Terpenoids represent one of the most diverse class of plant metabolites, being involved in numerous ubiquitous basic processes including photosynthesis, respiration, growth and development (Gershenzon and Kreis, 1999; Rodriguez-Concepción and Boronat, 2002). In addition to such vital molecules as sterols, carotenoids and the hormones gibberellins, strigolactones, abscisic acid and brassinosteroids, this class of metabolites includes monoterpenes, sesquiterpenes and diterpenes, which play important roles in plant defense against herbivores and pathogens, as well as in plant reproduction by attracting pollinators and seed dispersers (Dudareva et al., 2006). All terpenoids are derived from the universal five-carbon building blocks isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which are synthesized in plants by two alternative pathways that are localized in different subcellular compartments (Ashour et al., 2010; Hemmerlin et al., 2012). The classical mevalonic acid (MVA) pathway, which is localized in the cytosol and partially in peroxisomes, gives rise to IPP and via enzymatic isomerization to DMAPP, both of which serve as substrates (at a 2:1 ratio) for cytosolic farnesyl diphosphate (FPP) synthase (FPPS) to form FPP. In contrast, the plastidic methylerythritol phosphate (MEP) pathway directly produces both IPP and DMAPP (at a 6:1 ratio) for downstream formation of geranyl diphosphate (GPP) and geranylgeranyl diphosphate (GGPP) by the plastidic enzymes GPP synthase (GPPS) and GGPP synthase (GGPPS), respectively. While FPP in the cytosol serves as a precursor for sterols/brassinosteroids, plastidic GGPP is utilized for chlorophyll, carotenoid, strigolactone, abscisic acid and gibberellin biosynthesis. Independently, cytosolic sesquiterpene synthases use FPP and plastidic mono- and diterpene synthases use GPP and GGPP, respectively, as substrates. Although separated in two subcellular compartments, there is substantial evidence for metabolic interaction between the MVA and MEP pathways, with IPP exchange potentially in both directions (Kasahara et al., 2002; Nagata et al., 2002; Hemmerlin et al., 2003a; Laule et al., 2003; Schuhr et al., 2003; Dudareva et al., 2005; Furumoto et al., 2011). It was shown that the metabolic flux through the MEP pathway often exceeds that of the MVA pathway (Dudareva et al., 2005; Wu et al., 2006), and export of MEP pathway-derived IPP is of particular importance for cytosolic terpene biosynthesis in some plants (Adam et al., 1999; Steliopoulos et al., 2002; Dudareva et al., 2005; Hampel et al., 2005a,b; Orlova et al., 2009). Trafficking of IPP across the inner envelope membrane of plastids is mediated by an as yet unidentified metabolite transporter (Soler et al., 1993; Bick and Lange, 2003; Flügge and Gao, 2005).
While plastidic GGPPSs are homodimeric (Dogbo and Camara, 1987; Laferrière and Beyer, 1991; Burke and Croteau, 2002), GPPSs have either homodimeric or heterodimeric architectures depending on the plant species (Nagegowda, 2010). Heterodimeric GPPSs, like the one found in snapdragon (Antirrhinum majus), contain a catalytically inactive small subunit (GPPS-SSU) that interacts with a bona fide GGPPS subunit to form a heterodimer that catalyzes GPP formation (Tholl et al., 2004; Wang and Dixon, 2009). Moreover, inactive GPPS-SSUs interact in vitro and in planta with GGPPSs from distant plant species to form functional heterodimeric GPPSs (Burke and Croteau, 2002; Tholl et al., 2004; Orlova et al., 2009; Wang and Dixon, 2009). With the exception of Lithospermum erythrorhizon (Sommer et al., 1995; Li et al., 1998), all known plant GPPS enzymes appear to be plastidic. However, some cytosolic sesquiterpene synthases, in addition to FPP, can use GPP as a substrate to form monoterpenes (see the comprehensive list in Table S1). In cultivated strawberry (Fragaria ananassa) fruits, the cytosolic terpene synthase FaNES1 produces roughly similar amounts of the sesquiterpene nerolidol and the monoterpene linalool (Aharoni et al., 2004). Moreover, over-expression in the cytosol of monoterpene synthases (Ohara et al., 2003; Wu et al., 2006) and a sesquiterpene synthase possessing monoterpene synthase activity (Davidovich-Rikanati et al., 2008) resulted in formation of monoterpenes in transgenic plants, suggesting the existence of a GPP pool in this cellular compartment. These data raise questions about the origin of cytosolic GPP and its potential transport between plastids and the cytosol. To address these questions, a metabolic engineering approach was used to co-express snapdragon GPPS-SSU with the geraniol synthase gene GES, encoding a plastidic monoterpene synthase (Davidovich-Rikanati et al., 2007), and the zingiberene synthase gene ZIS, encoding a cytosolic terpene synthase that has been shown to possess both sesqui- and monoterpene synthase activities (Davidovich-Rikanati et al., 2008) in ripening tomato (Solanum lycopersicum) fruits under the control of the polygalacturonase (PG) promoter. A significant increase in monoterpenes was observed in both cases, suggesting that plastid-produced GPP is not only used for plastidic monoterpene formation, but is also exported to support cytosolic monoterpene biosynthesis.