## 3 Discussion  
Terpenes represent one of the largest groups of natural compounds and are ubiquitous in almost all kingdoms of life. In plants, terpen play important roles in defense against insects and pathogens. Poplars, for example, produce volatile terpen in response to insect herbivory and the released terpen have been discussed as attractants for herbivore enemies, as antimicrobial compounds, and as inter- and intra-plant defense signals [20; 24; 25]. To investigate the formation of herbivory-induced terpen in poplar, we identified and characterized _trans_-IDS enzymes that provide the precursors for mono- and sesquiterpenes in the model poplar species _P. trichocarpa_. _P. trichocarpa_ possesses a mid-sized _trans_-IDS gene family with 16 members, a number similar to that reported from Arabidopsis, which has been shown to contain 17 _trans_-IDS genes [28]. Based on sequence comparisons and dendrogram analysis, the individual poplar genes could be assigned to subfamilies of functionally-related _trans_-IDSs (Figure 1). Two out of the 16 putative _trans_-IDS genes, _PtGPPS1.LSU_ and _PtGPPS2.LSU_, grouped together with _GPPS_, _GGPPS_, and _GFPPS_ from other plants and were thus considered as potential GPPS genes in this study. Another group of three related poplar genes (Potri.017g124700, Potri.017g124600, and Potri.004g090600) clustered in the same clade of the phylogenetic tree; however, the low bootstrap value for this branching does not support a  
Figure 4: The small subunit PtGPPS.SSU1 enhances the GPPS activity of PtGPPS1.LSU and PtGPPS2.LSU. Purified recombinant PtGPPS1.LSU and PtGPPS2.LSU were incubated with IPP and DMAPP either in the presence (black bars) or absence (grey bars) of purified recombinant PtGPPS.SSU1. Product formation was analyzed by LC-MS/MS and quantified using authentic standards. Means \(\pm\) SE of triplicate assays are shown. Asterisks indicate statistical significance in Student’s t-tests and from Mann–Whitney Rank Sum Tests, respectively. PtGPPS1.LSU \(\pm\) PtGPPS.SSU1 GPP (\(p\) = 0.010, t = \(-\)4.067); PtGPPS1.LSU \(\pm\) PtGPPS.SSU1 GGPP (\(p\) = 0.075, t = 2.241); PtGPPS2.LSU \(\pm\) PtGPPS.SSU1 GPP (\(p\) = 0.003, t = \(-\)5.379); PtGPPS2.LSU \(\pm\) PtGPPS.SSU1 GGPP (\(p\) = 0.009, t = 4.091). reliable prediction of enzymatic function. More importantly, Potri.017g124700, Potri.017g124600, and Potri.004g090600 were not expressed in undamaged and herbivore-damaged poplar leaves and so were excluded from the list of candidates potentially involved in herbivore induced mono- and sesquiterpene formation in this organ. The two remaining GPPS candidates PtGPPS1.LSU and PtGPPS2.LSU were similar to the large subunit of a heterodimeric GPPS from hops [17] and produced GPP and GGPP when tested with IPP and DMAPP as substrates (Figure 3). Notably, each protein showed a different product specificity. While PtGPPS1.LSU showed a preference for the formation of GPP, PtGPPS2.LSU formed more GGPP than GPP (Figure 4). Catalytic promiscuity in vitro has been reported for other short-chain IDS enzymes [14; 29; 30; 31; 32]. The multifunctional PaIDS1 from Norway spruce, for example, was shown to produce GPP and GGPP when assayed with IPP and DMAPP [31], while PaIDS3 from the same species formed GPP, FPP, and GGPP in vitro [30; 31]. However, whether such catalytic promiscuity also occurs in vivo and has biological relevance is still unclear. While FPPSs and GGPPSs usually function as homodimers, GPPSs can form heterodimers consisting of a large subunit containing all amino acid motifs necessary for catalytic activity and a small subunit that is catalytically inactive [14]. Biochemical characterization of several heterodimeric GPPS complexes in different plants revealed that the small subunits can modulate the activity and chain-length specificity of the enzyme complex [14; 16; 17; 18; 33]. The large subunit of hops G(G)PPS, for example, was shown to produce GPP, FPP, and GGPP from IPP and DMAPP in vitro.