## 3 Discussion
Coexpression of G(G)PPS with the small subunit GPPS.SSU, however, yielded a heterodimeric complex exhibiting enhanced catalytic formation of GPP [17]. Among the 16 putative _trans_-IDSs identified in _P. trichocarpa_, two (PtGPPS.SSU1 and PtGPPS.SSU2) showed significant sequence similarity to small GPPS subunits from other plants (Figure 1). One of them, _PtGPPS.SSU1_, was found to be induced upon herbivory (Figure 2) and the recombinant protein modified the chain-length specificity of PtGPPS1.LSU towards increased GPPS activity in vitro (Figure 4). Because _PtGPPS1.LSU_ was also highly upregulated upon herbivory, we propose that both proteins form a heterodimeric GPPS complex that provides GPP as a precursor for the herbivory-induced formation of monoterenes in planta. The GPP-forming activity of PtGPPS2.LSU was also enhanced by PtGPPS.SSU1 in vitro and it is thus conceivable that PtGPPS2.LSU-PtGPPS.SSU1 complexes may also contribute to herbivory-induced GPP formation. In the absence of PtGPPS.SSU1 in undamaged leaves, PtGPPS2.LSU by itself could form GGPP and GPP for other metabolic processes. Transcript accumulation of the second small subunit PtGPPS.SSU2 was not influenced by herbivory (Figure 2). In contrast to PtGPPS.SSU1, recombinant PtGPPS.SSU2 did not influence the chain-length specificity of PtGPPS1.LSU and PtGPPS2.LSU in vitro. Notably, PtGPPS.SSU2 possessed both the FARM and the SARM motifs but showed no IDS activity (Figure S1). It is conceivable that PtGPPS.SSU2 functions as an interaction partner for other constitutively expressed _trans_-IDS genes and modulates their activities. The elucidation of its exact role in preyl diphosphate formation would be a worthwhile aim for future studies. _FPPS_ genes have been identified and characterized in a number of plant species from diverse families. Database surveys and detailed investigations with _Arabidopsis thaliana_, _Zea mays_, _Artemisia tridentata_, and the tree species _Euomnia ulmoides_ suggest that plants usually possess small _FPPS_ gene families with two to five members [34; 35; 36; 37; 38]. The different _FPPS_ gene copies of a plant species can be differentially regulated, resulting in distinct spatial and temporal expression patterns [36; 39]. The maize _fprps1_ gene, for example, is strongly expressed in roots and has been suggested to provide the precursor for the formation of constitutive sesquiterpenes and triterpenes in this organ. Maize _fprps3_, however, is induced upon caterpillar feeding in leaves and likely controls the herbivory-induced production of defense sesquiterpenes [36]. In contrast to maize and other plants, _P. trichocarpa_ possesses only one functional _FPPS_ gene _PtFPPS1_, which is constitutively expressed in leaves and not induced upon herbivory (Figure 2). Biochemical characterization of purified recombinant PtFPPS1 confirmed its activity as FPPS (Figure 3). Another poplar gene (Potri.016g004100) that grouped together with _PtFPPS1_ in the FPPS clade of plant _trans_-IDS genes (Figure 1) was characterized as a pseudogene. Its ORF encodes a protein lacking significant parts of typical FPPS sequences such as the FARMmotif essential for catalytic activity (Figure S1). Moreover, RNAseq data as well as our unsuccessful attempts to amplify the ORF from cDNA suggest that Potri.016g004100 is not expressed in poplar leaves (Figure 2). Since no other obvious _FPPS_ genes could be identified in the _P. trichocarpa_ genome, it is likely that PfFPPS1 acts as the sole FPPS and provides a constitutive pool of FPP for both primary and specialized metabolism. In this scenario, the herbivory-induced formation of volatile sesquiterpenes may be controlled by the upregulation of poplar sesquiterpene synthases as already reported in previous studies [(20; 21)] and/or by an increase of metabolic flux through the mevalonate pathway.