Potri.019G000400
93
98
Potri.019G023100
66
Potri.T001100
95
Potri.019G016500
100
93
Potri.019G016900
61
Potri.019G045400
Potri.T072200
Potri.T072300
100
Potri.019G045300
81
62
31
Potri.019G045100
TPS-a
STS
Potri.007G074400
Potri.005G095500
98
Potri.001G415100
76
60
Potri.011G142800
Potri.015G085500
61
100
37
Potri.015G032100
NtEAS
Potri.007G119700
AmMYS
26
Potri.011G032300
100
99
Potri.004G030200
74
Potri.004G030400
100
TPS-g
MTS
Potri.007G118600
100
81
Potri.017G041700
Potri.011G031800
MsLS
80
55 Potri.001G308200
100
Potri.T073000
Potri.001G308300
56
96 Potri.T072900
72
99
84
TPS-b
MTS
Potri.019G023000
Potri.019G022900
Potri.T001300
100
Potri.T000800
99 Potri.T000300
61 Potri.T001000
Potri.004G037900
100
TPS-f, DTS
AtTPS4
AtKS
91
TPS-e, KS
Potri.008G082700
100
100
Potri.008G082400
AtCPS
Potri.002G052100
100
0.1
100
TPS-c, CPS
Potri.005G210300
Figure S1. Phylogenetic tree of putative PtTPS genes and representative TPS genes from 6
different TPS gene subfamilies. The tree was inferred with the neighbor-joining method
(nucleotides were treated as triplets) and n = 1000 replicates for bootstrapping. Bootstrap values
are shown next to each node. NtEAS (L04680), Nicotiana tabacum 5-epi-aristolochene synthase;
AtCPS, (NM_116512) copalyl diphosphate synthase; AtKS, (NM_106594) kaurene synthase and
AtTPS4, (AY059757) geranyllinalool synthase of Arabidopsis thaliana; MsLS, Mentha spicata
(L13459) (4S)-limonene synthase; AmMYS, Antirrhinum majus (AY195609) myrcene synthase;
STS, sesquiterpene synthase ; MT, monoterpene synthase ; CPS, copalyl diphosphate synthase ;
DTS, diterpene synthase ; KS, kaurene synthase
Potri.002G052100
Potri.004G037900
Potri.005G210300
Potri.008G082400
Potri.008G082700
ATCPS
ATKS
ATTPS4
MAS-----------------------------FSIGVWLYGARGKQDNFHAHSRCSAISKPRTQGYADLFHQQNGLPLINWPHDVVEDDTEEDAAKVSVA
-------------------------------------------------------------------------MEFSKSFN-IQALVKTIKEELLSDNNMSSHYSSIHLLSTVPSTPRSSFSTSNNIHPLPFPAGVWLYGAKDKKD--NIHTRCSAISKPRTQEYADLFQQQNGLPLINWPHDIVEDDAEEDAAKVSVA
---------------------------------------------------------------------MSC-SRPWFCPSSISATLTDPASKLVTGEFK
---------------------------------------------------------------------MSC-IRPWFCPSSISATLTDPASKLVTGEFK
MSLQYHVLN---SIPSTTFLSSTKTTISSSFLTISGSPLNVARDKSR-----SGSIHCSKLRTQEYINSQEVQHDLPLIHEWQQLQGEDAPQISVG-SNS
---------------------------------------------------------------------MSINLRSSGCSSPISATLERGLDSEVQ--TR
-----------------------------------------------------------------------MKSSYGSSSNDLHAFVNEIKGEIQLSNIN
71
25
98
30
30
91
29
29
Potri.002G052100
Potri.004G037900
Potri.005G210300
Potri.008G082400
Potri.008G082700
ATCPS
ATKS
ATTPS4
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
KEIDEHVKTIKAMLEMMEDGEISISAYDTAWVALVEDIN----GSGLPQFPSSLQWIANNQLP-DGSWG------------DAEIFLAHDRLINTLACVV
-DPYSFIAP---------------SAYDTAWLAMVPDMS----EPCQPMFKNCLDWVLNNQNV-EGFWG----------EYDGHGMPTIECLPATIACMI
KEISKRVRTIKAMLDMMEDGEISISAYDTAWVALVEDIN----GSGLPQFPSSLQWIANNQLP-DGSWG------------DAGIFLAHDRLINTLACVV
TTSLNFHGTKERIKKMFDKIELSVSSYDTAWVAMVPSPD----CPETPCFPECTKWILENQLG-DGSWSLP----------HGNPLLVKDALSSTLACIL
TTSLNFHGTKERIKKMFDKIELSVSSYDTAWVAMVPSPD----CPETPCFPECTKWILENQLG-DGSWSLP----------HGNPLLVKDALSSTLACIL
NAFKEAVKSVKTILRNLTDGEITISAYDTAWVALID--A----GDKTPAFPSAVKWIAENQLS-DGSWG------------DAYLFSYHDRLINTLACVV
ANNVSFEQTKEKIRKMLEKVELSVSAYDTSWVAMVPSPS----SQNAPLFPQCVKWLLDNQHE-DGSWGLDN---------HDHQSLKKDVLSSTLASIL
LDPYSFVSP---------------SAYDTAWLSMIEEDINVDDNELKPMFQGCLDWIMCNQNAREGFWMNSTSYTTVADGRDEDGEKDMCILTSTLACVV
154
94
181
115
115
172
115
114
Potri.002G052100
Potri.004G037900
Potri.005G210300
Potri.008G082400
Potri.008G082700
ATCPS
ATKS
ATTPS4
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
ALKSWNLHQEKCEKGMLFFRDNLCKLED---ENAEHMPIGFEVAFPSLLEIAKKLDIEVPYDS---PVLQEIYASRNLKLTRIPKDIMHNVPTTLLHSLE
ALKRWNAGEMIIDKGMAFIEANAEKLIGEIYDSN--CPRWFAIVFPAMVEMAQINGLEIIFPDRIKRVEMSIFYKREQILEREELVDKYHYPPLLSYLEA
ALKSWNLHQDKCEKGMLFFRDNLCKLED---ENAEHMPIGFEVAFPSLLAIAKELDIEVPDDS---TFLQEIYARRNLKLKRISKDIMHNVPTTLLHSLE
ALKRWGIGEEQINKGLRFIELNSASVT----DNEQHKPIGFDIIFPGMIEYAIDLDLNLPLKP---TDINSMLHRRALELTSGGGKNLEGRRAYLAYVSE
ALKRWGIGEEQINKGLRFIELNSASVT----DNEQHKPIGFDIIFPGMIEYAKDLDLNLPLKP---TDINSMLHRRALELTSGGGKNLEGRRAYLAYVSE
ALRSWNLFPHQCNKGITFFRENIGKLED---ENDEHMPIGFEVAFPSLLEIARGINIDVPYDS---PVLKDIYAKKELKLTRIPKEIMHKIPTTLLHSLE
ALKKWGIGERQINKGLQFIELNSALVT----DETIQKPTGFDIIFPGMIKYARDLNLTIPLGS---EVVDDMIRKRDLDLKCDSEKFSKGREAYLAYVLE
ALQKWNIGCFHLHKGTRYIERNTEMIIGKYINEEGSYPRWFAIKFTGILELAQKLGLHFVFSSRCIEMIKGMFYQRQEIIQREKLVHDCNYKPLLAYLEV
248
192
275
208
208
266
208
214
Potri.002G052100
Potri.004G037900
Potri.005G210300
Potri.008G082400
Potri.008G082700
ATCPS
ATKS
ATTPS4
XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
GMPGLEWKRLLKLQSQ-----DGSFLFSPSSTAFALSQTKDKNCMEYLNKAVQRFEGGVPNVYPV-DLFEHIWAVDRLQRLGISRYFESQIDECVNYIHR
LP-ALYNFDQEDALKH--LHADGSLFQSPSATASAFMATGNKDCLNYLQTLVQKCTNGVPQTYPMDEELLKLCMVNQLQRLGLAEHFNQEIEELLEQVYR
GMRGLDWKQLIKLQCL-----DGSFLFSPSSTAFALSQTKDKNCLEYLNKAVQRFKGGVPNVYPV-DLFEHIWVVDRLQRLGVSRYFVSEINECVDYIHR
GIGKLQDWEMAMKYQR----KNGSLFNSPSTTAAAFIHIQDAECLHYIRSLLQKFGNAVPTIYPL-DIYARLSMVDALERLGIDRHFRKERKFVLDETYR
GIGKLQDWEMAMKYQR----KNGSLFNSPSTTAAAFIHIQDAECLHYIRSLLQKFGNAVPTIYPL-DIYARLSMVDALERLGIDRHFRKERKFVLDETYR
GMRDLDWEKLLKLQSQ-----DGSFLFSPSSTAFAFMQTRDSNCLEYLRNAVKRFNGGVPNVFPV-DLFEHIWIVDRLQRLGISRYFEEEIKECLDYVHR
GTRNLKDWDLIVKYQR----KNGSLFDSPATTAAAFTQFGNDGCLRYLCSLLQKFEAAVPSVYPF-DQYARLSIIVTLESLGIDRDFKTEIKSILDETYR
LPSKLYVTNQEDIIVKSLDSMDGSLFQSPSATASAFMLTRNTKCLAYLQNLVQKCPNGVPQKYPLNEDLIKLSMVNLIESTGLGEFFGIEIEHVLEQVYS
342
289
369
303
303
360
303
314
Potri.002G052100
Potri.004G037900
Potri.005G210300
Potri.008G082400
Potri.008G082700
ATCPS
ATKS
ATTPS4
DxDD
YWTEDG-ICWARNSEVHDIDDTAMGFRVLRLNGHHVSADVFKHFEKGGEFFCFAGQ-STAAVTGMFNLYRASQLLFPGEKILEKAKEFSFKFLREKQAAN
NYMNRESWPKWTNSMATQLYKDSLAFCLLRMHGFRVSPWMFCWFLLEEEVQDQIESNHEYFSSVILNVYRATDLMFPGDHELEEARSFSRKLLEKTTSMG
YWTENG-ICWARNSNVHDIDDTAMGFRILRLHGHQVSADVFKHFEKGGEFFCFAGQ-STGAVTGIFNLYRASQVLFPGEKILEDAKEYSFEFLREKQAAN
FWLQGE------EEIFSDNATCALAFRILRLNGYDVSL--------EDHFSNSLGG-YLKDSGAALELYRALQLSYPDESLLEKQNSRTSYFLKQGLSNV
FWLQGE------EEIFSDNATCALAFRILRLNGYDVSLDTLNQFS-EDHFSNSLGG-YLKDSGAALELYRALQLSYPDESLLEKQNSRTSYFLKQGLSNV
YWTDNG-ICWARCSHVQDIDDTAMAFRLLRQHGYQVSADVFKNFEKEGEFFCFVGQ-SNQAVTGMFNLYRASQLAFPREEILKNAKEFSYNYLLEKRERE
YWLRGD------EEICLDLATCALAFRLLLAHGYDVSYDPLKPFAEESGFSDTLEG-YVKNTFSVLELFKAAQ-SYPHESALKKQCCWTKQYLEMELS-RYEEKDFERMPMSYLADQLHKDSLAFRMLRMHGRDVSPRSFCWFLNDQETRNHLERNIDSFLLVILSVYRATDLMFPGEHDLQEAREYTRNLLEKRRSIK
440
389
467
388
395
458
393
414
Potri.002G052100
Potri.004G037900
Potri.005G210300
Potri.008G082400
Potri.008G082700
ATCPS
ATKS
ATTPS4
ELLDKWLITKDLPGEVGFALEIPWHASLPRVESRFYIEQYGGEDDVWIGKTLYRMPYVNNNEYLQLARLDYNNCQALHRIEWANFQKWYEECNLRDFGIS
NEDQHTVPFPSFHSVIKHELRFPWMARLDHLEHRMWMEEKNSSGLWMGKTSFHRLSCLHNDKLKQLAVKNYEFRQTTYKSELEELTRWSKSWGLSDMGFG
ELLDKWIITKDLPGEVGFALEIPWYASLPRVETRFFIEQYGGEDDVWIGKTLYRMSYINNSEYLQLAKLDYNNCQALHRIEWENFQKWYEECNLRDFGIS
SLCGDRLR-KNIIGEVHDALNFSDHANLQRLAIRRRIKH-YATDDTRILKTSYRCSTIGNQDFLKLAVEDFNICQSIQREEFKHIERWVVERRLDKLKFA
SLCGDRLR-KNIIGEVHDALNFPDHANLQRLAIRGRIKH-YATDDTRILKTSYRCSTIGNQDFLKLAVEDFNICQSIQREELKHIERWVVERRLDKLKFA
ELIDKWIIMKDLPGEIGFALEIPWYASLPRVETRFYIDQYGGENDVWIGKTLYRMPYVNNNGYLELAKQDYNNCQAQHQLEWDIFQKWYEENRLSEWGVR
SWVKTSVRDKYLKKEVEDALAFPSYASLERSDHRRKILNGSAVENTRVTKTSYRLHNICTSDILKLAVDDFNFCQSIHREEMERLDRWIVENRLQELKFA
EK------------MIMHELSTPWIARLKHLDHRMWIEDKNSNVLSMEKASFLRLHSSYSDKLTHLAARNFEFQQAKYCRELEELTMWVKKWGLSDIGFG
540
489
567
486
493
558
493
502
Potri.002G052100
Potri.004G037900
Potri.005G210300
Potri.008G082400
Potri.008G082700
ATCPS
ATKS
ATTPS4
DDxxD
RKTLLYSYFLAAASVFEPERSNERLAWAKTTILLEMIHSYFHEDDDNSGAQRRTFVHEFSTGISINGR----------------RSGTKKTRKELVKMLL
REKTAYCYFAVAASTSLPQDSEIRMMVAKSAIVITVADDFYDMEGSLDDLEKITDAVQRWDATGLSGHS----KTIFDALDSLVNELARKYFRQHGTDIT
RRTLIFSYFLAAASIFEPERSKERLAWATTTVLLDIVGSYFPENHNNSSEQRRAFIHEFSYGISINGREEN--DYLCLILVCCRRSGRKKTRQELVKLLL
RQKEAYCYFSAAATLFAPELSDARMSWAKNGVLTTVVDDFFDVGGSEEELVNLIELIERWDVNGSADFCSEEVEIIYSAIHSTISEIGDKSFGWQGRDVK
RQKEAYCYFSAAATLFAPELSDARMSWAKNSVLATVVDDFFDVGGSEEELVNLIELIERWDVNGSADFCSEKVEIIYSAIHSTISEIGEKSFGWQGRDVK
RSELLECYYLAAATIFESERSHERMVWAKSSVLVKAISSSFGE----SSDSRRSFSDQFHEYIANARRSDH--HFNDRNMRLDRPGSVQASR--LAGVLI
RQKLAYCYFSGAATLFSPELSDARISWAKGGVLTTVVDDFFDVGGSKEELENLIHLVEKWDLNGVPEYSSEHVEIIFSVLRDTILETGDKAFTYQGRNVT
REKTTYCYFATVTSLPYEYAIKFGKLAAKTAILITIADDFFDEKGSFNDLEGLTKAVLRWEGEELKSYG----NIIFRALDDIVRETANTCRTHHKTDII
624
585
665
586
593
650
593
598
Potri.002G052100
Potri.004G037900
Potri.005G210300
Potri.008G082400
Potri.008G082700
ATCPS
ATKS
ATTPS4
GTLNQLSFGALEVHGRDISHSLRHAWERWLISWELEGDRRRGEAELLVQTIHLTAGYKVSEELLVYHPQYEQLADLTNRICYQLGHYQKNKVHDNGSYST
NSLRDIWSETFASWFTEAKWSKSGFIPAAEEYLETGMTSIASHTLVLPASCFLSPSIPDYKLNPVQYESITKLLMVIPRLLNDIQSYKKEQKEGKTNFVL
GTLNQLSLGALVVHGRDISHSLRHAWEKWLLIWELEGDRRQGEAELLVQTINLTAGYLVSEELLAHHPQYEQLVDLTNRICYQLDHYKKNKVHYNGSYST
SQVIKIWLDLLKSMLTEAQWSSNKSVPTLDEYMTTAHVSFALGPIVLPALYFVGPKLSEEVAGHPELLNLYKVTSTCGRLLNDWRSFKRESEEGKLNAVS
SHVIKIWLDLLKSMLTEAQWSSNKSVPTLDEYMTTAHVSSALGPIVLPALYFVGPKLSEEVAGHPELLNLYKVTSTCGRLLNDWRSFKRESEEGKLNAIS
GTLNQMSFDLFMSHGRDVNNLLYLSWGDWMEKWKLYGD--EGEGELMVKMIILMKNNDLTN--FFTHTHFVRLAEIINRICLPR-QYLKARRNDE----HHIVKIWLDLLKSMLREAEWSSDKSTPSLEDYMENAYISFALGPIVLPATYLIGPPLPEKTVDSHQYNQLYKLVSTMGRLLNDIQGFKRESAEGKLNAVS
VHLRNIWGETFESWLREAEWSKKGHTSSMDEYIRNGMISIAAHTIALSISCLMEPCFPHNKLKPGNYDSITTLLMIIPRLLNDLQSYQKEQEQGKMNSVL
724
685
765
686
693
740
693
698
Potri.002G052100
Potri.004G037900
Potri.005G210300
Potri.008G082400
Potri.008G082700
ATCPS
ATKS
ATTPS4
ITG----------STDRITTPQIESDMQELMQLVIQKTSDGIDPKIKQTFLQVAKSFYYTAFCDPGTINY-----HIAKVLFETVA-------------LHLKENPE-ADIEDSIAYAREILDKKKKELLEHALMDGFNDFSKPCRHLHLSCVKVFQMFFDSSNRYDSNTEMFQDIQKAFYIPVEVGAPKPLPPHP--ITS----------NTDRITTPQIESDMQELVQLVVQNPSDGIDSNIKQTFLQVAKSFYYSAICDPGTINY-----HIAKVLFERVP-------------LYMIHSGGASTEEEAIEHFKGLIDSQRRQLLQLVLQEKDSIIPRPCKDLFWNMIKLLHTFYMKDDGFTSN-EMRNVVKAIINEPISLDEL---------LYMIHSGGASTEEETIEHFKGLIDSQRRQLLQLVLQEKDSIIPRPCKDLFWNMIKLLHTFYMKDDGFTSN-EMRNVVKAIINEPISLDEL------------------------KEKTIKSMEKEMGKMVELALSE-SDTFR-DVSITFLDVAKAFYYFALCG-DHLQT-----HISKVLFQKV--------------LHMKHERDNRSKEVIIESMKGLAERKREELHKLVLEEKGSVVPRECKEAFLKMSKVLNLFYRKDDGFTSN-DLMSLVKSVIYEPVSLQKESLT------LHMKNHPG-LEIEDSIAHIEKIIDSKRKEFLEHVLVDGLSDLPKPCKEIHMSCCKVFEMFFNKKNRYDSNTEMLHDIKKALYDPINVYELSEMEPMPLMA
795
781
836
775
782
802
785
797
Potri.002G052100
Potri.004G037900
Potri.005G210300
Potri.008G082400
Potri.008G082700
ATCPS
ATKS
ATTPS4
----------------------------------------------------------------------------------------------------GSKQRYPTVVASYHFNQRYKNRIIRLAANSVVSHP---ISGNPYMKMPMAP-KLKFCFM
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------HGDEYMILPLLLNSLPNILEFKRKDGYGAMKTSMCFGRSYRVNKRVMASQLDDQHKPLKIVASQRKPVPMMQSIFAPCFY
795
836
836
775
782
802
785
877
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 Nterminal stretch are labeled by X. AtCPS, (Q38802) copalyl diphposphate synthase; AtKS,
(Q9SAK2) kaurene synthase and AtTPS4, (Q93YV0) geranyllinalool synthase of Arabidopsis
thaliana.
Chromosome 19
20k
80k
50k
110k
019G000400
1890k
5310k
1950k
1920k
019G016500
5340k
019G045100
019G000600
019G016700
5370k
2610k
019G016900
019G022900
5400k
019G045300
2640k
019G023000
5430k
5590k
2670k
019G023100
5620k
019G045400
Figure S3. PtTPS genes located on P. trichocarpa chromosome 19. The analysis was
performed with the version 3 assembly of the P. trichocarpa genome (www.phytozome.net). 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-b
100
TPS-a
TPS-g
TPS-e
TPS-c
TPS-f
0.1
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
PtTPS7
PtTPS5
PtTPS4
PtTPS3
PtTPS2
PtTPS1
PtTPS6
PtTPS9
PtTPS11
PtTPS12
PtTPS13
PtTPS14
PtTPS15
PtTPS8
RRxxxxxxxxW
----------------------------------MSTQVSQEVVPKAHHNEIIRRTANYHPSIWGDQFISHLPKDKVHEAIELQEIEKLREQ------------FKRELL
----------------------------------MALQESTP-------KAQFRRTAEFHPSVWGDYFINKPPCDEMLFSAWIKEVEVLKEE------------VRTMLT
------------------------------------------MSNE------ERRSADYHPSVWELQLIESL-STPYSYELHANRLEELKQE------------AKRALV
--------------------------MALSCSVSLTAASGWPFPQNRNSERVKPILKEFKPTLPSTQKWSVSQKQTLAFGPTKQYPITINNDDSDTGYAEKLQTFKHILR
---------------MEYK----------QQVQVVQNSFQCQNNSEDIDRRQERRSANYKPNIWKYDFLQSL-SSKYDEEQYRRVTEKLREE------------VKSIFV
----------------------------------MSVEGSAIFSTATVEPNVSRRSASFPPSIWGDHFLSYA-TDSMETSD-KAEHKKLKEE------------VKRELM
-MALCLSTNLPSVNSMNWKDMPRHPLPRAAHRTCINLQFTCSVSLKESHEVSKRRLANYQPTTWSCDFLQSL-KNDNADKIYKDKAMELEEE------------VRCMIN
---------------------------------MSSHVSAAPNSIRKAIPDVTRRSANFHPSIWGDYFACHT-FNSMFDDHFEQEFIRLKEE------------VKEMLY
-------------------------------------------MENTNQQNHSRPEANFPPSVWGCSFASFS-FPQTEFESYSRQVEVLKEN------------VKDMLM
MAPTHLDSSFSTLPTLASPSRPLVESVSSKNSRSVPTKGRCKVTTGDDDQ-IVRRSANYQTSMWDYDFVQSL-TSKYKGEPYIARSEKLKAN------------IRMMLA
MALTHFASSFCTLPTLASPSRALAGPVSSKNYRSLPTKVRCMVATEAAGQ-IVRRSANYQTSIWEYDFVQSL-TSKYKGEPYTARSEKLKAN------------IRMMLA
----------------------------------METITTLRLTANTNQQNHSRPEANFPPSVWGCSFASFS-FPQTEFESYSRQVEELKEN------------MKDMLM
--------------------------MAFPINIDGNFSASFHLP-------------------------SLENELCLRHGKMVKEAGCILSN-------------------------------------------------------MALQTDLPSNSMQNFRPLADFPPTEWGFSFVSFS-FPEMEFESYNRQVEELKNI------------VMGMLM
64
57
49
84
72
62
96
64
54
96
96
63
41
59
PtTPS7
PtTPS5
PtTPS4
PtTPS3
PtTPS2
PtTPS1
PtTPS6
PtTPS9
PtTPS11
PtTPS12
PtTPS13
PtTPS14
PtTPS15
PtTPS8
AAASNSSQQLDLIDAIQRLGVAYHFETEIEEALQHIY--------NNRIDMEDDDLYNTALGFRLLRQHGYNVSCDIFNKFKDDK-GYFKQ--SNDVRGILGLYEAAHLA
SATLKPSEKLKLMDIVLRLGIGYHFEGEFNDIIEHAYN-------TYHGNNFDDDLFTVALRFRLLRQYGYNVSSDIFNEFKDGK-GNFKDNLIDDVEGLLSLYEASFLG
ST-NEPRAKLKLIDSIQRLGVAYHFEREIEEAIKLT------------ELDVNGDLHTTSLHFRLLRQHAFSVSTDVFGKFRSNRDGKFKDSIRTDVAGLLSLYEASYLG
KEGEEPIQGLAMIDAIQRLSIDYHFQEEIDSILTRQSML------LST-IHSDNNLYEVALRFRLLRQQGYHVSAGVFDNFKDNE-GRFKQQLSSDIMGLVSLYEASQLS
EA-VDLLAKLKLVDSVIKLGLGSYFEEEIKQSLDIIAA-----SIKNKNLKVEENLYVTALRFKLLRLHGYEVSQGVFNGFFD---GTSDKSKCTDVRGLIELFEASHLA
TTINKPSQTLDFIDAIQRLGISYHFEMEIDEILREMYRSPCDFDNGDDDDHHHNDLYAISLKFRLLRQQGYKISCDVFGKFKNSQ-GNFNDSLVNDTRAILSFYEATHLR
SGDMEMLTMLEMIDDIQRLGLGHRFEKDIKRKLDRISS-----SEQS-YVEAEKSLRATALCFRLLRQHGYEISQDIFHGFLDDQ-GNFMASLHNDIEGMLSLYEASHLA
ATTNKLSQQLNFIDVIQRLGVSYLFESEIDEMLRDLY-----YQGSGGAYVHDHDLQMVALRFRLLRQQGHYVSCDVFKKFKDTE-GNYKVCLANDIQGMLSLYEAIHLR
ASKKDTVEHIEFINLLCRLGVSYHFDDEIENSLKEIFD-----DLPHLLEKHDFDLYTLSLLFRVLRQHGFKMPCVVFDKFKDTN-GEFKKTIINDVKGILSLYEASFLS
NA-SKPLDQLELIDTLERLGLSYHFVDEIKSILKSLFD-----ENHIENTETVHDLYATALEFRLLRQRGYHVPQEVFNHFKDEQ-GNFRACIHDDLKGMLNLYEASYFL
NA-SKPLDQLELIDALQRLGLSYHFVDEIKSTLKSLFY-----KNHIENTKTVHDLYATALEFRLLRQHGYKVPQEVFNHFKDEQ-GNFRAWIHDDLKGMLFLYEASYFL
ASKKDTVEHIEFINLLCRLGVSYHFDDEIENSLKEIFD-----DLPHLLEKHDFDLYTLSLLFRVLRQHGFKMPCVVFDKFKDTN-GEFKKTIINDVKGILSLYEASFLS
TAGKDPLEGLVMIDTLQRLGIDYHLREEIEAFLNTQYMN------FSSPNHLPLDVFGVALRFRLLRQEGYNVSQEVFNNFKNEE-GNFHLIQENDVKGLMALYEASQLS
ASKKDPVENVEFINLLCRLGVSYHFETEIEDQVDYVHD-----ALPSLLENNDHDLHTVALLFRVLRQYGCKVSSDVFKKFKDTN-GEFKKTITSDVKGNLSLHEAAHLS
163
159
146
186
173
171
199
168
158
199
199
167
144
163
PtTPS7
PtTPS5
PtTPS4
PtTPS3
PtTPS2
PtTPS1
PtTPS6
PtTPS9
PtTPS11
PtTPS12
PtTPS13
PtTPS14
PtTPS15
PtTPS8
Rx
VHGE-DILDEALAFTTIHLKSMASSP-NCP-LTAKVSHALKQPIQRGVPRLESRRYISIYQDEPSCNKTLLRLAKLNFNLVQELHKEELAEITRWWKGLDFARRLPFARD
GHGE-DTLDKALSFCKTHLESAVAHL-VSP-LADKVSHALKRPLLKGVPKHEQWHHILIYQQDEACTGAVLKLAKLDFNVVQKCYQDELRIISRWWIDLDFATKLPFARD
VPGEDHVLEEAKNFSSKHLKSLMLETIKDEVLAKQVKQSLEVPLHWKMPRIEARDFIDIYSSDNTRNLDLLELAKLDYNLVQSQHQRELKDLARWWGALGFKKKLSFSRD
IRGE-DVLDEAGDYSYQLLRSSLTHL--DYNQARLVRNSLDHPHHKSLASFTAKYFFN--DEPNGWIGELQELAKTEFKRVQSQHQHEIVEIFKWWKDLGLSTELRFARD
YEGE-ATLDDAKAFSTR-ILTGINCSAIESDLAKHVVHVLELPSHWRVMWFDVKWHINAYENDKQTNRHLLALAKVNFNMVQATLQKDLGDVSRWWRNLGIIENLSFTRD
VHGD-EVLEEALVFTTSHLEFLATHS-SSP-LRAKINHALKQPIRKNIPRLEARNYFSVYQEDPSCSEVLLNFAKLDFNILQKQHQKELSDI-----------------A
CEGE-EILNKANKQTS--IYLRNHLGNSDSMTAERVSHALEVPLHHKMIMLEARWHIESYGKREDANPKLLQLSKLDFNMMQSVLQRDLQDMSRWWHDLGLKNKLRFSRD
VHGE-DILEDALTFATTHLKSITTDM-CPPPLLVKLRHALDQPIHKDLPWLGAKHYISIYEQEASHSEVLLKFAKLNFNFLQNMHQKELADMTMWWKKVDLSKKLPFARD
VHGE-QILDDALVFTKANLESSAMQ--SSPRLADHIRNALIRPFHKGVPRIEARKYISFYEEEESRNDTLLKFAKIDFNRVQLIHRQELSILSRWWNDLNFAEEFPYARD
VDGE-NILEDARDFTTKNLENYVKKCNTTEYLSELVSHALELPLAWRMLRLEAHWFINLYETKTDMEPVLLELAKLDFNMVQAVYQEDLKDSSRWWKMTGLGEKLDFARD
VEGE-SILEDARDFTTKNLEKYFKKCNPSEYLSKMVSHALELPLAWRMLRLESNWFINVYETKTDMEPVLLELAKLDFNMVQALHQEDLKHSSRWWKRTGLGEKLDFARD
VHGE-QILDDALVFTKANLESSAMQ--SSPRLADHIRNALIRPFHKGIPRIEARKYISFYEEDESHMDTLLKFAKIDFNRVQLLHRQELSILSRWWNDLNFAEEFPYARD
MESE-DILDEAGEFSAKLLN---------HHESEIVANTLKHPYHKSLARFMVKNFLNNIDIRNENIKVFSELAKIDCEIVRSIHQKEILQISNWWKDLGLAKELKFARN
VNGE-QILDEALEFSRTNLESLATQ--SGPRLARHIKYALIRPIHKTVQRLEAREYISFYEEEDFRNETLLKFAKLDFNRVQLLHQQELSTLSSWWKDLNLVEELPYARD
270
266
256
291
281
261
306
276
265
308
308
274
244
270
PtTPS7
PtTPS5
PtTPS4
PtTPS3
PtTPS2
PtTPS1
PtTPS6
PtTPS9
PtTPS11
PtTPS12
PtTPS13
PtTPS14
PtTPS15
PtTPS8
R
DDXXD
RVVECFFWIVGVYFEPQYSLARKILTKVIAMTSIIDDIYDVYGTLEELELFTEAIDRWDTKSMDQLP-DYMKICYEALLNVFSEIEEKVAKEGWSY-RVHYGKDAMKVLV
RVIECFFWGLGAFLEPQFVLARRFITKVLIFLSILDDIYDVHGTIEELELFTEKIERWDTS-MEDLP-DYMKLFFEALIGFFDEIEQETGKEGRPY-CVHYSREMLKNQA
RLMENYLWAMGMVFEPQFSKCRIGLTKFVCILTAIDDIYDVYGLPEELELFTKLVNRWDSMAIDDLP-DYMKICYLALFNFVNEMAYDVMRDH-GLFVLPYLVEEWANLC
QPLKWYMWSMSCLTDPSLSEQRIELTKPVSMIYIIDDIFDVHGTLNELVCFTEVINRWDIAAAEQLP-DYMKICFKALNNITNEISYKIYKEHGWN-PVESLRKAWASLC
RLVESFLCTVGLVFEPKYSSFRKWLTKVIIMILIIDDVYDVYGSLHELQQFTKAVSRWDTGEVQELP-ECMKICFQTLYDITNEMALEMQREKDGSQALPHLKKVWADFC
KVIECYFWILGVYFEPEHFLARRMLTKVIAMTSVIDDIYDVYGKPEELELFTDAIERWEITAVDLLP-EYMKVTYKALLDVYTEIEENMVNEERSY-RVYYAKEAMKNQV
RLMECFFWTVGMAFEPEFNSCRKGLTKVTSFITTIDDVYDVYGVLDELEAFTEAVERWDVSAVRNLP-DYMKLCFLALFNTVNEMAYDHLKEQ-GEDAIPCLTKAWADLC
RLVECYFWILGVCFEPQYSFARIIMTKVIAMTSVMDDVYDVYGTMEELVLFTDAIERWDISNIDHLP-EYMKFFYKQLSDVYKEIETELAAQGRSY-RVDYAKEAMKKQV
RIVEIYFWANGVHFEPQYAFSRMVVTKYTKIVSLLDDTCDAYASFEEIQHFTNAIERCCMDAIDQLPAEYLKVLYRALLNLFSETESDMGKQGRSY-ALYYVKEAFKELA
RLVVYLLWSVGIIFEPQFGNIRRMITKLNSLITTIDDVYDVYGTLDELELFTDAVVRWDLNFMDHLP-DYMKLCFFALFNSINEIAYDILRDQ-GVDNLPYLKKTWADLC
RLVENFLWTVGVIFEPQFGNCRRMLTKVNSLITTIDDVYDVYGTLDELELFTDAVVRWDLNYMDRLP-DYMKLCFLALFNSVNEMAYNILKYQ-GVDILPYLKKAWADLC
RIVEIYFWVNAIHFEPQYAFSRMVVTKYTKFVSLLDDTYDAYASFEEIQHFTNAIERCCMDAIDQLPAEYLKVLYRALLNLFSETESDMGKQGRSY-ASYYLKEAFKELA
QPLKWHMWSMSVLIDPNLSEQRVELTKPISLVYIIDDIFDLYGTLNDLSIFTEAVNEWDLTPANQLP-ESMKISLMALFDITESISTKILEKHGWN-PIESLQKSWKKLC
RIVEMYFWVNAMHFEPQYALARILSTKLGALITVIDDTYDAYSTYEELQHFTKAVIRCNIDAIDQLPTDSMKALYRALLSYFDDVANEVSKNGKSFTAVNYVKEEMKEMI
378
373
364
399
390
369
414
384
374
416
416
383
352
380
PtTPS7
PtTPS5
PtTPS4
PtTPS3
PtTPS2
PtTPS1
PtTPS6
PtTPS9
PtTPS11
PtTPS12
PtTPS13
PtTPS14
PtTPS15
PtTPS8
NDxxTxxxE
DDxxSxxxE
HAYFNEAKWFHENHIPTMEEYMQVALVT-SGYSMLTTVSFIGMGDMVTKQAFDWVFNHPK-IIRASETIGRLLDDVKSHKFEQERGHAASGVECYIRQYG-LSEQEVYKE
RAYLIEARWFNQDCVPQLEEYRRGGVYT-SCYPMAAVAWLCGMAETGSKEVFEWMLKNPK-IVVASSDIGRLMDDITSHEFEQERGHVASAVECCMKQYG-VSKKEAYDM
GSYLVEARWFSSKYSPTLSEYLENARTSIGSPAALAHAYMLLGSPIAQSSLMDCFKHGSDQLIYWSSLITRLSDDLGTYTAESERGDVTKSIQCYMIEKG-ASEKESKEH
RAFLVEARWFASGKFPSGEEYLKNGIVS-SGVHVVLVHIFFLLGQGISKENVELISNFPP-IISSTATILRLWDDLGSAKDENQDGHDGSYVECYLRENEGSSLEDARKQ
KAMFMEAKWFNEGYTPSLQEYLSNAWVSSSGTVISVHSFFSVMTELETGEISNFLEKNQD-LLYNISLIIRLCNDLGTSVAEQERGDAASSVACYMREVN-VSEEVARNH
RAYYLESKWFHQKHTPTMEEYMAVALVT-SAYAMLAATSFVGMGRVVTKDSFDWLFRGPK-ILKASEIICRLMDDIVSHKFEQKRGHVASSIECYMKQHG-TTEQETVHE
KAFLQEAKWSYNNITPSFEEYLENAWRSVSGTVILIHAYF-LMGENISKQALDYLVNYDE-LLRWPSIIFRLSNDLATSSAEIARGETANSISCYMYETG-ASEAEARKH
QAYFVEARWLHENYMPTMDEYMRISLIS-SGYPLLTCISFVGMGDIVTKDAFEWLNKDPK-IVKAASLIARLMDDIVSHKFEQERGHVASAVECYMNQHE-VSEEQAYDE
RAYRVEAQWADEGHVPTFDEYVRNGLTT-SAYGVITAVSFVGMDEVAGQEEYKWLKSNPK-IMKAGKMICRLVNDIVGHEDEQKRGDCASGVECFMKQYD-VSEKKAIEE
KSHLLEAKWYYSGYTPTLQEYLDNAWISVGAPLAIVHAYF-YASNPTTKEASHFMEEYPD-IIRWSSIILRLADDLGTSSDEMKRGDVSKSIQCYMYESE-ASEEEARDH
KSYLLEAKWYFSGYTPTLQEYMDNAWISISAPVILVHAYF-YVSNPTTEEASQFMEEYPD-IIRWSSMILRLADDLGTSTDELKRGDISKSIQCYMHEAG-VSEEKAREH
RAYQVEAQWADEGHVPTFDEYVRNGLAT-SSYGVTTAASFVEMDEVAGREEYEWLNSNPK-IIKAGKMIGRLMNDIAGHEDEQKRGDCASGVECYMKQYD-ASEKKAIEE
NAFLEEAKWFASGKLSKPEEYLRNGIVS-SGVHVVLVHMFFLLGQGINKETVDFVDGFPP-IISFTATILRLWDDLGTAKDENQNGHDGSYLECYIREHPNVTVERAREH
RTYIVEAQWCNDRFVPPLNEYVRNGKIS-IGFMATTTVFFV--VETARIKELEWLTSKAK-ISEAGCLFLRLMNDIVTHEFEQKREHCASAIECYMKEYG-VSMNEAVKE
485
480
473
507
498
476
521
491
481
523
523
490
460
485
PtTPS7
PtTPS5
PtTPS4
PtTPS3
PtTPS2
PtTPS1
PtTPS6
PtTPS9
PtTPS11
PtTPS12
PtTPS13
PtTPS14
PtTPS15
PtTPS8
FHMQVVNAWKDINEECLKP--TAVPTPLLERILNLSRVIDVIYKEEDGYTHVGKV-MKNNVASLLINSVPI-------LNKMVESDWKDINEELLKP--STVPRQVLILMLNLARIIDVVYKDYDGYTEAKNA-TKEMLTAFLVDPLPVVA-----IKGLINQAWKELNKENSK---CSLPKPLVNLSLNMARTAQCIFQFGDGIGT-STGVTRDRLTSLIVEPIPVKEP----VLHLISDAWKQLNQECLSP--NPFPSTFSKASLNIARMVPLMYDYDDNHRLPSLEEHMKSLLYENVSP----------INNIVKKTWKKINGHCFT--KSPTLQLLVNINTNMARVVHNLYQHGDGFGVQDRHENKKQILTLLVEPFKLDLPEFSFFRKQVTDAWKDVNEEFLHP--TAVPMPLLTRMLNLARVIDVVYKDEDGYTNAGTA-LKDLVSALLIDPVPM-------IEKLIQKAWRNMNKCQIDN-ETPFARSFVGTAINLARIAQCTYQHGDGHGAPDSR-SKNRISSLIIEPISFH------LRRQVVEAWKDINEELLIGPEDHVPIPLLTRVLNLARVMDVMYKDGDGYTNAKGK-VRNYITSLLIEPVQLATPSLLAS
IQKMVANGWKDINEDCMMP--TNAPMLLLQHIVNLVRVTEVTYGHNDDAYTIPLS-LKDYVTLLYVEQVPLYE-----IRKLISNAWKKINAYQFS--DPHISQTIIGVVVNLARAAQCIYQYGDGHAI-EHLETKDRVMSLLIKPL---------IRNLIENTWKKINDYQFD--NPRISQTFIGIAMNLARMAQCMYQYGDGHGV-GHLETKDRVKSLLIKPL---------IQNMVANGWKDINEDCMRP--TNAPMLLLQHIVNLVRVTDVMYGDDDDAYTIPLS-LKDYVTLLYVEQVPMCE-----VSQLICDAWKKLNQECLSR--SPFSPSFTKACLNVARMIPLMYSYDDNPGLASLKEHMRSLAAHLESKPF--------LQKTCADAWKDINEDCLKP--TAISMNLLNVCVNNARATDVVYESND-AYTNASC-LKGRISLLFVEKISL--------
553
550
543
573
574
544
591
569
551
589
589
560
528
552
Figure S5. Amino acid sequence comparison of the characterized poplar monoand 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.
injector
temperature
230°C
1
120
2
80
3
Relative abundance
(TIC x 10,000 ions)
4
40
16
injector
temperature
150°C
Relative abundance
(TIC x 10,000 ions)
160
PtTPS7
17
18
Retention time (min)
2
30
5
4
20
10
3
1
16
17
18
Retention time (min)
Relative abundance
(TIC x 10,000 ions)
Relative abundance
(TIC x 10,000 ions)
PtTPS11
40
8
30
cont.
20
1
10
0
7
cont.
6
15
16
17
18
19
20
Retention time (min)
cont.
16
12
7
8
6
8
cont.
9
5
4
1
0
15
16
17
18
19
20
Retention time (min)
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, αselinene; 4, unidentified sesquiterpene; 5, germacrene A; 6, (E)-β-caryopyhyllene; 7, α-humulene;
8, elemol; 9, hedycaryol.
B
A
C
18
20
22
24
26
24
26
18 (-)-caryophyllene
12
6
18
20
22
60
PtTPS9
product
40
20
18
20
22
30
P. trichocarpa
20
volatiles
10
18
20
22
24
24
26
PtTPS15
sesqui80 terpene
40 products
46
40
80
49
47
48
49
(+)-nerolidol(+)-(3S)
40
26
46
47
48
60
40
20
PtTPS15
monoterpene
products
22
(+)-(3S)
23
24
60 (±)-linalool (+)-(3S)
40
20
(-)-(3R)
60
40
20
(-)-linalool
22
23
24
(-)-(3R)
22
49
Retention time (min)
23
24
Retention time (min)
E
PtTPS12 (-)-(3R)-linalool
(+)-(3S)monolinalool
terpene
product
20
22
(±)-linalool
(-)-(3R)
30
24
26
(+)-(3S)
20
10
20
22
24
PtTPS11
3 sesqui2 terpene
1 products
4
26
10 (-)-linalool (-)-(3R)
8
6
4
2
Relative abundance (TIC x 10.000)
Relative abundance (TIC x 10.000)
48
(-)-(3R)
46
120
D
40
47
(±)-nerolidol(+)-(3S)
80
Retention time (min)
60
45
30
15
(+)-(3S)
120
120
Relative abundance (TIC x10.000)
(-)
(+)
Relative abundance (TIC x10.000)
Relative abundance (TIC x 10.000)
(±)-caryophyllene
9
6
3
Time-->
4
14.5
(-)-β-elemene
15.5
16.5
17.5
18.5
17.5
18.5
17.5
18.5
(±)-β-elemene
(+)
3
(-)
2
1
14.5
60
15.5
16.5
(-)-β-elemene
45
(-)
30
15
20
22
24
Retention time (min)
26
14.5
15.5
16.5
Retention time (min)
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 GCMS. Retention times and spectra were compared to those of the pure standards and standard
mixtures shown.
3
5
7
9
1
2
4
6
8
10
basal herbivory
3
5
7
9
1
2
4
6
8
10
apical herbivory
3
5
7
9
1
2
4
6
8
10
control
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.
geranial
500
neral
nerol
0
Relative abundance
(TIC x 10,000 ions)
300
5.00
7.00
9.00
11.00
100
30
13.00
empty vector control + FPP
200
0
Relative abundance
(TIC x 1,000 ions)
geraniol
empty vector control + GPP
(Z)-farnesol
(Z,Z)-farnesal
(E)-farnesol
(E,E)-farnesal
Relative abundance
(TIC x 1,000 ions)
1000
15.00
17.00
19.00
21.00
empty vector control + GGPP
20
10
0
19.00
21.00
23.00
25.00
Retention time (min)
27.00
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.