Figure S2. Amino acid sequence comparison of putative diterpene synthases of P. trichocarpa with characterized diterpene synthases from A. thaliana. Identical amino acids are marked by black boxes and amino acids with similar side chains are marked by gray boxes. The highly conserved DDxxD and DxxDD motifs are labeled. Amino acids belonging to a conserved N- terminal stretch are labeled by X. AtCPS, (Q38802) copalyl diphposphate synthase; AtKS, (Q9SAK2) kaurene synthase and AtTPS4, (Q93YV0) geranyllinalool synthase of Arabidopsis thaliana.

Chromosome k 20k50k 80k 019G G k1920k 1950k 019G G G k2640k2670k 019G G G k5340k 5370k 019G G k 5430k 019G k5620k Figure S3. PtTPS genes located on P. trichocarpa chromosome 19. The analysis was performed with the version 3 assembly of the P. trichocarpa genome ( Big arrows indicate putative functional monoterpene synthase genes (gray) and sesquiterpene synthase genes (black). Small arrows show TPS gene fragments. Arrow direction is in accordance to gene direction. Black boxes show other non TPS genes.

TPS-g TPS-c PtTPS1 PtTPS9 93 PtTPS7 93 NtEAS 58 PtTPS5 69 PtTPS8 PtTPS11 PtTPS PtTPS4 MsLS PtTPS2 PtTPS6 69 PtTPS12 PtTPS AmMYS PtTPS3 PtTPS Potri.005G Potri.002G AtCPS 100 PtTPS10 AtTPS4 100 AtKS Potri.008G Potri.008G TPS-a TPS-b TPS-e TPS-f Figure S4. Phylogenetic tree of characterized PtTPS enzymes and representive TPS from 6 different TPS subfamilies. The tree was inferred with the neighbor-joining method and n = 1000 replicates for bootstrapping. Bootstrap values are shown next to each node. NtEAS (Q40577), Nicotiana tabacum 5-epi-aristolochene synthase; AtCPS, (Q38802) copalyl diphposphate synthase; AtKS, (Q9SAK2) kaurene synthase and AtTPS4, (Q93YV0) geranyllinalool synthase of Arabidopsis thaliana; MsLS, Mentha spicata (AAC37366) (4S)-limonene synthase; AmMYS, Antirrhinum majus (AAO41727) myrcene synthase

Figure S5. Amino acid sequence comparison of the characterized poplar mono- and sesquiterpene synthases. Identical amino acids are marked by black boxes and amino acids with similar side chains are marked by gray boxes. Conserved motifs are labeled.

Relative abundance (TIC x 10,000 ions) Relative abundance (TIC x 10,000 ions) Retention time (min) Retention time (min) injector temperature 230°C injector temperature 150°C PtTPS cont cont. 8 Retention time (min) Relative abundance (TIC x 10,000 ions) Relative abundance (TIC x 10,000 ions) PtTPS7 Figure S6. GC-MS analysis of PtTPS7 and PtTPS11 products. The enzymes were expressed in E. coli, extracted, partially purified, and incubated with the substrate FPP. Products were collected with a solid-phase microextraction (SPME) fiber and analyzed by GC-MS with different injection temperatures to visualize heat induced rearrangements. 1, β-elemene; 2, eremophilene; 3, α- selinene; 4, unidentified sesquiterpene; 5, germacrene A; 6, (E)-β-caryopyhyllene; 7, α-humulene; 8, elemol; 9, hedycaryol.

Retention time (min) Relative abundance (TIC x ) (+) (-) (±)-caryophyllene (-)-caryophyllene PtTPS9 product P. trichocarpa volatiles Relative abundance (TIC x10.000) (+)-nerolidol (±)-nerolidol (-)-(3R) (+)-(3S) PtTPS15 sesqui- terpene products (±)-linalool (-)-(3R) (+)-(3S) PtTPS15 mono- terpene products (+)-(3S) Relative abundance (TIC x10.000) Retention time (min) (-)-(3R) (-)-linalool Retention time (min) Retention time (min) Relative abundance (TIC x ) PtTPS12 mono- terpene product (-)-linalool (±)-linalool (-)-(3R) (+)-(3S) (+)-(3S)- linalool (-)-(3R) (-)-(3R)-linalool Retention time (min) (±)-β-elemene (-)-β-elemene (-) (+) Relative abundance (TIC x ) Time--> (-)-β-elemene PtTPS11 sesqui- terpene products A B C DE Figure S7. Chiral analysis of PtTPS enzyme products and poplar volatiles. The recombinant enzymes were incubated with the substrate GPP and FPP. Enzyme products and volatile collections from herbivore-induced poplar leaves were run on a chiral column and analyzed by GC- MS. Retention times and spectra were compared to those of the pure standards and standard mixtures shown.

apical herbivory basal herbivorycontrol Figure S8. Experimental set up. Herbivory was applied to a single leaf of intact trees either apically (LPI3), basally (LPI10), or not at all (control). Volatiles of eight single leaves (LPI3 to LPI10) were individually measured from all trees.

(Z)-farnesol (Z,Z)-farnesal (E)-farnesol (E,E)-farnesal empty vector control + FPP empty vector control + GPP Relative abundance (TIC x 1,000 ions) nerol neral geraniol geranial Retention time (min) Relative abundance (TIC x 10,000 ions) Relative abundance (TIC x 1,000 ions) empty vector control + GGPP Figure S9. GC-MS analysis of protein extracts from E. coli expressing an empty vector. The empty vector was expressed in E. coli and the raw protein extract was incubated with GPP, FPP and GGPP, respectively. Products were collected with a solid-phase microextraction (SPME) fiber and analyzed by GC-MS. Substrate hydrolysis products are labeled, peaks not labeled are non-terpenoide contaminations.