We also describe a detailed quantitative expression of these genes in different parts of the plant. In addition, we establish that the expression of the geranyllinalool synthase genes correlates well with the induced emission of
TMTT in tomato leaves after alamethicin and methyl jasmonate (
MeJA) treatments and with the total concentrations of
HGL-DTGs in
N. attenuata leaves and floral organs.
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