Supporting Information
Volatile analysis
Analysis of P. nigra volatiles was carried out using GC-MS/FID analysis an Agilent 6890
series gas chromatograph (Agilent, Santa Clara, CA, USA) coupled either to an Agilent 5973
quadrupole mass selective detector (interface temp. 270 °C, quadrupole temp. 150 °C, source
temp. 230 °C; electron energy 70 eV) or a flame ionization detector (FID, temp. 300 °C). The
constituents were separated with a DB-5MS column (30 m x 0.25 mm x 0.25 μm) and He
(mass detector) or H2 (FID) as carrier gases. 1 µL of the sample was injected without splitting
at an initial oven temperature of 40 °C (2 min hold) followed by a ramp to 155 °C (1 °C min 1
), a ramp to 300 °C (60 °C min-1), and a hold for 3 min. To confirm identification of
coeluting compounds, samples were also run on an HP Innowax column (Agilent, 30 m x 0.25
mm x 0.25 μm) under the same conditions, except the final temperature gradient ended at 260
°C.
Compounds were identified by comparison of retention times and mass spectra to those of
authentic standards obtained from Fluka (Seelze, Germany), Roth (Karlsruhe, Germany),
Sigma (St. Louis, MO, USA) or Bedoukian (Danbury, CT, USA) or to reference spectra in the
Wiley and National Institute of Standards and Technology libraries and in the literature
(Joulain and König, 1998). Many standards not commercially available were provided by
Wilfried A. König, University of Hamburg (essential oils of Oreodaphne porosa and Aloysia
sellowii) and DMNT was kindly synthesized by Stefan Bartram (MPI-ICE). The quantity of
each compound was determined from its peak area in the FID trace in relation to the area of
the internal standard using the effective carbon number concept (Scanion and Wills, 1985).
Chiral analysis was performed using a RtTM-ßDEXsm column (Restek, Bad Homburg,
Germany) with a temperature program from 50 °C (2 min hold) to 220 °C (1 min hold) with a
gradient of 2 °C min-1. While formation of artifacts is known during volatile analysis,
1
decomposition was minimized by the use of solvent desorption from a Super Q adsorbent
(Tholl, D., et al. 2006, Plant Journal 45, 540-560). No evidence was found for the
decomposition of any major terpene, green leaf volatile, aromatic or nitrogen-containing
compound during volatile collection and analysis.
Description of the aldoxime synthesis
For testing 2- and 3-methylbutyraldoxime, these compounds were synthesized by
condensation of the respective aldehyde with hydroxylamine (Schwetlick et al., 2009).
Briefly, 10 ml 50% NaOH were added dropwise to a mixture of 50 mmol aldehyde, 55 mmol
hydroxylamine hydrochloride, 12.5 ml water, 2.5 ml EtOH and approximately 20 g ice. After
stirring for 1 h, the mixture was extracted with 20 ml diethylether and the separated aqueous
phase was adjusted to pH = 6 with HCl and extracted twice with 20 ml diethylether. The two
latter ether extracts were combined, dried over CaCl2 and the solvent was evaporated.
Aldoximes were separated from the residue by vacuum distillation (2-methylbutraldoxime, 15
mbar, 73-74 °C; 3-methylbutryaldoxime, 96 mbar, 99-102 °C). Product identity was verified
by LC-MS/MS, GC-MS and 1H NMR. The compounds were obtained as mixtures of the (E)and (Z)-isomers as follows: 2-methylbutyraldoxime, E:Z, 3:1; 3-methylbutyraldoxime, E:Z,
2:1). Both isomers of each compound were also found in the natural blend of P. nigra.
2
Table S1. Volatile compounds collected from Lymantria dispar caterpillars and faeces.
Compounds
Mean
± SEM
(Z)-3-Hexenol
0.281
0.096
(E)-2-Hexenol
0.190
0.082
Non-identified
0.716
0.309
Benzaldehyde
0.124
0.027
Non-identified
0.611
0.152
p-Cymene
0.142
0.057
Non-identified
0.088
0.020
Benzyl alcohol
0.393
0.051
Salicylaldehyde
8.875
2.274
1,2-Cyclohexanediol
4.248
1.344
Nonanal
0.349
0.088
Benzene ethanol
1.247
0.178
Benzyl cyanide
0.066
0.032
Benzoic acid
0.166
0.062
N-decanal
0.079
0.011
Non-identified
0.063
0.008
benzenacetonitrile
5.282
1.324
Eugenol
3.237
0.355
4-Hydroxy-
3
Germacrene D
0.181
0.043
δ-Cadinene
0.176
0.055
1,2-(Z)-Calamanene
0.080
0.019
Nerolidol
0.178
0.065
Caryophyllene oxide
0.207
0.057
Humulene oxide
0.400
0.121
Non-identified
0.347
0.101
Non-identified
0.088
0.029
Non-identified
0.320
0.084
Non-identified
0.050
0.018
Non-identified
0.076
0.030
Non-identified
0.089
0.038
Non-identified
0.048
0.019
4
Table S2. Source, purity and vapor pressure of the standards used for electroantennogram
recordings (EAG). Compounds are arranged in decreasing order according to their vapor
pressure values. (E)-2-Hexenal was used as reference compound.
Compound
Source
Purity
Vapor pressure
mm HG (25°C)
Isoamyl acetate
Fluka
98%
5
(E)-2-Hexenal (Reference)
Fluka
97%
4.62
(Z)-β-Ocimene
Chemos
80%
1.970
2-Methylbutyraldoxime
Chemical synthesis
98%
1.409
Chemical synthesis
98%
1.409
(Z)-3-Hexenyl acetate
SAFC Global
98%
1.220
(Z)-3-Hexenol
Bedoukian
98%
1.040
(E)-DMNT
Chemical synthesis
99%
0.582
Salicylaldehyde
Acros
99%
0.328
Sigma-Aldrich
97%
0.17
Benzyl cyanide
Sigma-Aldrich
95%
0.055
(E)-β-Caryophyllene
Sigma-Aldrich
80%
0.012
(E,E)-α-Farnesene
Chemos
80%
0.007
(E:Z, 3:1)
3-Methylbutyraldoxime
(E:Z, 2:1)
(R)-(
)-Linalool
5
Table S3: List of oligonucleotides used in this study. QPCR, primer were used for qRT-PCR analysis; IBA, primer were used for cloning of the
ORF into the vector pASK-IBA7; TOPO100, primer were used for cloning of the ORF into the vector pET100/D-TOPO.
name
sequence
usage
Ubi_f
GTTGATTTTTGCTGGGAAGC
QPCR
Ubi_r
GATCTTGGCCTTCACGTTGT
QPCR
PnTPS1-1
GTCTGTCCTCATAGATCCTAACC
QPCR
PnTPS1-2
CATTGAGCGTCCCGTAAAGAT
QPCR
PnTPS2-1
GGCGCTCTGGAAATTATCCC
QPCR
PnTPS2-2
CAGCATCCAATGGTTTCTCAAG
QPCR
CYP79D6/7-1
GAGAGACTTGTCCAAGAATCAG
QPCR
CYP79D6/7-2
GAAGTAGTTGGCAACTGTTGT
QPCR
PnTPS1fwd
ATGGTAGGTCTCAGCGCATGGTAGCGACCGAAACTTGTGA
IBA
PnTPS1rev
ATGGTAGGTCTCATATCATATATCTTGGAGAGGAATGGGCTT
IBA
PnTPS2fwd
CACCATGGCATTTCCCATCAATATTGATGGCAAC
TOPO100
PnTPS2rev
CTAGAAAGGTTTGCTTTCTAAATGAGCAGC
TOPO100
6
Table S4: Volatile compounds of Populus nigra released from leaves fed upon by gypsy moth larvae for 41 h and adjacent undamaged leaves as
well as the corresponding leaves from unattacked 1 m tall trees. Herbivory was carried out on basal leaves, designated as “herbivore-damaged” and
the apical leaves on these trees were designated as “adjacent undamaged” (Figure 1). Basal and apical leaves from a tree without herbivory were
used as controls. Emission rates are presented as means ± SE in ng g-1 fw h-1 (n = 20). Information about collection, identification and quantification
of volatiles is given in the supplemental methods section.
Compound
Control tree
Herbivore damaged
Control tree
Adjacent
(basal leaves)
(basal leaves)
(apical leaves)
undamaged
(apical leaves)
Monoterpenoids
1,8-Cineole
2.95
±
1.51
10.56
±
1.98
2.92
± 0.85
5.40
±
1.16
α-Pinene
1.29
±
0.32
4.12
±
0.87
2.95
± 0.77
5.16
±
1.12
α-Terpineol
0.11
±
0.11
0.29
±
0.19
0.06
± 0.04
0.03
±
0.03
β-Pinene
1.61
±
0.44
5.87
±
1.36
4.14
± 1.17
7.65
±
1.75
Borneol
0.00
±
0.00
3.06
±
0.97
0.02
± 0.02
0.40
±
0.22
Camphene
1.95
±
0.48
5.51
±
1.59
4.71
± 1.37
8.22
±
1.95
Camphor
2.12
±
0.29
7.73
±
1.62
4.15
± 0.83
6.40
±
0.98
(Z)-β-Ocimene
1.45
±
0.76
16.20
±
5.64
3.68
± 1.43
5.05
±
1.34
(Z)-Linalool oxide
0.12
±
0.07
13.95
±
4.06
0.23
± 0.12
1.45
±
0.44
7
(E)-Epoxyocimene
0.15
±
0.08
6.79
±
1.48
0.40
± 0.17
1.33
±
0.30
Epoxylinalool
0.00
±
0.00
0.00
±
0.00
0.00
± 0.00
0.00
±
0.00
Limonene
0.69
±
0.17
3.89
±
0.64
1.64
± 0.40
3.16
±
0.54
Linalool
2.11
±
0.85
109.48
± 39.66
3.25
± 1.42
15.36
±
5.57
Linalool oxide
0.00
±
0.00
1.29
±
0.53
0.00
± 0.00
0.04
±
0.04
Myrcene
0.78
±
0.16
4.97
±
0.68
1.19
± 0.18
2.34
±
0.35
Sabinene
1.39
±
0.29
8.27
±
1.20
1.99
± 0.33
4.53
±
0.92
Terpinen-4-ol
0.06
±
0.06
1.35
±
0.27
0.03
± 0.03
0.29
±
0.10
(E)-β-Ocimene
4.73
±
2.13
144.23
± 53.12
9.72
± 2.61
25.37
±
5.80
Alloaromadendrene
1.55
±
0.36
2.68
±
0.67
1.81
± 0.58
2.16
±
0.64
α-Cadinene
0.95
±
0.28
2.53
±
0.54
0.86
± 0.25
1.26
±
0.29
α-Calacorene
0.27
±
0.10
0.59
±
0.18
0.20
± 0.11
0.17
±
0.09
α-Copaene
1.02
±
0.25
5.07
±
0.82
0.96
± 0.30
2.26
±
0.41
α-Cubebene
0.05
±
0.03
3.92
±
0.69
0.11
± 0.06
1.18
±
0.31
α-Humulene
5.01
±
1.34
26.70
±
4.72
10.99
± 2.96
19.56
±
3.20
(E)-β-Caryophyllene
10.51
±
1.82
81.96
± 12.99
17.05
± 3.00
36.75
±
4.22
β-Cubebene
0.07
±
0.04
10.51
±
2.08
0.20
± 0.09
4.07
±
1.00
Caryophyllene oxide
1.81
±
0.36
5.41
±
1.56
1.75
± 0.35
2.32
±
0.33
δ-Cadinene
5.98
±
1.26
19.70
±
3.48
6.80
± 1.81
11.79
±
2.90
Sesquiterpenoids
8
(E,E)-α-Farnesene
1.32
±
0.35
30.92
±
6.00
1.61
± 0.38
7.45
±
4.27
γ-Cadinene
3.77
±
0.82
9.04
±
1.78
4.03
± 1.09
5.83
±
1.49
Germacrene D
1.57
±
0.42
94.00
± 18.14
3.39
± 1.17
46.32
±
11.11
Humulene oxide
2.89
±
0.60
7.40
±
2.52
2.20
± 0.56
2.57
±
0.46
Nerolidol
0.82
±
0.49
4.08
±
1.53
0.11
± 0.07
0.30
±
0.10
unidentified ST11
0.04
±
0.03
1.31
±
0.27
0.13
± 0.07
0.61
±
0.13
unidentified ST2
0.20
±
0.11
2.26
±
0.60
0.43
± 0.21
1.56
±
0.46
unidentified ST3
1.42
±
0.33
5.22
±
1.01
1.40
± 0.40
2.34
±
0.47
unidentified ST4
1.00
±
0.24
2.71
±
0.60
0.75
± 0.22
1.25
±
0.21
unidentified ST5
0.29
±
0.12
3.43
±
0.51
0.58
± 0.19
1.56
±
0.36
unidentified ST6
0.00
±
0.00
5.16
±
1.27
0.00
± 0.00
1.07
±
0.57
unidentified ST7
2.17
±
0.48
5.34
±
1.03
2.19
± 0.61
3.34
±
0.81
unidentified ST8
0.20
±
0.07
1.21
±
0.24
0.30
± 0.09
0.63
±
0.18
unidentified ST-OH12
0.09
±
0.05
3.32
±
0.83
0.20
± 0.10
0.72
±
0.14
unidentified ST-OH2
0.56
±
0.15
1.36
±
0.48
0.50
± 0.17
0.55
±
0.16
unidentified ST-OH3
2.62
±
0.93
3.68
±
1.46
1.97
± 0.52
2.82
±
0.68
unidentified ST-OH4
2.44
±
0.59
5.94
±
1.77
2.52
± 0.74
2.86
±
0.68
unidentified ST-OH5
1.26
±
0.73
3.46
±
1.70
0.70
± 0.49
2.14
±
1.09
unidentified ST-OH6
2.04
±
0.46
5.21
±
1.34
1.65
± 0.39
2.74
±
0.71
unidentified ST-OH7
0.00
±
0.00
1.69
±
0.33
0.03
± 0.03
1.09
±
0.29
9
Homoterpenoids
1.67
±
0.29
514.60
± 82.95
1.60
± 0.34
50.55
±
14.94
0.00
±
0.00
2.90
±
0.83
0.00
± 0.00
0.20
±
0.09
1-Hexylacetate
0.03
±
0.03
3.85
±
2.31
0.11
± 0.06
0.17
±
0.08
(Z)-3-Hexenol
0.03
±
0.02
30.29
± 13.15
0.25
± 0.10
3.32
±
3.10
(Z)-3-Hexenyl acetate
1.59
±
0.41
129.10
± 75.12
2.47
± 0.64
5.57
±
2.75
(Z)-3-Hexenyl
0.00
±
0.00
5.09
±
3.57
0.00
± 0.00
0.00
±
0.00
0.00
±
0.00
11.55
±
4.09
0.03
± 0.03
0.10
±
0.06
(E)-2-Hexenyl acetate
0.00
±
0.00
2.25
±
1.48
0.03
± 0.03
0.00
±
0.00
(Z)-3-Hexenyl
0.00
±
0.00
17.41
± 14.28
0.00
± 0.00
0.00
±
0.00
(E)-2-Methylbutyraldoxime
0.12
±
0.06
38.26
±
9.19
0.08
± 0.06
0.60
±
0.47
(Z)-2-Methylbutyraldoxime
0.03
±
0.03
11.50
±
2.22
0.05
± 0.05
0.00
±
0.00
2-Phenylnitroethane
0.09
±
0.09
4.39
±
1.52
0.16
± 0.16
0.00
±
0.00
(E)-3-Methylbutyraldoxime
0.05
±
0.05
5.98
±
3.70
0.00
± 0.00
0.61
±
0.54
(E)-DMNT
(Z)-DMNT
Green leaf volatiles
isobutyrate
(Z)-3-Hexenyl
isovalerate
benzoate
Nitrogenous
10
(Z)-3-Methylbutyraldoxime
0.00
±
0.00
5.82
±
1.32
0.00
± 0.00
0.00
±
0.00
Benzyl cyanide
0.00
±
0.00
56.28
± 24.12
0.00
± 0.00
0.10
±
0.07
Indole
0.00
±
0.00
3.14
±
0.91
0.00
± 0.00
0.02
±
0.02
(E)-Phenylacetaldoxime
0.00
±
0.00
0.48
±
0.22
0.03
± 0.03
0.00
±
0.00
(E)-Phenylacetaldoxime
0.00
±
0.00
0.53
±
0.17
0.00
± 0.00
0.00
±
0.00
Amyl benzoate
0.05
±
0.05
1.13
±
0.33
0.11
± 0.11
0.46
±
0.40
Benzaldehyde
0.70
±
0.15
3.12
±
1.00
0.68
± 0.16
1.08
±
0.14
cis-Jasmone
0.02
±
0.02
4.20
±
0.87
0.12
± 0.08
1.17
±
0.44
Eugenol
0.04
±
0.04
4.90
±
2.16
0.05
± 0.04
0.19
±
0.13
Methylsalicylate
0.08
±
0.08
5.64
±
1.19
0.11
± 0.08
0.96
±
0.38
Salicylaldehyde
0.17
±
0.09
4.56
±
1.76
0.03
± 0.03
0.34
±
0.34
1,2-Cyclohexanediol
0.05
±
0.05
0.99
±
0.24
0.00
± 0.00
0.22
±
0.10
2-Hydroxy-cyclohexanone
0.00
±
0.00
2.07
±
1.11
0.00
± 0.00
0.25
±
0.17
2-Phenylethyl acetate
0.00
±
0.00
0.44
±
0.22
0.00
± 0.00
0.00
±
0.00
4-Pentene-1-yl acetate
1.16
±
0.34
3.22
±
1.22
1.36
± 0.46
1.66
±
0.44
Isoamyl acetate
0.02
±
0.02
3.42
±
0.95
0.05
± 0.04
0.06
±
0.04
Nonanal
2.04
±
0.32
4.02
±
1.12
1.57
± 0.30
2.14
±
0.27
Aromatics
Others
11
Phenylmethyl acetate
0.17
±
0.10
2.01
±
0.78
0.81
± 0.57
1.05
±
0.32
Prenyl acetate
1.78
±
0.49
3.11
±
0.75
1.73
± 0.47
2.15
±
0.37
1
ST, sesquiterpene
2
ST-OH, sesquiterpene alcohol
12
13
Figure S1: Four-field arena for parasitoid preference tests.
14
Figure S2 Hourly release rates of dispensers with P. nigra herbivore-induced volatiles used
for field tests of parasitoid attraction. Compounds were diluted in hexane at a concentration of
10 μg μl-1, during an 11 day period. Release rates are displayed as means ±SEM in μg/h.
Daily measurements were carried out in a chamber with controlled humidity (70%) and
temperature (25°C) resembling early summer field conditions. During the first seven days
release rates for all compounds remained relatively homogeneous at an average of around 5
μg h-1 at 25°C.
15
Figure S3. Design of the traps used for field experiments on parasitoid attraction. Photograph
shows a dispenser pasted at the bottom of a rectangular yellow sticky trap (10 x 15 cm) which
was in turn pasted vertically onto a “hat trap”. The whole apparatus was attached to a tree
branch.
Hat trap
Yellow sticky trap
Dispenser
16
Figure S4: Sequence comparison of PnTPS1 and PnTPS2 with a monoterpene synthase
(PtTPS3) and a sesquiterpene synthase (PtTPS2) from Populus trichocarpa. The sequence
alignment
was
performed
with
the
software
BioEdit
(http://www.mbio.ncsu.edu/bioedit/page2.html) using the ClustalW algorithm. Amino acid
motives involved in metal cofactor binding are labeled DDxxD and
DDxxTxxxE/NDxxTxxxE. Amino acids identical in all four sequences are shown in black
boxes and amino acids with similar side chains are shaded in gray boxes. The putative Nterminal signal peptides are underlined and italicized.
PnTPS1
PnTPS2
PtTPS2
PtTPS3
MALYQLAPFPISTVTKRTFSRRTSLGSSRNGCFPSEVRCMVATETCDQSIARRSGNYPTPFWDHKFLQSLTSEYVGEPYT
----------------------------------------------MAFPINIDGNFSASF----HLPSLENELCLRHGK
-----------------------MEYKQQVQVVQNSFQCQNNSEDIDRRQERRSANYKPNIWKYDFLQSLSSKYDEEQYR
MALSCSVSLTAASGWPFPQ----NRNSERVKPILKEFKPTLPSTQKWSVSQKQTLAFGPT---KQYPITINNDDSDTGYA
80
30
57
73
PnTPS1
PnTPS2
PtTPS2
PtTPS3
GQANKLKETVRDMLEKPLDAVYQLELIDNLQRLGVAYHFELEIKSILESRWTDYKKDNREMKEDLYATSVEFRLLRQHGY
MVKEAGCILSNTAGKDPLEGLV---MTDALQRLGIDYHFREEIEAFLNTQYMNLSSPNHP-PLDVFGVALRFRLLRQEGY
RVTEKLREEVKSIFVEAVDLLAKLKLVDSVIKLGLGSYFEEEIKQSLDIIAASIKNKNLKVEENLYVTALRFKLLRLHGY
EKLQTFKHILRKEGEEPIQGLA---MIDAIQRLSIDYHFQEEIDSILTRQSMLLSTIHSD--NNLYEVALRFRLLRQQGY
160
106
137
148
PnTPS1
PnTPS2
PtTPS2
PtTPS3
NVPQDVFNSFKDEQGNFKNCLRDDVKGMLNLYEASYYLGNGESILEEARDFSEKHLKEYSKEQNEDHYLSLLVNHSLELP
NVSQEVFNNFKNEEGNFHLIQENDVKGLMALYEASQLSMESEDILDEAGEFSAKLL---------NHHESEIVANTLKHP
EVSQGVFNGFFDGTSDKSKC--TDVRGLIELFEASHLAYEGEATLDDAKAFSTRIL-TGINCSAIESDLAKHVVHVLELP
HVSAGVFDNFKDNEGRFKQQLSSDIMGLVSLYEASQLSIRGEDVLDEAGDYSYQLL--RSSLTHLDYNQARLVRNSLDHP
240
177
214
226
PnTPS1
PnTPS2
PtTPS2
PtTPS3
LHWRMQRMEARWFIDAYGRKRDLNPILLEFAGLDFNMVQAKYQEDIRHASRWWTSMDLGNKLFYTRDRLMENTLWAVGEV
YHKSLARFMVKNFLNNIDIGNENIKVFSELAKIDCEIVRSIHQKEILQISNWWEDLGLAKELKFARDQPLKWHMRSMSVL
SHWRVMWFDVKWHINAYENDKQTNRHLLALAKVNFNMVQATLQKDLGDVSRWWRNLGIIENLSFTRDRLVESFLCTVGLV
HHKSLASFTAKYFFN--DEPNGWIGELQELAKTEFKRVQSQHQHEIVEIFKWWKDLGLSTELRFARDQPLKWYMWSMSCL
320
257
294
304
PnTPS1
PnTPS2
PtTPS2
PtTPS3
DDxxD
FEPQFGYYRKMATRVNALITTLDDAYDVYGTLEELEVFTDVIERWDINALDQLPYYMKISFFALFQSINEIGYNILKEQIDPNLSEQRVELTKPISLVYIIDDIFDLYGTLNDLSIFTEAVNEWDLTAANQLPESMKISLKALFDITASISTKIFEKHFEPKYSSFRKWLTKVIIMILIIDDVYDVYGSLHELQQFTKAVSRWDTGEVQELPECMKICFQTLYDITNEMALEMQREKD
TDPSLSEQRIELTKPVSMIYIIDDIFDVHGTLNELVCFTEVINRWDIAAAEQLPDYMKICFKALNNITNEISYKIYKEH-
399
336
374
383
PnTPS1
PnTPS2
PtTPS2
PtTPS3
GINVVPSLKKLWGDLCRAFLKEAKWYYAAYTPTLQEYLDNAWLSISGQVILGHAFFLVTNQL-TEEAVRCCMEYPDLIRY
GWNPIESLQKSWKKLCNAFLEEAKWFASGKLPKPEEYLRNGIVSSGVHVVLVHMFFLLGQGI-NKETVDFVDGFPPIISF
GSQALPHLKKVWADFCKAMFMEAKWFNEGYTPSLQEYLSNAWVSSSGTVISVHSFFSVMTELETGEISNFLEKNQDLLYN
GWNPVESLRKAWASLCRAFLVEARWFASGKFPSGEEYLKNGIVSSGVHVVLVHIFFLLGQGI-SKENVELISNFPPIISS
478
415
454
462
PnTPS1
PnTPS2
PtTPS2
PtTPS3
NDxxSxxxE
DDxxTxxxE
SSTILRLADDLGTSSDEIARGDNPKSIQCYMHET-GATEQEAREHVRYLIHETWKKLNAEILKPYPFSKKFMGIPMDLAR
TATILRLWDDLGTAKDENQDGHDGSYLECYIREHPNVTVERAREHVSHLICDAWKKLNQECLSPSPFSPSFTKACLNVAR
ISLIIRLCNDLGTSVAEQERGDAASSVACYMREV-NVSEEVARNHINNIVKKTWKKINGHCFTKSPTLQLLVNINTNMAR
TATILRLWDDLGSAKDENQDGHDGSYVECYLRENEGSSLEDARKQVLHLISDAWKQLNQECLSPNPFPSTFSKASLNIAR
557
495
533
542
PnTPS1
PnTPS2
PtTPS2
PtTPS3
TAQCFYEAGDAYGIQDQETHGRLASLFVKPIPLQDI-----MIPLMYSYDDNPSLASLKEHMRSLAAHLESKPF--------VVHNLYQHGDGFGVQDRHENKKQI-LTLLVEPFKLDLPEFSF
MVPLMYDYDDNHRLPSLEEHMKSL-LYENVSP----------
17
593
528
574
573
Figure S5: GC-MS analysis of enzyme products from recombinant PnTPS1 and PnTPS2. The
enzymes were expressed in E. coli, partially purified, and incubated with the potential
substrates GPP and FPP. Products were collected with a solid-phase microextraction (SPME)
fiber and analyzed by GC-MS. 1, α-pinene; 2, camphene; 3, β-pinene; 4, limonene; 5, αterpinolene; 6, (E)-nerolidol; cont, contamination.
18