SUPPLEMENTAL MATERIALS
Supplemental Materials and Methods
Identification of capsidiol by 1H- and 13C NMR
1
H- and
13
C-NMR spectroscopy were performed on a Bruker Biospin 500 spectrometer
equipped with a cryoprobe in CDCl3 solution. Chemical shifts are reported in ppm relative to
CHCl3 ( = 7.260 ppm) for 1H-NMR and CDCl3 ( = 77.16 ppm) for 13C-NMR spectroscopy.
13
C signal assignments were made according to Stillman et al. (1981) for capsidiol and
Marshall et al. (1990) for α-cembratrienediol. All solvents of analytical grade were redistilled
in a glass apparatus. For labeling experiments, leaves including their petioles were cut from 2
month-old tobacco plants (Nicotiana tabacum L. var. xanthi) grown in greenhouse. A 1%
water solution (10 mL per 3 leaves) of (1-13C)-D-glucose (99% isotope abundance) was
supplied and absorption was supported by a transpiration stream using a horizontal laminar
flow hood. Once all visible liquid was absorbed, entire leaves were elicited for 16 h and
capsidiol was adapted from the protocol described above (capsidiol production, extraction and
analysis). A control experiment was performed without addition of any glucose to obtain a 13C
natural abundance sample.
Proteins interfering with the extraction procedure were precipitated with one volume of icecold acetone, removed from the collected aqueous/acetone solution by centrifugation and the
water/acetone phase after elicitation was evaporated and the remaining aqueous solution was
extracted with ethyl acetate (3 x 200 mL). The combined extracts were dried over anhydrous
MgSO4, filtered and evaporated to dryness. This crude extract proved by 1H-NMR to contain
mainly capsidiol and -cembratrienediol. As there was no overlapping of the
13
C signals of
capsidiol and -cembratrienediol, the two terpenoids were not separated and the sample
directly analyzed by NMR spectroscopy.
13
C isotope abundances of each carbon were
determined by comparing the relative intensities of the signals 13C labeled sample with those
of the 13C natural abundance sample (1.1 %) using as internal reference the C-11 signal in the
capsidiol spectrum and the C-15 signal for the -cembratrienediol spectrum following the
procedure previously described reported (Rohmer et al., 1993).
Identification of capsidiol by GC-MS
Capsidiol was identified by GC-MS with a 6890 gas chromatograph (Agilent) equipped with a
HP5-MS column (Agilent J & W; 30m long, 0.32mm i.d., 0.25 mm film thickness) coupled
1
with a 5973 mass selective detector. Injections were made in splitless mode with ratio of 50;
and the temperature of the injector was set at 250°C.The temperature program was a ramp
from 60°C to 300°C at 10°C per min followed by a 5 min plateau. He as a carrier gas was set
at 2mL/min. Mass spectra obtained for capsidiol at an electron ionization of 70eV were in
agreement with that of an authentic sample (Mialoundama et al., 2009).
Identification of capsidiol by UHPLC-ESI- MS/MS
Extracts were analyzed on an ultra high performance liquid chromatography coupled to
tandem mass spectrometry (UHPLC-MS/MS) at MS and MS/MS mode. The analyses were
performed on a Waters Quattro Premier XE (Waters, Mildorf, MA USA) equipped with an
Electrospray Ionisation (ESI) source and coupled to an Acquity UPLC system (Waters) with
diode array detector (DAD). UV spectra were recorded from 190 to 500 nm. Chromatographic
separation was achieved using an Acquity UPLC BEH C8 column (100 x 2.1 mm, 1.7µm;
Waters), coupled to an Acquity UPLC BEH C8 pre-column (2.1 x 5 mm, 1.7µm; Waters). The
two mobile phases were water (MS grade, Sigma) with 1% 1M NH4Ac, 0.1% acetic acid
(Buffer A), and acetonitrile: isopropanol (7:3, UHPLC grade Sigma) containing 1% 1M NH4Ac, 0.1% acetic acid (Buffer B). The gradient separation, which was performed at a flow rate
of 400 μL min-1, was as follow: 1 min 45% of buffer A, 3 min linear gradient from 45% of
buffer A to 35% of buffer A, 8 min linear gradient from 25 to 11% of buffer A, 3 min linear
gradient from 11% A to 1% of buffer A. After washing the column for 3 min with 1% of
buffer A the buffer was set back to 45% of buffer A and the column was re-equilibrated for 4
min (22 min total run time). To determine capsidol best ionization parameters, positive and
negative electrospray scan modes were used. Capsidiol was detected only at [M+ H-H2O] and
[M+ H-2H2O] parent ions as described by Literakova et al. (2010). Capsidiol with adducts of
(Cl, Na, K) were searched and not detected. The selected Ion recording (SIR) MS mode was
then used to determine parent mass transition conditions. For the SIR mode the [M+ H-H2O]
and the [M+ H-2H2O] parent ion were selected, (m/z 219 and m/z 201) as described by
Literakova et al. (2010). Daughter scan (DS) MS/MS mode was then used to determine the
fragmentation pattern of capsidiol. For fragmentation the cone voltage was set to 25 eV and
collision energy after optimization was set to 18 eV. The fragmentation pattern of capsidiol
was compared to published data and was similar to that obtained by Literakova et al. (2010).
Acquisition and analysis were performed with the MassLynx software (version 5.1) running
under Windows XP professional on a Pentium PC.
2
Cloning and heterologous expression of tobacco protein farnesyltransferase and protein
geranylgeranyltransferase
Total RNA was isolated from 3-day-old tobacco BY-2 cells using a Trizol-based protocol and
cDNAs were prepared following the manufacturer’s instructions (Invitrogen). The cDNA
encoding the alpha subunit of tobacco protein prenyltransferase was PCR amplified using
primers from the full length Nicotiana benthamiana EST clone (TC18998). The full cDNAs
coding for the β subunits (β-farnesyltransferase and β-geranylgeranyltransferase) were not
available. Thus, a cDNA fragment was amplified as described by Courdavault et al., (2005).
5’-RACE-PCR and 3’-RACE-PCR were realized to determine the sequence of each extension
of the cDNA according to the protocol described by Scotto-Lavino et al. (2006). Finally, after
reconstitution of the sequence, a further PCR amplification was performed for isolation of the
full-length cDNA to make sure that the reconstituted sequence belongs to the same gene. All
primers utilized to isolate the full-length cDNAs coding for the three subunits are listed in
Table S5.
For heterologous expression in E. coli, the sequence coding for the α-subunit was cloned into
the same vector as the β subunit to allow coexpression and reconstitution of the heterodimers
constituting the protein prenyltransferases. The XhoI site in NtβF was removed by sitedirected mutagenesis using primers listed in Table S5. Open-reading frames were PCRamplified to introduce required restriction sites and the products were cloned into pRSFDuet1 (Novagen). Primers are listed in Table S5. The generated plasmids pRSF-NtPGGT
(containing the Ntα and the NtβGG genes) or pRSF-Nt-PFT (containing the Nt-alpha gene and
the NtβF) were transformed in E. coli BLRA cells (BL21DE3 cells (Novagen), containing the
pRARE plasmid (Novagen)). Overnight cultures were diluted 100-fold in Luria Bertani (LB)
medium containing kanamycin (30 µg mL-1). They were cultivated at 32°C and induced with
isopropyl-β-D-thiogalactoside to a final concentration of 0.1 mM when the A600 reached 0.7,
and further cultivated for 18 h before being harvested and stored at -20°C.
Determination of inhibition of cell growth, cell death, toxicity, AOS production induced
by chemical treatment
The cell growth rate was evaluated by determining the fresh weight of cells collected by
suction filtration after 24 h culturing. The rate is expressed in percentage of growth of treated
cells, compared to the non-treated cell suspension. Following the protocol based on a double
staining technique using propidium iodide and fluorescein diacetate and described by
Hemmerlin and Bach (1998), cell viability was evaluated by fluorescence microscopy. Active
3
oxygen species (AOS) were detected by the DAB-uptake method using 3,3’diaminobenzidine (1 mg mL-1) and described by Thordal-Christensen et al. (1997).
Supplementary REFERENCES
Courdavault V, Burlat V, St-Pierre B, Giglioli-Guivarc’h N (2005) Characterization of
CaaX-prenyltransferases in Catharanthus roseus: relationships with the expression of genes
involved in the early stages of monoterpenoid biosynthetic pathway. Plant Sci 168: 1097-1107
Literakova P, Lochman J, Zdrahal Z, Prokop Zbynek, Mikes V, Kasparovsky T (2000)
Determination of capsidiol in tobacco cells culture by HPLC. J Chromatogr Sci 48: 436-440
Mialoundama AS, Heintz D, Debayle D, Rahier A, Camara B, Bouvier F (2009) Abscisic
acid negatively regulates elicitor-induced synthesis of capsidiol in wild tobacco. Plant Physiol
150: 1556-1566
Marshall JA, Robinson ED, Lebreton J (1990) Synthesis of the tumor inhibitory tobacco
constituents α- and β-2,7,11-cembratriene-4,6-diol by diastereoselective [2,3] Wittig ring
contraction. J Org Chem 55: 227-239
Rohmer M, Knani M, Simonin P, Sutter B, Sahm H (1993) Isoprenoid biosynthesis in
bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem J
295: 517-524
Scotto-Lavino E, Du G, Frohman MA (2006) 5’ end cDNA amplification using classic
RACE. Nat Protoc 1: 2555-2562
Stillman MJ, Stothers JB, Stoessl A (1981) Capsidiol and 1-epicasidiol: absolute
configuration, NMR, and optical spectra of the dibenzoates. Can J Chem 59: 2303-2035
Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997) Sub-cellular localization of
H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barleypowdery mildew interaction. Plant J 11: 1187-1194
4
Table S1. 1H-NMR (500 MHz, CDCl3) and 13C-NMR (125 MHz, CDCl3) of capsidiol and
(1S,2E,4S,6R,7E,11E)-2,7,11-cembratriene-4,6-diol.
Metabolite
Capsidiol
1
H-NMR  = 5.93 (1H, dd, J = 6.8, 2.1 Hz, 9-H), 4.72 & 4.69 (2x1H, 2
br. s, 12-Ha, 12-Hb), 4.57 (1H, dt, J = 12.3, 4.6 Hz, 3-H), 4.34
(1H, dd, J = 3.8, 2.5 Hz, 1-H), 1.72 (3H, s, 13-H), 1.35 (3H, s,
15-H), 0.86 (3H, d, J = 6.9 Hz, 14-H).
C-NMR  = 149.6 (C-11), 140.5 (C-10), 129.2 (C-9), 108.9 (C-12),
13
75.2 (C-1), 65.6(C-3), 47.8(C-4), 45.0 (C-6), 40.2(C-7),
39.2(C-5), 36.3(C-2), 32.2 (C-15), 30.5 (C-8), 21.2 (C-13), 9.1
(C-14).
α-Cembratrienediol
1
H-NMR  = 5.33 (3H, m, 2-H, 3-H, 7-H), 5.04 (1H, t, J = 6.0 Hz, 11H), 4.48 (1H, ddd, J = 9.5, 7.9, 2.6 Hz, 5-H), 1.67 (3H, d, J =
1.2 Hz, ), 1.52 (3H, br. d, J = 1.2 Hz, ), 1.35 (3H, s, H-18),
0.82 & 0.79 (2x3H, 2 d, J = 6.7 Hz, 6.7 Hz, 16-H/17-H).
C-NMR  = 137.6 (C-3),137.0 (C-8), 133.6 (C-12), 130.7 (C-7), 127.9
13
(C-2), 124.5 (C-11), 72.6 (C-4), 66.5 (C-6), 52.3 (C-5), 46.5
(C-1), 39.0 (C-9), 36.9 (C-13), 33.1 (C-15), 30.3, (C-18), 28.1
(C-14), 23.7 (C-10), 20.8 (C-17), 19.5 (C-16), 16.2 (C-19),
15.2 (C-20).
5
Table S2. 13C Isotopic abundances of isoprene units from capsidiol after incorporation of (113
C)glucose. The signal in C-11 (that is labeled neither by the MVA, nor by the MEP
pathway) was utilized as internal reference. When indicated (4 or 5), the corresponding
isoprene units can not be discriminated.
Corresponding
Capsidiol
carbon
δ (ppm)
atoms
isoprene units
Isotope
IPP and DMAPP
abundance
numbering
11
149.6
3
1.1
10
140.5
3
1.2
9
129.2
4
5.9
12
108.9
4 or 5
6.5
1
75.2
2
6.1
3
65.6
4
6.3
4
47.8
3
1.2
6
45
1
1.2
7
40.2
2
6.5
5
39.2
2
5.7
2
36.3
1
1.3
15
32.2
5
5.6
8
30.5
1
1.6
13
21.2
4 or 5
6.2
14
9.1
5
6.5
6
Table S3.
13
C isotope abundance of isoprene units of α-cembratrienediol (α-CBT) after
incorporation of (1-13C)glucose. The signal of C-15 (that is labeled neither by the MVA, nor
by the MEP pathway) was utilized as internal reference
Corresponding
α-CBT
carbon
δ (ppm)
atoms
isoprene units
Isotope
IPP and DMAPP
abundance
numbering
3
137.7
4
1.2
8
137.1
3
1.1
12
133.6
3
1.2
7
130.7
4
1.1
2
127.9
1
1.4
11
124.5
4
1.2
4
72.6
3
1.2
6
66.5
1
1.4
5
52.3
2
1.2
1
46.5
2
1.0
9
39
2
Overlap
13
36.9
2
1.2
15
33.1
3
1.1
18
30.3
5
1.4
14
28.1
1
1.5
10
23.4
1
Overlap
17
20.8
5
1.3
16
19.5
4
1.3
19
16.2
5
1.4
20
15.2
5
Overlap
.
7
Table S4. Effect of carvone and known inhibitors of protein isoprenylation on capsidiol production in cellulase-elicited tobacco leaf-disks.
Capsidiol was extracted from 3 mL culture-medium used to treat the leaf-disks during 18 h.
Compound
Formula
FW
Structure
Conc.
Control
S-Carvone
Percentage
(capsidiol)
100%
C10H14O
O
150.21756
2 mM
0%
2 mM
0%
2 mM
0%
2 mM
0%
O
R-Carvone
C10H14O
150.21756
OH
S-Perillyl alcohol
C10H18O
154.24932
OH
R-Perillyl alcohol
C10H18O
154.24932
8
Limonene
PFT Inhibitor I
GGti-2133
C10H16
136.23404
NH
C19H29NNa3O6P
O
467.37918
C27H28N4O3
456.53622
COONa
PO 3Na2
H3C
O
NH
H3C
HO
NH
2 mM
100%
60 µM
100%
60 µM
49%
50 µM
42%
50 µM
100%
N
NH
O
O
AFC
C20H33NO3S
367.54592
N
H
COOH
S
O
AGGC
C25H41NO3S
435.66294
N
H
COOH
S
9
Table S5. Oligonucleotides used in this study. Oligonucleotides were custom synthesized (Sigma-Aldrich).
Name
Sequence (5’-3’)
Comments
Ntα-F
GTCCGATCAGATCCCCGAGATCGA
Isolation of Ntα
Ntα-R
TTCCCAGCAGATTCCGACGTG
Isolation of Ntα
NtβFdeg-F
GACCWTGGCTKTGYTACTGG
Cloning of a specific DNA fragment coding for NtβF
NtβFdeg-R
ARGTATACCCRCCRTGAGC
Cloning of a specific DNA fragment coding for NtβF
NtβGGdeg-F
CAACAGSMWGATGGAAGYTTTATGC
Cloning of a specific DNA fragment coding for NtβGG
NtβGGdeg-R
AAARCCACCRTCRTAWGAYTGACA
Cloning of a specific DNA fragment coding for NtβGG
NtβF-F1
CATTCAATCGCTTTGTTGGGAGAA
3’-RACE for NtβF
NtβF-F2
CGCAACAACTTATGCTGCAGTCAA
3’-RACE for NtβF
NtβF-R1
AACCTGGTTCGCCAGCAATTCCAC
5’-RACE for NtβF
NtβF-R2
ATGCATCCTGAAGCCACCACTTGTG
5’-RACE for NtβF
NtβGG-F1
AGAGACAGACCTCCGCTTTGTA
3’-RACE for NtβGG
NtβGG-F2
AACTGGAGTGGCATTGACAAGGAG
3’-RACE for NtβGG
NtβGG-R1
TCCAACATGGAAGAGATTGCAGC
5’-RACE for NtβGG
NtβGG-R2
TACAAAGCGGAGGTCTGTCTCTG
5’-RACE for NtβGG
Q0
CCAGTGAGCAGAGTGACG
3’- and 5’- RACE
QT
CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTTT
3’- and 5’- RACE
QI
GAGGACTCGAGCTCAAGC
3’- and 5’- RACE
NtβF-F3
ATGGAGTCGAGGAGAGTAACGAAGACGCTGG
Site-directed mutagenesis of NtβF to remove the XhoI-RS
NtβFmutXhoI-R3
GAGACCCCGAGTGAGATAATCG
Site-directed mutagenesis of NtβF to remove the XhoI-RS
BamHI-Ntα-F
CGGGATCCGATGGATACTGGCGAAGATAAGCG
Construction of the pRSF-His6α plasmid
Ntα-HindIII-R
CCCAAGCTTCTACAAGTTATCCTGCATGC
Construction of the pRSF-His6α plasmid
NdeI-NtβGG-F
GGGTTTCATATGGCGGAGGAAGATGATGAATTTCGGAGC
Construction of the pRSF-His6α-βGG plasmid
NtβGG-XhoI-F
CCGCTCGAGTCAAAGTCTAGTGCGTGTACCA
Construction of the pRSF-His6α-βGG plasmid
10
NdeI-NtβF-F
GGGTTTCATATGGAGTCGAGGAGAGTAACGAAGACGCTGG
Construction of the pRSF-His6α-βF plasmid
NtβF-XhoI-F
CCGCTCGAGTCACAAGCATGAGAAGAACCTGCG
Construction of the pRSF-His6α-βF plasmid
NdeI-NtHMGR2-F
GGAATTCCATATGCATCACCATCACCATCACGATAATAATGATGAATGTTGGGAT
Construction of the pBAD-H6-CatHMGR2 plasmid
NtHMGR2-EcoRI-R
CGGAATTCCGGTTAGGAGGATGCCTTTGTGAC
Construction of the pBAD-H6-CatHMGR2 plasmid
N6
NNNNNN
Reverse transcription random hexamers
dT18
TTTTTTTTTTTTTTTTTTVN
Reverse transcription
Q-actin-F
TGATAACGGAACAGGAATGG
Quantitative RT-PCR
Q-actin-R
TCGAGGTCGACCAACAATAC
Quantitative RT-PCR
Q-EF2-F
TGCTGGTACACAAGCTCATCAA
Quantitative RT-PCR
Q-EF2-R
AGTCACTGCCTGCTTCAAACC
Quantitative RT-PCR
Q-α-F
GCTATTCCGTTAAACCCTGGAAA
Quantitative RT-PCR
Q-α-R
CCCAGCAATGCGATCAACA
Quantitative RT-PCR
Q-βF-F
CGTCAAGTTCGGGAGATATACGA
Quantitative RT-PCR
Q-βF-R
GTGCTTATCACGTTGAAGCTCTAAGAT
Quantitative RT-PCR
Q-βGG-F
CTGGAGTGGCATTGACAAGG
Quantitative RT-PCR
Q-βGG-R
GTGGCACCACCATGTGATTC
Quantitative RT-PCR
Q-HMGR1-F
GGCATTAGTCATGTGGAGAAAG
Quantitative RT-PCR
Q-HMGR1-R
CTCACATGCCTTCATAGAAAGAATACA
Quantitative RT-PCR
Q-HMGR2-F
TCAGGACCCAGCTCAGAACATA
Quantitative RT-PCR
Q-HMGR2-R
TCAATGGAAGGCATTGTAACAGAA
Quantitative RT-PCR
11
Figure S1. GC-MS spectra of (1S,2E,4S,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (A) and
capsidiol (B).
12
Figure S2. Ultra-performance liquid chromatography coupled with tandem mass
spectrometry (UHPLC-MS/MS) spectra of capsidiol. A. Single ion recording
chromatograms of untreated leaf-disks or elicited for 4 h or 18 h. The accumulation of
capsidiol is indicated. B. MS fragmentation spectrum. ES+ scan of the compound with a
retention time of 3.99 min corresponding to capsidiol (FW: 236.34986).
13
Figure S3. Subcellular distribution of the fluorescence observed by confocal microscopy
in tobacco BY-2 cells expressing GFP-BD-CVIL and treated with different inhibitors
used in this study. Illustrated pictures are representative of the majority of observed cells
under each condition. Control, untreated cells; S-Ca: S-carvone (1 mM); Fol: farnesol (20
µM); GGol: geranylgeraniol (20 µM); PFT-I: PFT Inhibitor I (60 µM); GGti: GGti-2133 (60
µM); Fos: fosmidomycin (100 µM); MV: mevinolin (10 µM). Cell suspensions were shacked
(130 rpm) for 18 h in six well-plates at 26°C in the dark. White bar = 20 µm.
14
Figure S4. Evaluation of IC50 values for the inhibition of Nt-PPTases initial velocities by
S-carvone. SDS-PAGE (Coomassie blue stained) of total protein extracts of E. coli cells in
which PPTases were overexpressed. The corresponding purified heterodimeric enzymes are
also illustrated with the subunit Nt-α (41 kDa), Nt-βF (51 kDa) and Nt-βGG (40 kDa). S:
supernatant; E250: elution with 250 mM imidazole. The fraction S was used to determine
initial velocities (V0) of PPTases in the presence of increasing concentrations of S-carvone
using a fluorimetric assay. PFT activity was measured with Dansyl-GCVIM and FPP as
substrates, while PGGT-I activity was measured with Dansyl-GCVIL and GGPP as
substrates. Percentages of residual activities were calculated and IC50 determined (IC50PFT =
1.4 mM). The IC50PGGT-I could not be determined, as it appeared that for concentrations < 4
mM, V0 were activated. Radio assays using untreated tobacco BY-2 protein extracts were
performed as described in the Materials and Methods section using His6-GFP-BDCVIM/[3H]FPP as substrates for PFT or His6-GFP-BD-CVIL/[3H]GGPP to evaluate PGGT-I
activity. Increasing concentrations of S-carvone were added in vitro.
15