1
Methods S1. A manual for ST/TD-GC-QMS analysis of PVs.
2
3
Preparation and conditioning of silicone laboratory tubing (ST)
4
Prior to analysis, silicone laboratory tubing (ST, 1 mm i.d. x 1.8 mm o.d., Carl Roth) is
5
prepared in batches by cutting into pieces of uniform size using a scalpel and a handmade
6
cutting mask, and soaking in 4/1 (v/v) acetonitrile/methanol for 3 h (Figure 1a). (We use
7
0.5 cm pieces corresponding to an 8.8 μL volume, but larger pieces may also be used to
8
increase sensitivity as shown in Figure S1, keeping in mind that larger pieces will cause larger
9
silicone peaks from the STs which may need to be excluded from measurement in the MS
10
method.) Solvent is decanted, and up to 2,000 STs are poured into two 50 mL glass columns
11
each containing a glass frit to disperse gas flow. The glass columns are connected to N2 gas
12
flow at 5 L/min to flush solvent and contaminants, and transferred into a modified heating
13
oven (Figure 1a). After 3 h of heating at 210 °C, STs are allowed to cool to RT under N2 flow,
14
then transferred to glass vials and stored in aliquots under Ar for subsequent use.
15
Alternatively, STs can be soaked in solvent overnight, which does not noticeably alter the
16
cleanliness of STs or the recovery of plant volatiles (PVs) compared to 3 h soaking. Within
17
each experiment, we always use STs from a single batch.
18
19
Sampling of volatiles from standard solutions
20
For headspace sampling of volatiles from standard solutions, 1 to 5 clean STs are placed in
21
either 4 mL or 20 mL glass screw-cap vials, except in recovery experiments, for which glass
22
screw-cap bottles of 30, 300, or 600 mL are used (glass bottles with volume markers for 25,
23
250, or 500 mL, Schott) to include sampling volumes from flower and leaf headspaces (30 or
24
600 mL, Figure 2 insets and described below). The maximum amount of liquid volume added
25
to containers is estimated by the ideal gas law to allow full solvent evaporation within the
26
sampling volume: Vimax = Vs*MW*-1*[22,400 mL/mol]-1 at RT, with Vimax, maximum
1
27
injection volume; Vs, sampling volume, MW, molecular weight of the solvent; and , solvent
28
density. We add different amounts of standard dissolved in 1 to 4 µL of dichloromethane
29
(maximum 2 µL in 4 mL vials and 4 µL in 20 mL vials) to the glass containers with STs,
30
being careful to add solvent to the wall of the vial and not directly to any STs. Containers are
31
closed immediately and STs are usually transferred after 3 h to clean 1.5 mL screw-cap glass
32
vials and stored at -20 °C for subsequent TD-GC-QMS analysis. Although dichloromethane
33
and other nonpolar solvents are known to swell the PDMS matrix (Lee et al., 2003), we obtain
34
80-90% recovery of PVs from STs when a standard in dichloromethane is either directly
35
applied, or added into the headspace of 1.5 mL vials, compared to injection of the same
36
standard by a liquid autosampler (Table S2). Thus the solvent does not greatly interfere with
37
the absorption of PVs in the standard by STs, and we obtain low limits of detection
38
comparable to liquid injection of standards (LoDs Figure 1 C, Figures S4, S5).
39
40
Sampling of PVs from leaf and flower headspaces with STs
41
Poly(ethylene terephthalate) (PET) containers (Stewart-Jones and Poppy, 2006) are used to
42
enclose leaves and flowers for headspace sampling with STs (see insets, Figure 2). For leaves,
43
we use 300 mL PET cups with detachable lids (Iso-pack, total volume of full cup and lid is
44
600 mL). The lids have a hole in the center with a diameter of 2.5 cm, which is sufficient to
45
pass over a gently rolled leaf without causing damage; additionally, we make holes of ca.
46
1 cm diameter at the base of the cup to allow transpiration and airflow across the headspace,
47
as well as easy addition and removal of STs over time course measurements. For leaf
48
headspace samples, a similar, fully expanded, mature, non-senescent stem leaf is chosen from
49
each plant for treatment and volatile sampling (Halitschke et al., 2000). For flowers, 30 mL
50
PET soufflé cups (Solo) are used. We cut one hole at the edge of the cup’s base, and another,
51
in the opposite edge of the lid, sufficiently large to slip over a flower. STs can be easily
52
inserted and removed through the hole in the base of the cup. As for containers used with
2
53
leaves, the holes are offset such that PVs must diffuse from the open flower into the container
54
before diffusing out. Flowers are enclosed before opening, on the first night of opening
55
(Kessler et al., 2010). STs in otherwise empty trapping containers dispersed among plants are
56
used to monitor background levels of PVs.
57
To measure PVs released after damage, leaves are treated with 6 rows of wounds from
58
a pattern wheel as a standardized damage treatment. To measure herbivore-induced PVs,
59
20 µL of Manduca sexta oral secretions diluted 1:5 with distilled water are added to wounds
60
(W+OS) to mimic feeding by this solanaceous specialist, as described previously (Schittko et
61
al., 2001; Schuman et al., 2012).
62
To reveal differences among plants in the jasmonate-mediated emission of PVs, one
63
leaf per plant is sprayed with the synthetic JA-Ile analogue I-indanoyl-Ile-Me (Lauchli and
64
Boland, 2003; Svoboda and Boland, 2010) at a concentration of 100 μM in 0.8% EtOH.
65
Leaves are allowed to dry for 1 h after spraying and then enclosed in PET containers and
66
exposed to STs as described above.
67
68
TD-GC-QMS analysis
69
TD-GC-QMS analysis is performed on a TD-20 thermal desorption unit (Shimadzu)
70
connected to a quadrupole GC-MS-QP2010Ultra (Shimadzu). Individual STs are placed in
71
89 mm glass TD tubes (Supelco) and desorbed under a stream of nitrogen at 60 mL min-1 for
72
8 min at 200 °C; after desorption, less than 0.2% of the original signal remains on STs
73
(Table S2). All substances desorbed from the ST are cryo-focused at -20 °C onto a Tenax®
74
adsorbent trap in front of the column. After desorption, the Tenax® trap is heated to 230 °C
75
within 10 s, and analytes are injected using a 1 to 20 split ratio onto an Rtx-5MS column
76
(30 m long, 0.25 mm i.d., 0.25 µm film thickness; Restek) with He as the carrier gas at a
77
constant linear velocity of 40 cm s-1. The TD-GC interface is held at 230 °C. We use two
78
different GC oven gradients for the profiling of leaf and flower headspaces. For leaf samples,
3
79
the oven is held at 40 °C for 5 min, then ramped to 185 °C at 5.0 °C min-1, and finally to
80
280 °C at 30 °C min-1, where it was held for 0.83 min. For analysis of benzyl acetone
81
emission from flower samples, the oven is held at 60 °C for 1 min, then ramped to 150 °C at
82
10 °C min-1, and finally to 250 °C at 30 °C min-1. Electron impact (EI) spectra are recorded at
83
70 eV in scan mode from 33 to 400 m/z using a scan speed of 2,000 Da s-1.The transfer line is
84
held at 240 °C and the ion source at 220 °C. Data processing against our reference compounds
85
(Table 1) is performed using the Shimadzu GCMS solutions software (v2.72). Compounds in
86
Table 1 were identified by comparison of spectra and Kovats retention indices to libraries and,
87
when possible, by comparison to pure standards.
88
89
Comparison of PV measurements from ST vs. Poropak Q sampling
90
Leaf positions +1 and +2 (the two youngest fully-expanded leaves) are treated with W+OS
91
and enclosed in PET containers as described above. One container is connected to a Poropak
92
Q filter attached to a vacuum manifold pulling air through the headspace at 300 mL/min, and
93
the other recieves a 0.5 cm ST. Headspace samples are collected every 4 h by exchanging
94
Poropak Q filters and STs. Background levels of PVs are monitored using empty PET
95
containers distributed among plants and either connected to Poropak Q filters attached to a
96
vacuum manifold pulling air through the headspace at 300 mL/min, or containing a 0.5 cm ST
97
(n = 3 background monitors of each type, sample n given in Figure 4).
98
99
STs are analyzed by TD-GC-QMS as described above. Poropak Q filters are spiked
with 320 ng tetralin (Sigma-Aldrich) as an elution standard, eluted with 250 μL
100
dichloromethane into a 1.5 mL clear glass screw-cap GC vial containing a 250 μL microinsert
101
as previously described (Halitschke et al., 2000; Schuman et al., 2012), and analyzed on the
102
same instrument as described for STs, except that a liquid autosampler (AOC-20i, Shimadzu)
103
injects 1 µL of sample, splitless, into the GC inlet held at 230 °C. Both ST and Poropak
104
samples are analyzed using the 35 min program described above. Results are analyzed using
4
105
the reference list in Table 1. Peaks found in the background controls are used to set a
106
threshold for quantification prior to statistical analysis: within each sampling interval, for each
107
compound processed, the maximum peak area measured in the background controls is
108
subtracted from the peak areas of samples; any sample peak areas which are less than the
109
maximum in the background controls are set to zero. Background-corrected peak areas from
110
Poropak Q samples are additionally normalized to the elution internal standard, tetralin, and
111
expressed as percentage tetralin peak area (Gaquerel et al., 2009; Schuman et al., 2009).
112
Statistical analyses are described in the Experimental procedures section of the main text.
113
114
Supporting References
115
116
117
118
Gaquerel E, Weinhold A, Baldwin IT (2009) Molecular interactions between the specialist
herbivore Manduca sexta (Lepidoptera, Sphigidae) and its natural host Nicotiana
attenuata. VIII. An unbiased GCxGC-ToFMS analysis of the plant’s elicited volatile
emissions. Plant Physiol, 149, 1408-1423.
119
120
121
Halitschke R, Keßler A, Kahl J, Lorenz A, Baldwin IT (2000) Ecophysiological
comparison of direct and indirect defenses in Nicotiana attenuata. Oecologia, 124, 408417.
122
123
Lee JN, Park C, Whitesides GM (2003) Solvent compatibility of poly(dimethylsiloxane)based microfluidic devices. Anal Chem, 75, 6544–6554.
124
125
Kessler D, Diezel C, Baldwin IT (2010) Changing pollinators as a means of escaping
herbivores. Curr Biol, 20, 237-242.
126
127
128
Schuman MC, Barthel K, Baldwin IT (2012) Herbivory-induced volatiles function as
defenses increasing fitness of the native plant Nicotiana attenuata in nature. eLife, 1,
e00007.
129
130
131
Schuman MC, Heinzel N, Gaquerel E, Svatos A, Baldwin IT (2009) Polymorphism in
jasmonate signaling partially accounts for the variety of volatiles produced by Nicotiana
attenuata plants in a native population. New Phytol, 183, 1134-1148.
132
133
Stewart-Jones A, Poppy GM (2006) Comparison of glass vessels and plastic bags for
enclosing living plant parts for headspace analysis. J Chem Ecol, 32, 845-864.
5