1 Running head: Guard cells regulate sesquiterpene emission.
Accepted Article
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Emission of herbivore elicitor-induced sesquiterpenes is regulated by stomatal aperture in maize (Zea mays) seedlings. 1 Seidl-Adams, Irmgard1, Annett Richter2, KB Boomer3, Naoko Yoshinaga1,4, Joerg Degenhardt2, James H. Tumlinson1. 1
Center of Chemical Ecology, Entomology Department, The Pennsylvania State University, University Park PA 16802, USA, 2 Pharmaceutical Biotechnology, Martin Luther Universität, D-06120 Halle (Saale), Germany, 3 Mathematics Department, Bucknell University, Lewisburg PA 17837, USA, 4 Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan Corresponding author: Irmgard Seidl-Adams, Center for Chemical Ecology, Department of Entomology, The Pennsylvania State University, University Park, PA 16802, Tel. 814 863 1791, E-mail
[email protected] This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/pce.12347
This article is protected by copyright. All rights reserved.
2
Accepted Article
22 23
Abstract
24
Maize seedlings emit sesquiterpenes during the day in response to insect herbivory.
25
Parasitoids and predators use induced volatile blends to find their hosts or prey. To
26
investigate the diurnal regulation of biosynthesis and emission of induced sesquiterpenes
27
we applied linolenoyl-L-glutamine (LG) to maize seedlings in the morning or evening
28
using a cut-stem assay and tracked farnesene emission, in-planta accumulation, as well as
29
transcript levels of Farnesyl Pyrophosphate Synthase3 (ZmFPPS3) and Terpene
30
Synthase10 (ZmTPS10) throughout the following day. Independent of time of day of LG
31
treatment, maximum transcript levels of ZmFPPS3 and ZmTPS10 occurred within 3-4
32
hours after elicitor application. The similarity between the patterns of farnesene emission
33
and in-planta accumulation in light-exposed seedlings in both time courses suggested
34
unobstructed emission in the light. After evening induction, farnesene biosynthesis
35
increased dramatically during early morning hours. Contrary to light-exposed seedlings
36
dark-kept seedlings retained the majority of the synthesized farnesene. Two treatments to
37
reduce stomatal aperture, dark exposure at midday, and ABA treatment before daybreak,
38
resulted in significantly reduced amounts of emitted and significantly increased amounts
39
of in-planta accumulating farnesene. Our results suggest that stomata not only play an
40
important role in gas exchange for primary metabolism but also for indirect plant
41
defenses.
42
Keywords: Zea mays, herbivore-induced plant volatiles, farnesene, linolenoyl-l-
43
glutamine, stomata, nocturnal emission, diurnal emission, ZmFPPS3, ZmTPS10, GC-
44
FID, quantitative Realtime PCR.
45
This article is protected by copyright. All rights reserved.
3
Introduction
48
Plants defend themselves against pathogens and herbivores directly by producing either
49
constitutive or inducible barriers, toxins, and deterrents, and indirectly by attracting
50
enemies, i.e. predators or parasites of the attackers. Terpenoids have been shown to
51
perform all these defense roles in numerous representatives of the plant kingdom
52
(Gershenzon & Dudareva 2007). The antimicrobial properties of terpenoids have been
53
demonstrated in members of monocots and dicots, herbaceous and woody plants, (Raffa
54
et al. 1985; Huffaker et al. 2011; Huang et al. 2012). In various species in the pine family
55
terpenoids function as barriers, toxins and deterrents against insect attackers (Alfaro
56
1995; Werner 1995; Martin et al. 2002). And finally, in both monocots and dicots volatile
57
terpenoids act above and below ground as attractants for predators and parasites of
58
different life stages of diverse insect herbivores (Turlings, Tumlinson & Lewis 1990;
59
Turlings et al. 1991; Turlings 2000; Kessler & Baldwin 2001; Hoballah & Turlings 2005;
60
Rasmann et al. 2005; Tamiru et al. 2011).
61
The biosynthesis pathways for terpenes are well documented (Ashour, Wink &
62
Gershenzon 2010). Therefore we will only give a brief summary here; all terpenoids are
63
synthesized from the same basic building blocks, isopentenyl pyrophosphate (IPP) and
64
dimethylallyl pyrophosphate (DMAPP), which are produced in the cytosol from acetyl-
65
CoA via the mevalonate (MAV pathway) and in plastids from pyruvate, thiamine
66
pyrophosphate, and glyceraldehyde 3-phosphate via methylerthyritol 4-phosphate (MEP
67
pathway). As a general rule, sesquiterpenes are synthesized by terpene synthases (TPS) in
68
the cytosol from farnesyl pyrophosphate (FPP), which in turn is made by FPP synthase
69
(FPPS) from IPP and DMAPP produced via the MVA pathway. Mono- and diterpenes
70
are made in plastids from geranyldiphosphate and geranylgeranyl diphosphate
71
respectively, produced from MEP pathway derived IPP and DMAPP, but exceptions have
72
been reported (Dudareva et al. 2005; Nagegowda, 2010).
73
Maize is a model system to study induced volatile emissions (Degenhardt 2009); the
74
volatile terpenoid bouquet is not only emitted in response to lepidopteran larvae feeding,
75
but also to treatments with larval regurgitants, and fatty acid amino acid conjugate (FAC)
76
elicitors like linolenoyl-L-glutamine (LG) (Turlings et al. 1990; Schmelz, Alborn &
Accepted Article
46 47
This article is protected by copyright. All rights reserved.
4 Tumlinson 2001; Yoshinaga et al. 2010). Furthermore, FPP synthases and several terpene
78
synthases are well characterized. Recently, Richter et al. (submitted) characterized the
79
only insect herbivore-inducible farnesyl pyrophosphate synthase (ZmFPPS3) in maize.
80
Transcript levels of ZmFPPS3 increase within 1 hour after caterpillar feeding, and
81
caterpillar feeding mimics like wounding and applications of the FAC elicitors volicitin
82
and LG to the wounding site (Richter et al. submitted). FPP is the substrate for the
83
terpene synthases ZmTPS23 and ZmTPS10, whose major products are caryophyllene
84
(Koellner et al. 2008), and bergamotene and farnesene (Koellner, Gershenzon &
85
Degenhardt 2009), respectively. Just as for ZmFPPS3, transcript levels of these two
86
terpene synthases increase within hours of insect feeding or FAC elicitor treatments
87
(Koellner et al. 2008; 2009). While the components of the terpenoid blend vary in
88
different cultivars of maize, the sesquiterpenes caryophyllene, bergamotene and
89
farnesene are common in most volatile profiles (Gouinguene, Degen & Turlings 2001;
90
Degen et al. 2004).
91
Terpene synthases are thought to be key regulators of terpene biosynthesis, because
92
developmental (Dudareva, Pichersky & Gershenzon 2004) as well as induced terpene
93
emissions (Koellner et al. 2008; 2009) are correlated with terpene synthase expression.
94
Furthermore, upon having been fed on by beet armyworm caterpillars, cotton plants
95
emitted de novo synthesized terpenes, which were different from the constitutive volatiles
96
released from damaged glands (Pare & Tumlinson 1997). Yet, it is not clear, how well
97
terpene synthase transcripts are correlated with terpene synthase activity, and whether
98
whatever is synthesized is emitted at a similar rate without any regulatory step between
99
synthesis and emission.
Accepted Article
77
100
Induced volatile emissions in general follow a diurnal pattern (Loughrin et al. 1994; De
101
Moraes, Mescher & Tumlinson 2001). Yet, it is unclear whether, and if so how, light
102
and/or the circadian clock regulate biosynthesis, or emission, or both. In order to identify
103
points of regulation of volatile emissions, both biosynthesis and the mechanics of
104
emission have to be well understood. While the biosynthetic pathway of terpenoids has
105
been studied extensively, studies of the physical path volatiles take within and through
106
plant tissues are mostly studied for isoprenes and monoterpenes (Kesselmeier & Staudt
107
1999). Isoprenes are clearly emitted from stomata in oak and aspen leaves but
This article is protected by copyright. All rights reserved.
5 surprisingly emission rates are not affected by aperture of stomata (Fall & Monson 1992).
109
The authors explain these seemingly contradictory findings by postulating that the high
110
internal build-up of isoprenes in leaves compensates quickly for the decreased diffusion
111
rate due to decreased stomatal aperture. This compensation happens somewhat slower in
112
Pinus pinea for oxygenated monoterpenes (Niinemets et al. 2002).
113
Maize stomata, in particular, respond within minutes to a change in light conditions
114
(Raschke & Fellows 1971; Pallaghy 1971). Flaccid guard cells close stomatal aperture in
115
the dark (Humble & Hsiao 1969; Humble & Hasiao 1970; Humble & Raschke 1971). In
116
preliminary experiments LG-induced sesquiterpenes accumulated during the dark period
117
in the tissue of maize seedlings suggesting that closed stomata restrict emission of
118
sesquiterpenes for several hours. We investigated this phenomenon through detailed time
119
course experiments, monitoring transcript levels of ZmFPPS3 and ZmTPS10, as well as
120
emitted and in-planta accumulated farnesene amounts. More specifically, we tested the
121
following hypotheses (1) the patterns of induced transcript levels of ZmFPPS3 and
122
ZmTPS10 depend on the time of day when the elicitor was applied and the subsequent
123
light availability; (2) gene expression patterns of ZmFPPS3 and ZmTPS10 predict
124
farnesene biosynthesis and emission; (3) farnesene biosynthesis is light-dependent, and
125
finally; (4) stomata play a role in the observed emission patterns for farnesene.
Accepted Article
108
126 127
Materials and methods
128
Plant growth conditions
129
Delprim seeds (Delley Switzerland) were treated with FLINT fungicide (Bayer
130
CropScience Kansas City, MO) according to the manufacturer’s recommendations.
131
Fungicide treated seeds were planted directly into autoclaved moistened Sunshine MVP
132
(Sun Grow Horticulture, Agwam MA) and a quarter teaspoon of slow release fertilizer,
133
Osmocote Plus 15-9-12 (Scotts®) was sprinkled on the surface of the soil. Seedlings
134
were grown in growth chambers under a 17/7 day/night cycle, with lights on between
135
4:30 AM and 9:30 PM. Temperature from 6:30 AM until 9:30 PM was set at 25°C, and
136
from 9:30 PM to 6:30 AM was set at 23°C. All light conditions were produced using an
137
equal number of metal halide and high pressure sodium lamps with an output of 400W
138
each. One hour of dawn between 4:30 AM and 5:30 AM was simulated at a
This article is protected by copyright. All rights reserved.
6 photosynthetic photon flux of 130 mol m-2 s-1. Full daylight between 5:30 AM and 6:30
140
PM was simulated with 270 mol m-2 s-1. A prolonged dusk period between 6:30 PM and
141
9:30 PM was simulated with 2 hours at 130 mol m-2 s-1 and one hour at 70 mol m-2 s-1.
Accepted Article
139
142 143
Time course experiments
144
All day and night time courses were conducted similarly. Since transcript level and in-
145
planta terpenoids measurements are destructive, seedlings were harvested at the
146
respective time points and gene expression and accumulated farnesene amounts were
147
determined for each seedling. To measure emissions a separate experiment was
148
conducted where emissions from the same set of seedlings were measured in 90 minute
149
intervals throughout the course of the experiment.
150
Morning induction: At 9:00 AM, 14-day old maize seedlings, stage v3, were cut off 1 cm
151
above the first leaf, and the cut end of the stem was immersed in 250 L of either elicitor
152
solution (25 mM phosphate buffer pH7.8, 4 L of LG at 100 ng L-1 in 100 mM
153
phosphate buffer pH7.8) or 25 mM phosphate buffer, pH7.8. At 9:30 AM, after the
154
seedlings had taken up the treatment solution, seedlings were transferred to ddH2O. For a
155
total of seven time points, four buffer treated and five LG-treated seedlings were
156
harvested every 90 min between 10:30 AM and 6 PM into liquid N2 for subsequent
157
extraction of RNA and in-planta sesquiterpenes. Similarly, for base line establishment
158
three buffer treated and three LG-treated seedlings were harvested at 9:30 AM into liquid
159
N2.
160
Emitted volatiles: At 9 AM, six seedlings each were induced with LG and buffer as
161
described above. After having taken up all the treatment solution the stem of each
162
individual seedling was inserted through the lid into 10 mL glass vials containing ddH2O.
163
At 9:30 AM these seedlings were then inserted into volatile collection chambers, which
164
were placed at a 15° angle from the horizontal. Charcoal filtered clean air entering the
165
chambers at 10 psi was pulled over the seedlings and volatiles were drawn onto SuperQ
166
filter traps at a flow of 0.5 L min-1. Filter traps were replaced every 90 min and eluted
167
with two aliquots of 50 L 1:1 dichloromethane: hexane containing 4 ng L-1
168
nonylacetate, internal standard. At 6 PM all seedlings were weighed.
This article is protected by copyright. All rights reserved.
7 Evening induction: Since it took seedlings more time to take up the elicitor solution the
170
evening induction experiment was started already at 8:30 PM, 1 hour before dark, to
171
insure that by 9:30 PM all seedlings were induced. Otherwise the induction procedure
172
was conducted in the same manner as in the morning induction experiment. 14-day old
173
maize seedlings, stage v3, were cut off 1 cm above the first leaf, and the cut end of the
174
stem was immersed in either elicitor solution or phosphate buffer. They were transferred
175
to 25 mL beakers with ddH2O after they had taken up the elicitor solution (around 9:30
176
PM) and placed into the dark growth chamber. One third of the LG-treated seedlings
177
were placed under plexiglass tubes covered with aluminum foil, to keep these seedlings
178
in the environmental conditions of the growth chamber, but in the dark, when the lights
179
came on the following morning (LG+DARK). A diagram of the treatments is shown in
180
Supplemental Figure 1. We verified that temperature reads inside these tubes did not
181
differ from growth chamber temperature and that no light entered the tubes.
182
Every 90 min three seedlings from each treatment were harvested, corresponding to six
183
nocturnal (9:30 PM, 10:30 PM, midnight, 1:30 AM, 3 AM, 4:30 AM) and six diurnal
184
samplings (6 AM, 7:30 AM, 9 AM, 10:30 AM, noon, 1:30 PM) for subsequent extraction
185
of RNA and internal sesquiterpenes. During the night three buffer treated and three LG-
186
treated seedlings were harvested per time point, while during the daytime hours three
187
buffer-treated seedlings and three seedlings of both the LG treatment and the LG+Dark
188
treatment were harvested. Seedlings were harvested directly into liquid N2 immediately
189
after cutting off an additional centimeter from the base to eliminate the tissue that had
190
been in immediate contact with the elicitor solution. To establish a baseline three
191
seedlings per treatment were harvested immediately after the treatment solution had been
192
taken up at 9:30 PM.
193
Emitted volatiles: At 8:30 PM 12 seedlings were induced with LG and six seedlings with
194
buffer as described above. After having taken up all the treatment solution the stem of
195
each individual seedling was inserted through slits in the lid into 10 mL glass vials
196
containing ddH2O. At 9:30 PM these seedlings were then inserted into volatile collection
197
chambers, which were placed at a 15° angle from the horizontal. The LG-treated
198
seedlings designated to remain in the dark were placed into collection chambers wrapped
199
with aluminum foil. Emitted VOC were entrained in clean air and trapped on SuperQ
Accepted Article
169
This article is protected by copyright. All rights reserved.
8 filters as before. Filters were changed every 90 min and eluted as before. At 1:30 PM all
201
seedlings were weighed.
Accepted Article
200 202 203
Tests of stomatal involvement in volatile release
204
Midday dark treatment: Eleven seedlings were treated with LG at 9 AM. At 9:30 AM all
205
seedlings had taken up the LG solution and had been transferred to individual vials
206
containing ddH20. Subsequently seedlings were placed into individual volatile collection
207
chambers as described above. At 1:30 PM the chambers of five of the LG-treated
208
seedlings were wrapped with aluminum foil to block the light. Filters were exchanged
209
every 90 min from 10:30 AM through 6 PM. At 6 PM all seedlings were weighed and
210
harvested into liquid N2 for subsequent extraction and measurements of in-planta
211
terpenoids.
212
ABA treatment: Eighteen 14 day-old Delprim seedlings were induced with LG at 8:30
213
PM outside of the growth chamber as described above for the night time course
214
experiment. After elicitor solution was taken up, seedlings were transferred to individual
215
vials containing ddH2O, and inserted into the tubular volatile collection chambers. Six
216
collection chambers were wrapped in aluminum foil to block the light. At 3 AM the
217
ddH20 of six other seedlings was exchanged for vials containing 250 M ABA in ddH2O.
218
Volatiles were collected on SuperQ filters as described above. Filters were exchanged at
219
3 AM, 4:30 AM (just prior to the lights coming on), 6 AM, 7:30 AM, 9 AM and 10:30
220
AM. At 10:30 AM seedlings were harvested into liquid nitrogen for in-planta terpenoids
221
measurements.
222 223
Internal terpene concentration measurements
224
Terpene extraction from ground plant tissue was adapted from a published protocol
225
(Koellner et al. 2004): Frozen samples were ground in a Genogrinder with three metal
226
balls ( 6mm) for 2 min at 1200 strokes per min. About 500 mg of frozen tissue powder
227
was suspended in 2 mL of pentane containing 400 ng humulene as internal standard.
228
Glass vials were shaken in the refrigerator at 10
229
centrifugation at 10
230
glass vial with a screw cap containing a Teflon/silicone septum. The pentane was
for 75 min. Tissue was precipitated by
for 5 min at 3000 g. Then, supernatant was transferred to a 4 mL
This article is protected by copyright. All rights reserved.
9 evaporated under a stream of nitrogen to dryness. Afterwards a slit was cut into the
232
septum with a clean razor blade. Through this slid a SuperQ filter attached to vacuum and
233
a thin metal tube attached to a nitrogen manifold were inserted. By heating vials for 7
234
min at 80
235
0.4 L min-1. Volatile organic compounds were eluted from the SuperQ filters with two
236
aliquots of 50 L 1:1 dichloromethane: hexane each containing 4 ng L-1 nonylacetate a
237
second internal standard. One L of the eluent was analyzed on an Equity-5 column (30
238
m x 0.2 mm x 0.2 μm film thickness; Supelco, Bellefonte, PA) by GC-FID. Terpenoids
239
were quantified relative to the square root of the product of both internal standards.
Accepted Article
231
volatiles were drawn under nitrogen gas onto SuperQ filters at a flow rate of
240 241
Chromatographic and spectroscopic analysis and identification
242
Components of volatile blends emitted or extracted from plants were identified by
243
comparison of GC retention times and mass spectra with those of authentic synthetic
244
compounds, and by matching spectra to those from the NIST 02 mass spectral libraries.
245
Samples were analyzed in an Agilent 6890 gas chromatograph-flame ionization detector
246
system (GC-FID) equipped with an Equity-5 column (30 m x 0.2 mm x 0.2 μm film
247
thickness; Supelco, Bellefonte, PA). Helium was used as carrier gas at an average linear
248
velocity of 26 cm s-1. Samples, 1 μL, were injected in the splitless mode and the injector
249
was changed to split mode after 0.75 min. The initial oven temperature was held at 40°C
250
for 1 min, then programmed to increase at 8°C min-1 to 180°C; followed by ramping at
251
30°C min-1 to 300°C, and holding at 300°C for 5 min. The injector and the detector
252
temperatures were set to 280°C and 300°C, respectively. Selected samples were analyzed
253
in a GC-MS system consisting of an Agilent 6890N gas chromatograph interfaced with
254
an Agilent 5973N mass selective detector. The capillary column and GC conditions were
255
equivalent to those used in the GC-FID. The MS was used in electron impact (EI)
256
ionization mode with the default temperature settings (ion source: 230°C, and
257
quadrupole: 150°C).
258 259
Reverse transcriptase quantitative PCR (RT-qPCR)
260
RNA extraction and cDNA synthesis: Total RNA was extracted from 100 mg ground
261
tissue according to the manufacturer’s protocol with the RNeasy Plant mini kit (Qiagen)
This article is protected by copyright. All rights reserved.
10 including the shredder step. Genomic DNA in 3 g total RNA was digested in a 50 L
263
reaction with the Turbo DNA-free™ kit (Applied Biosystems) according to the
264
manufacturer’s protocol. 550 ng of DNase treated total RNA was reverse transcribed in a
265
20 L reaction with SMART™ MMLV Reverse Transcriptase (Clontech) according to
266
the manufacturer’s protocol using a mix of anchored 18mer polydT and random 8mer
267
oligo primers (Genomics Core Facility PennState University) at a final concentration of
268
3.75 M.
269
Quantitative PCR: cDNA was diluted 1:10 and used in 5 L aliquots as template in 20 l
270
PCRs. Final primer concentration was 0.5 M. All other components necessary for qPCR
271
were contained in the SsoFast™ EvaGreen® Supermix (BioRad). All PCRs were run in
272
triplicate. A water control and a standard curve were run on each of the 96-well plates.
273
The standard curve was generated using five three-fold serial dilutions of pooled cDNA
274
from highly expressing samples as template. The highest concentrated template for the
275
standard curves (3x) was obtained by diluting 1.5 L undiluted pooled cDNA into 3.5 L
276
H2O and scaled up correspondingly so that on all plates standard curves were generated
277
from the same templates. Similarly, enough cDNA of individual samples was diluted so
278
that all reactions on all plates were from the same template pool thus guaranteeing that
279
the same amount of template was used in all reactions. Standard curves were constructed
280
assigning 81 arbitrary units as the starting amount of the transcript of interest when the 3x
281
pooled cDNA was used as template. To control for the amount of cDNA in each of the
282
samples adenine phosphate transferase 1 (ZmAPT1) was used as reference gene. Its
283
expression is invariant during our experimental conditions. All transcript levels of the
284
genes of interest were expressed as the ratio of their and ZmAPT1 starting quantities
285
based on the respective standard curves.
286
Primer sequences: ZmTPS10 F363: AGGGAACTTCGTGGTGGATGATAC, ZmTPS10
287
R476: TGGCGTCTGGTGAAGGTAATGG; ZmFPPS3 F1044:
288
CCTGGCTAGTTGTGCAAGCT, ZmFPPS3 R1262: GAAAACAGTTTGGACTGCCT;
289
ZmAPT1 F380: AGGCGTTCCGTGACACCATC, ZmAPT1 R541:
290
CTGGCAACTTCTTCGGCTTCC.
Accepted Article
262
291 292
Statistical analysis
This article is protected by copyright. All rights reserved.
11 All statistical analyses were conducted in SAS ver. 9.3. General and generalized linear
294
mixed models were used to model the effects of treatment, harvest time, and their
295
interaction. Based on the Bayesian information criterion (BIC) and the Akaikie
296
information criterion (AIC), an unstructured covariance structure was selected as the best
297
structure within in the repeated measures analyses. Bonferroni adjusted p-values for
298
multiple comparisons were used based on a priori comparisons of interest; the respective
299
adjusted level of significance for each analysis are listed in Table I. We tested two
300
separate hypotheses for each time course: (Hypothesis1) mean treatment effects at any
301
particular time point do not differ significantly, and (Hypothesis 2) mean treatment
302
effects for a particular treatment do not differ between time points.
303
Analyses of emission measurements from the morning induction were modeled with a
304
repeated measures general linear model. None of the other emission measurements nor
305
in-tissue measurements (gene expression and tissue-extracted farnesene measurements)
306
satisfied the requirements of normality or equal variance, even after transformations.
307
Therefore the data from these experiments were analyzed using a generalized linear
308
mixed model assuming a lognormal distribution, with the main factors treatment and
309
harvest time and a linear interaction term of treatment and harvest time. All pairwise
310
interactions were significant except for extracted farnesene after morning induction and
311
during the dark period after evening induction. After testing for significant interaction of
312
treatment and harvest time within light/dark period we split the analysis of evening
313
induction time courses into separate analyses for the light and dark period.
314
The percent emitted farnesene was analyzed with a generalized linear model assuming a
315
binomial distribution.
Accepted Article
293
316 317
Results
318
Time course after morning induction
319
After morning induction transcript levels of ZmFPPS3 (Figure 1A) and ZmTPS10 (Figure
320
1B) increased rapidly in response to wounding with both buffer (wounding control) and
321
LG treatment. Within 1-2 hours of treatment transcript levels of ZmFPPS3 and ZmTPS10
322
increased at least eight-fold compared to background levels at 9:30 AM. About 3-4 hours
323
after induction, at noon, LG-induced ZmFPPS3 and ZmTPS10 transcript levels reached This article is protected by copyright. All rights reserved.
12 their maximum level, and then declined until they reached close to background levels at 6
325
PM. Buffer treatment had a similar effect on ZmFPPS3 and ZmTPS10 transcript levels,
326
with maximum amounts at noon, about 50% of LG-induced maximum levels, and a more
327
rapid decline to background levels (Figure 1 A, B).
328
After morning induction the dynamics of farnesene accumulations within the tissue
329
(Figure 1C) and emissions (Figure 1D) were similar: both increased steadily until they
330
reached a plateau during the measurement interval between 1:30 PM and 3 PM. Extracted
331
and emitted amounts were of the same order of magnitude at all time points; maximum
332
in-planta accumulation of farnesene was 1 g per g freshweight (g g-1) in LG-induced
333
and 0.4g g-1 in buffer-treated seedlings (Figure 1C), compared to maximum emission of
334
1.3g g-1 hr-1 by LG-induced and 0.75 g g-1 hr-1 by buffer-treated seedlings (Figure 1D).
Accepted Article
324
335 336
Time course after evening induction
337
When seedlings were treated with LG in the evening during the last hour of the light
338
cycle (between 8:30 PM and 9:30 PM), the dynamics of transcript accumulation (Figure
339
1E, F), despite happening in the dark, were similar to the morning induction: a rapid
340
increase until midnight (3 to 4 hours after induction) and for ZmFPPS3 a slow and steady
341
decline continuing into the following morning (Figure 1E). ZmTPS10 transcript levels
342
appeared to reach a plateau around midnight (Figure 1F). This plateau persisted until 7:30
343
AM when transcript levels started to decline rapidly in light-exposed seedlings (Figure
344
1F). Interestingly, after evening induction transcript levels of ZmTPS10 in the seedlings
345
that were kept in the dark during the following light period (dark-kept) were higher than
346
transcript levels of seedlings exposed to light (Figure 1F).
347
Buffer-induced transcript levels of ZmFPPS3 declined more rapidly than LG-induced
348
transcript levels (Figure 1E); buffer-induced relative to LG-induced transcript levels of
349
ZmTPS10 never reached levels as high as after morning induction. (Figure 1B, F).
350
Transcript levels of ZmFPPS3 were more than twice as high for corresponding time
351
points after evening induction (i.e. midnight) compared to plants induced in the morning
352
(i.e. noon) (Figure 1A, E). This comparison is legitimate because the same template was
353
used to generate the standard curves for both qPCR assays.
This article is protected by copyright. All rights reserved.
13 Although transcript levels of ZmFPPS3 declined steadily after midnight (Figure 1E),
355
farnesene amounts extracted from the tissue (Figure 1G) began increasing at 1:30 AM.
356
By the end of the dark cycle, at 4:30 AM, 0.63 g g-1 had accumulated on average in
357
induced seedlings (Figure 1G). With the beginning of the light cycle farnesene began
358
accumulating rapidly in the tissue independent of whether the seedlings were exposed to
359
light (Figure 1G) suggesting an increase in biosynthesis with daybreak. By 7:30 AM
360
when light-exposed seedlings had experienced their first 90 min interval exposed to full
361
light, light-exposed seedlings reached with 2.3g g-1 their maximum in-planta
362
accumulation of farnesene. At the same time point in-planta accumulations in dark-kept
363
seedlings was with 3.17g g-1 of the same order of magnitude (Figure 1G). In contrast,
364
farnesene emissions (Figure 1H) from light-exposed and dark-kept seedlings differed
365
dramatically. During the collection interval from 6 to 7:30 AM light-exposed seedlings
366
reached their maximum emission of farnesene with 3.01 g g-1 hr-1, while dark-kept
367
seedlings emitted only 0.12g g-1 hr-1 in the same time interval (Figure 1H). After 7:30
368
AM emitted and retained farnesene amounts decreased steadily in light-exposed seedlings
369
(Figure 1G, H), while emissions by dark-kept seedlings increased slowly throughout the
370
morning to reach 0.3g g-1 hr-1 during the last collection interval from noon to 1:30 PM
371
(Figure 1H). But in-planta amounts of farnesene in dark-kept seedlings had increased by
372
10:30 AM to 6.62g g-1 and stayed at this level until the end of collections at 1:30 PM
373
(Figure 1G).
374
Dark-kept seedlings produced overall less than 60% of the total amount of farnesene
375
produced by seedlings in the light (Supplemental Figure 2).
376
Bergamotene, the other major product of ZmTPS10, was produced to a lesser amount
377
then farnesene but followed the same patterns for in-planta accumulation and emission in
378
all time courses (Supplemental Figure 3).
379
In summary, ZmFPPS3 and ZmTPS10 transcript levels accumulated in LG-induced
380
seedlings following a similar schedule independent of the time of induction or availability
381
of light (Figure 1A, B, E, F). ZmFPPS3 transcript levels after evening induction seemed
382
to be at all time points higher than after morning induction (Figure 1A, E). Farnesene
383
biosynthesis calculated as the sum of emission and accumulation in the tissue happened
384
at low levels at night but increased dramatically with the beginning of the daylight cycle
Accepted Article
354
This article is protected by copyright. All rights reserved.
14 whether the seedlings were actually exposed to light or not. Finally, light-exposed
386
seedlings emitted most of the farnesene they synthesized whereas dark-kept seedlings
387
retained the bulk of their produced farnesene (Figure 1H). The accumulation and
388
emission pattern of bergamotene, the other main product of TPS10, was the same as
389
farnesene, albeit at lower amounts (Supplemental Figure 3).
Accepted Article
385
390 391
Role of stomata in volatile release
392
The fact that terpenoids accumulated inside dark-kept seedlings, while emissions were
393
severely reduced, suggests that there are regulated exit ports, and light is a key regulator
394
of the opening of these ports. Since guard cells respond to light signals by opening or
395
closing stomata we hypothesized that most of the farnesene emissions are regulated by
396
stomatal aperture. To test this hypothesis we manipulated the stomatal status by two
397
different treatments. First seedlings that had been induced with LG in the morning and
398
exposed to the normal light cycle were placed in the dark at 1:30 PM, the time of
399
maximum emission. Given that stomata in maize close within minutes after darkening
400
(Raschke & Fellows 1971) we expected that darkening would result in a more or less
401
rapid closure of most of the stomata and thereby increase the amount of retained
402
farnesene at the expense of emitted amounts. In a second experiment we wanted to
403
prevent stomata from opening. Since ABA supplied through the transpiration stream
404
closes open stomata (Raschke & Hedrich 1985) or prevents closed stomata from opening
405
(Willmer, Don & Parker 1978) we treated evening-induced seedlings with 250 M ABA
406
through their cut stems at 3 AM (90 min before the light cycle started). Again, if our
407
hypothesis that stomata are the exit ports for volatile emission was correct, farnesene
408
emissions should decrease and its accumulation should increase in ABA-treated
409
seedlings.
410 411
Midday dark treatment
412
All seedlings were treated between 9:00 AM and 9:30 AM with LG and inserted into
413
individual flow through collection chambers. During the first two collection intervals the
414
two groups of seedlings did not emit significantly different amounts of farnesene (Figure
415
2A). At 1:30 PM collection chambers of half of the seedlings were wrapped in aluminum
This article is protected by copyright. All rights reserved.
15 foil to prevent light exposure. Dark treatment of LG-treated seedlings resulted in
417
significantly reduced emissions during all subsequent collection intervals (Figure 2A).
418
Seedlings were harvested at the conclusion of emission collections and in-planta
419
terpenoids were extracted and analyzed (Figure 2B). Total farnesene production was
420
estimated as the sum of all emitted amounts over the duration of the experiment and the
421
in-planta accumulated amount at 6 PM (the end of the experiment). Seedlings produced
422
similar total amounts (about 2.5 g g-1) independent of treatment (Figure 2B), however
423
the relative amounts of retained and emitted farnesene were significantly different
424
between treatments: seedlings that were not subjected to the darkening treatment emitted
425
in the course of the experiment 94% (Figure 2B); while seedlings that were darkened
426
after reaching their emission maximum emitted only 64% of their total farnesene
427
production with most of this emission occurring before the darkening treatment (Figure
428
2A, B).
Accepted Article
416
429 430
ABA treatment
431
Seedlings were induced with LG in the evening and their emissions were measured in
432
individual flow through collection chambers. The collection chambers of one third of the
433
seedlings were wrapped with aluminum foil to exclude light. At 3 AM (90 min before the
434
lights came on) the water for half of the seedlings in unwrapped collection chambers was
435
exchanged for a 250 M ABA water solution. Both ABA-treated and the remaining third
436
of the seedlings were exposed to the normal light cycle. Despite being exposed to light
437
ABA-treated seedlings emitted farnesene amounts intermediate between those farnesene
438
amounts emitted by dark-kept and light-exposed seedlings (Figure 3A). Correspondingly,
439
ABA-treated seedlings retained 29% of total farnesene produced, intermediate between
440
the 76% retained by darkened seedlings and 3% retained by light-exposed seedlings
441
(Figure 3B). Interestingly, despite being exposed to light in ABA-treated seedlings total
442
biosynthesis was suppressed to the amounts of dark-kept seedlings (Figure 3B).
443
In summary manipulation of stomatal opening by two independent methods, light
444
exclusion and ABA treatments, under two different induction regimes, morning and
445
evening induction, reduced the relative amount of emitted and increased the relative
446
amount of retained farnesene.
This article is protected by copyright. All rights reserved.
16
Accepted Article
447 448
Discussion
449
Schmelz et al. (2001) has already clearly demonstrated that maximum amounts of
450
induced VOC are emitted by plants in the light period following the application of FAC
451
elicitors, independent of whether the treatment happened the previous evening, at
452
midnight or in the morning before the lights came on. Furthermore, there is a positive
453
correlation between light intensity and VOC emission (Loughrin et al. 1994; Gouinguene
454
& Turlings 2002). However, it is not clear which of the two, VOC synthesis or their
455
release, is controlled by light, circadian rhythm, or some other factor. Since biosynthesis
456
of terpenes requires photosynthetic products and reduction equivalents (Shah & Rogers
457
1969) as well as energy, it seems likely that light in some way activates or controls at
458
least biosynthesis (Rodrigues-Conception et al. 2004). Measuring transcript levels of the
459
enzymes catalyzing the last two steps in the biosynthesis of farnesene and the
460
corresponding emitted and in-planta farnesene amounts every 90 minutes for 9 or 16
461
hours following treatment with LG we found that in maize the observed diurnal emission
462
of induced foliar terpenoids is not only the result of diurnally regulated biosynthesis but
463
also light-dependent opening of stomata. In fact, our data strongly supports the
464
hypothesis that the bulk of volatile sesquiterpenes is emitted through open stomata.
465
Therefore terpenoid biosynthesis is not necessarily synonymous with terpenoid emission.
466
Several observations support this conclusion. Seedlings induced in the evening and kept
467
in darkness throughout the next morning hardly emitted any sesquiterpenes (Figure 1H)
468
while in-planta accumulations continued to increase (Figure 1G), suggesting that in
469
induced dark-kept seedlings biosynthesis of sesquiterpenes occurs, albeit at reduced
470
levels (Supplemental Figure 2), but the sesquiterpenes produced accumulate within the
471
tissue rather than being emitted. Sesquiterpene emissions increased slightly in the course
472
of the following day suggesting that the increasing internal build-up of volatiles forces
473
small amounts out through almost closed exit ports; alternatively a small fraction of the
474
sesquiterpenes exits the leaf by diffusion through the epidermis (Figure 1H). On the other
475
hand, those seedlings exposed to the normal light cycle reached maximum emission rates
476
during the first collection interval conducted in full light (between 6 and 7:30AM) with
477
corresponding in-planta accumulations (Figure 1G, H).
This article is protected by copyright. All rights reserved.
17 Furthermore, two independent treatments, exclusion of light and ABA treatment that are
479
well documented to affect the movement of guard cells in plants in general (Tallman
480
2004; Chen et al. 2012) and maize in particular (Raschke & Fellows 1971; Raschke &
481
Hedrich 1985) resulted in reduced emission and increased retention of sesquiterpenes
482
within the tissue. When morning-induced maize seedlings were put into darkness at the
483
time of highest emission by wrapping their collection chambers with aluminum foil,
484
sesquiterpenes became trapped within the tissue rather than being emitted (Figure 2A).
485
While total farnesene amounts produced in darkened and light-exposed seedlings were
486
similar, darkened seedlings only emitted 64% of total farnesene compared to 94%
487
emitted by seedlings in the light (Figure 2B).
488
Similarly, ABA treatment affected the relative amounts of emitted farnesene: ABA-
489
treated plants emitted 71%, intermediate amounts compared to 24% emitted by dark-
490
treated plants and 97% emitted by light exposed plants (Figure 3B). Contrary to the
491
midday-darkening experiment the closure of the stomata and possibly the ABA treatment
492
itself affected severely the total amount of biosynthesis of farnesene (Figure 3B).
493
Although our experimental set-up was not designed to analyze the cause of this reduction,
494
the fact that the relative amounts that were emitted were significantly reduced in ABA-
495
treated seedlings supports the hypothesis that stomata are regulating the emission of
496
farnesene. While Gouinguene & Turlings (2002) did not specifically investigate the role
497
of stomata they speculated about their role in the regulation of induced VOC emissions
498
when they observed maximum emission of volatiles at 60% relative air humidity, the air
499
humidity levels to which the seedlings were acclimated; they also observed a slight
500
decrease in total induced volatile emission with increasing soil humidity. Especially the
501
observed decreased emissions at high soil water levels might be attributed to water-
502
logging conditions at which stomatal apertures are reduced (Parent et al. 2008).
503
The simplest model of sesquiterpene emission accommodating our data is that as
504
sesquiterpenes are synthesized in the interior of the leaf, they build up in the intercellular
505
spaces of the leaf, diffuse from there into the stomatal cavities and past open guard cells
506
into the atmosphere. If, as in evening-induced dark-kept seedlings, most guard cells are
507
closed, sesquiterpenes will build up inside the leaf and not be emitted. If stomata are
508
open, as in morning-induced seedlings, when light and water conditions do not interfere
Accepted Article
478
This article is protected by copyright. All rights reserved.
18 with the opening of guard cells, emission is unimpeded. As sesquiterpenes are
510
synthesized an equilibrium concentration within the leaf tissue is reached, where the rate
511
of synthesis and the rate of emission are similar and therefore in-planta concentration as
512
well as rate of emission are correlated with rate of biosynthesis. Consistent with this
513
model the pattern of emission and in-planta accumulations in morning-induced seedlings
514
was similar (Figure 1C, D).
515
This interpretation fits the model for VOC emissions developed by Niinemets &
516
Reichstein (2003a, b). The regulatory role of stomata according to these authors is
517
dependent on the time it takes for the intercellular concentration build-up to compensate
518
for reduced diffusion through closed stomata. For isoprenes this equilibrium is reached
519
within minutes (Fall & Monson 1992), and therefore the role of stomata in isoprene
520
emissions is negligible (Sharkey & Yeh 2001). For some monoterpenes it takes slightly
521
longer and becomes observable (Niinemets et al 2002). According to our results the
522
required amount of time for induced sesquiterpenes is strikingly longer, even after 9
523
hours only minimal amounts of the internal sesquiterpene pool were escaping into the
524
atmosphere, suggesting that stomata indeed play a significant role in the regulation of
525
induced foliar sesquiterpene emissions.
526
Biosynthesis of terpenoids ultimately depends on photosynthesis not only for the carbon
527
skeletons but also for the energy equivalents (Arimura et al 2008). Although biosynthesis
528
in dark-kept seedlings was clearly reduced by more than 40%, both carbon skeletons and
529
energy equivalents must have become available (Supplemental Figure 2).
530
The drastic increase in the morning of biosynthesis in dark-kept seedlings after evening
531
induction supports the general notion that induced terpenoid biosynthesis is under
532
circadian control. The high amounts of farnesene synthesized at 7:30 AM within 3 hours
533
of light exposure, despite decreased transcript levels of ZmFPPS3, suggest that ZmFPPS3
534
and ZmTPS10 are translated throughout the night and are stable for several hours.
535
However, our data does not allow identification of the critical step/s in the biosynthetic
536
pathway. Either activation of the enzymes ZmFPPS3 and/or ZmTPS10 is under circadian
537
regulation or ZmFPPS3 is substrate-limited at night. In this latter scenario it is possible
538
that enzymes catalyzing an earlier step in the biosynthetic pathway are under circadian
539
control and ultimately responsible for the dramatic increase in sesquiterpene biosynthesis
Accepted Article
509
This article is protected by copyright. All rights reserved.
19 in the early morning hours after evening induction. This is true for enzymes in the MEP
541
pathway (Cordoba, Salmi & Leon 2009). In particular, in snapdragon petals 1-deoxy-D-
542
xylose-5-phosphate synthase (DXS) facilitates the first committed step in the synthesis of
543
IPP in plastids and its transcripts accumulate following diurnal rhythm. The emission of
544
terpenoid floral volatiles from snapdragon petals tracks DXS transcript accumulations
545
(Dudareva et al. 2005). It remains to be seen whether, in maize, enzymes catalyzing
546
earlier steps in the MVA pathway, like 3-hydroxy-3-methyl-glutaryl-CoA reductase,
547
which catalyzes the rate-controlling step, are under diurnal control, i.e. not transcribed
548
during the dark cycle.
549
Incidentally, Koellner et al. (2013) detected ZmTPS10 transcripts only in the interior of
550
induced maize leaves but not in epidermal peels; suggesting that the final biosynthetic
551
step of farnesene happens in the interior of the leaf. This seems to be different from the
552
biosynthesis of methyl benzoate, the major floral compound in snapdragon. Benzoic acid
553
carboxyl methyltransferase (BAMT) the enzyme catalyzing the last step in the
554
biosynthesis of methyl benzoate was only found in epidermal cells of snapdragon petals
555
(Kolosova et al. 2001). Furthermore the special composition of the cuticle of snapdragon
556
petals could potentially allow the diffusion of methyl benzoate (Goodwin et al. 2003).
557
Our data, showing low emission but high build-up of induced sesquiterpenes within
558
plants under various conditions promoting the closure of stomata, suggest strongly that
559
diffusion through the cuticle plays at most a minor role in the emission of induced foliar
560
sesquiterpenes in maize.
561
Thus, at least in maize, typical diurnal sesquiterpene emission patterns after herbivory are
562
due to light dependent and diurnally regulated biosynthesis modulated by the light-
563
dependent opening of stomata.
564
In order for VOC release to be an effective indirect defense, emissions have to be
565
coordinated with periods of activity of members of the appropriate trophic level (Turlings
566
et al. 1995; Dicke 2009). Most of the parasitic wasps are foraging/looking for oviposition
567
sites during the day (Siekmann, Keller & Tenhumberg 2004; Joyce et al. 2009; Benelli et
568
al. 2012). Hence a circadian regulation of attractive volatile signals fits this postulate.
569
Yet, herbivores are not the only stress factor where the optimal defense response includes
570
a particular status of guard cells; drought (McAdam & Brodribb 2012), temperature
Accepted Article
540
This article is protected by copyright. All rights reserved.
20 (Azad et al. 2007; Pandey et al. 2007), elevated CO2 (Assmann 1999; Casson &
572
Hetherington 2010) and pathogen attack (Melotto et al. 2006), all affect stomatal
573
aperture. In fact, often plants are exposed to pathogens in addition to being attacked by
574
more than one herbivore. One of the general responses of plants to bacterial infection is
575
closing their stomata (Underwood, Melotto & He 2007), which does not only restrict
576
access for bacteria but - in light of our findings – should also increase concentrations of
577
antimicrobially active terpenoids (Voegeli & Chappell 1988; Huang et al. 2012) thereby
578
reducing the quality of environment encountered by entering bacteria. Conversely some
579
bacteria and fungi have evolved the capability to produce toxins like coronatine or
580
fusicoccin or other virulence factors to keep stomata open thus overcoming this first line
581
of defense (Guimaraes & Stotz 2004; Underwood et al. 2007). It therefore becomes a
582
multifactorial optimization problem for plants to adjust the status of stomatal aperture so
583
that they respond appropriately to the complex assemblage of biotic and abiotic factors,
584
which, when considered individually, demand competing stomatal settings. Stomata need
585
to be kept open to take in CO2, the ultimate prerequisite for growth, and for optimal
586
emission of signaling VOC, but reduced stomatal aperture is desirable under drought
587
conditions and pathogen attack. It helps that plants can adjust stomatal aperture locally
588
(van Gardingen, Jeffree & Grace 1989), which allows plants to avoid the complete shut
589
down of photosynthesis and transpiration and at the same time respond adequately to
590
local herbivory and pathogen attack.
591
Our findings have far reaching implications; stomata are not only involved in the
592
exchange of CO2, O2 and water vapor with the atmosphere but can also regulate the
593
emission of indirect defense signals, especially sesquiterpenes in maize, a rapidly
594
growing annual crop plant. Therefore, a reduction in stomatal densities or aperture will
595
affect the strength of this signal and could translate into poorer defense responses. Rising
596
CO2 concentrations, increasing temperature and drought conditions, all predicted
597
consequences of climate change, are associated with modified stomatal densities and/or
598
aperture (Casson & Hetherington 2010). Breeding crop plants to withstand extreme biotic
599
stresses predicted for the future by manipulating stomatal densities (Stecker 2012; Diehl
600
2013) need to not only consider yield and susceptibility to herbivory but also the intricate
601
communication of herbivore-damaged plants via volatiles with parasitoids and predators.
Accepted Article
571
This article is protected by copyright. All rights reserved.
21
Accepted Article
602 603
Acknowledgements
604 605 606 607 608 609
A.R. J.D. and I.SA. were supported by a grant from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 648 “Molecular mechanisms of information processing in plants”). N.Y. was the recipient of the Postdoctoral Fellowship for Research Abroad (no. 01212) from the Japan Society for the Promotion of Science for Young Scientists.
610
This article is protected by copyright. All rights reserved.
22
References
613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656
Alfaro R.I. (1995) An Induced Defense Reaction in White Spruce to Attack by the White-Pine Weevil, Pissodes strobi. Canadian Journal of Forest Research-Revue Canadienne De Recherche Forestiere, 25, 1725-1730. Arimura G.I., Koepke S., Kunert M., Volpe V., David A., Brand P., et al. (2008) Effects of feeding Spodoptera littoralis on lima bean leaves: IV. Diurnal and nocturnal damage differentially initiate plant volatile emission. Plant Physiology, 146, 965-973. Ashour M.W., Michael; Gershenzon, Jonathan. (2010) Biochemistry of Terpenoids: Monoterpenes, Sesquiterpenes, and Diterpenes. In Biochemistry of Plant Secondary Metabolism (ed. by M. Wink), pp. 258-303. Wiley-Blackwell, Oxford, UK. Assmann S.M. (1999) The cellular basis of guard cell sensing of rising CO2. Plant Cell and Environment, 22, 629-637. Azad A.K., Sawa Y., Ishikawa T. & Shibata H. (2007) Temperature-dependent stomatal movement in tulip petals controls water transpiration during flower opening and closing. Annals of Applied Biology, 150, 81-87. Benelli G., Bonsignori G., Stefanini C. & Canale A. (2012) Courtship and mating behaviour in the fruit fly parasitoid Psyttalia concolor (Szepligeti) (Hymenoptera: Braconidae): the role of wing fanning. Journal of Pest Science, 85, 55-63. Casson S.A. & Hetherington A.M. (2010) Environmental regulation of stomatal development. Current Opinion in Plant Biology, 13, 90-95. Chen C., Xiao Y.G., Li X. & Ni M. (2012) Light-Regulated Stomatal Aperture in Arabidopsis. Molecular Plant, 5, 566-572. Cordoba E., Salmi M. & Leon P. (2009) Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants. Journal of Experimental Botany, 60, 2933-2943. De Moraes C.M., Mescher M.C. & Tumlinson J.H. (2001) Caterpillar-induced nocturnal plant volatiles repel conspecific females. Nature, 410, 577-580. Degen T., Dillmann C., Marion-Poll F. & Turlings T.C.J. (2004) High genetic variability of herbivore-induced volatile emission within a broad range of maize inbred lines. Plant Physiology, 135, 1928-1938. Degenhardt J. (2009) Indirect defense responses to herbivory in grasses. Plant Physiology, 149, 96-102. Dicke M. (2009) Behavioural and community ecology of plants that cry for help. Plant Cell and Environment, 32, 654-665. Diehl P. (2013) Breeding versus engineering to make drought tolerant corn. http://biotech.about.com/od/Genetically-Modified-Organisms/a/Breeding-VersusEngineering-To-Make-Drought-Tolerant-Corn.htm. Dudareva N., Andersson S., Orlova I., Gatto N., Reichelt M., Rhodes D., et al. (2005) The nonmevalonate pathway supports both monoterpene and sesquiterpene formation in snapdragon flowers. Proc Natl Acad Sci U S A, 102, 933-938. Dudareva N., Pichersky E. & Gershenzon J. (2004) Biochemistry of plant volatiles. Plant Physiol, 135, 1893-1902. Gershenzon J. & Dudareva N. (2007) The function of terpene natural products in the natural world. Nature Chemical Biology, 3, 408-414. Goodwin S., Kolosova N., Kish C.M., Wood K.V., Dudareva N. & Jenks M.A. (2003) Cuticle characteristics and volatile emissions of petals in Antirrhinum majus. Physiologia Plantarum,
Accepted Article
611 612
This article is protected by copyright. All rights reserved.
23 119, 605-605. Gouinguene S., Degen T. & Turlings T.C.J. (2001) Variability in herbivore-induced odour emissions among maize cultivars and their wild ancestors (teosinte). Chemoecology, 11, 916. Gouinguene S.P. & Turlings T.C.J. (2002) The effects of abiotic factors on induced volatile emissions in corn plants. Plant Physiology, 129, 1296-1307. Guimaraes R.L. & Stotz H.U. (2004) Oxalate production by Sclerotinia sclerotiorum deregulates guard cells during infection. Plant Physiol, 136, 3703-3711. Hoballah M.E. & Turlings T.C. (2005) The role of fresh versus old leaf damage in the attraction of parasitic wasps to herbivore-induced maize volatiles. J Chem Ecol, 31, 2003-2018. Huang M., Sanchez-Moreiras A.M., Abel C., Sohrabi R., Lee S., Gershenzon J., et al. (2012) The major volatile organic compound emitted from Arabidopsis thaliana flowers, the sesquiterpene (E)-beta-caryophyllene, is a defense against a bacterial pathogen. New Phytologist, 193, 997-1008. Huffaker A., Kaplan F., Vaughan M.M., Dafoe N.J., Ni X.Z., Rocca J.R., et al. (2011) Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize. Plant Physiology, 156, 2082-2097. Humble G.D. & Hsiao T.C. (1969) Specific Requirement of Potassium for Light-Activated Opening of Stomata in Epidermal Strips. Plant Physiology, 44, 230-234. Humble G.D. & Hsiao T.C. (1970) Light-Dependent Influx and Efflux of Potassium of Guard Cells during Stomatal Opening and Closing. Plant Physiology, 46, 483-487. Humble G.D. & Raschke K. (1971) Stomatal Opening Quantitatively Related to Potassium Transport - Evidence from Electron Probe Analysis. Plant Physiology, 48, 447-453. Joyce A.L., Bernal J.S., Vinson S.B. & Lomeli-Flores R. (2009) Influence of Adult Size on Mate Choice in the Solitary and Gregarious Parasitoids, Cotesia marginiventris and Cotesia flavipes. Journal of Insect Behavior, 22, 12-28. Kesselmeier J. & Staudt M. (1999) Biogenic volatile organic compounds (VOC): An overview on emission, physiology and ecology. Journal of Atmospheric Chemistry, 33, 23-88. Kessler A. & Baldwin I.T. (2001) Defensive function of herbivore-induced plant volatile emissions in nature. Science, 291, 2141-2144. Koellner T., Lenk C., Schnee C., Koepke S., Lindemann P., Gershenzon J., et al. (2013) Localization of sesquiterpene formation and emission in maize leaves after herbivore damage. BMC Plant Biology, 13, 15. Koellner T.G., Gershenzon J. & Degenhardt J. (2009) Molecular and biochemical evolution of maize terpene synthase 10, an enzyme of indirect defense. Phytochemistry, 70, 1139-1145. Koellner T.G., Held M., Lenk C., Hiltpold I., Turlings T.C.J., Gershenzon J., et al. (2008) A maize (E)-beta-caryophyllene synthase implicated in indirect defense responses against herbivores is not expressed in most American maize varieties. Plant Cell, 20, 482-494. Koellner T.G., Schnee C., Gershenzon J. & Degenhardt J. (2004) The sesquiterpene hydrocarbons of maize (Zea mays) form five groups with distinct developmental and organspecific distribution. Phytochemistry (Amsterdam), 65, 1895-1902. Kolosova N., Sherman D., Karlson D. & Dudareva N. (2001) Cellular and subcellular localization of S-adenosyl-L-methionine: Benzoic acid carboxyl methyltransferase, the enzyme responsible for biosynthesis of the volatile ester methylbenzoate in snapdragon flowers. Plant Physiology, 126, 956-964. Loughrin J.H., Manukian A., Heath R.R., Turlings T.C.J. & Tumlinson J.H. (1994) Diurnal cycle
Accepted Article
657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702
This article is protected by copyright. All rights reserved.
24 of emission of induced volatile terpenoids herbivore-injured cotton plants. Proceedings of the National Academy of Sciences of the United States of America, 91, 11836-11840. Martin D., Tholl D., Gershenzon J. & Bohlmann J. (2002) Methyl jasmonate induces traumatic resin ducts, terpenoid resin biosynthesis, and terpenoid accumulation in developing xylem of Norway spruce stems. Plant Physiology, 129, 1003-1018. McAdam S.A.M. & Brodribb T.J. (2012) Stomatal innovation and the rise of seed plants. Ecology Letters, 15, 1-8. Melotto M., Underwood W., Koczan J., Nomura K. & He S.Y. (2006) Plant stomata function in innate immunity against bacterial invasion. Cell, 126, 969-980. Nagegowda D.A. (2010) Plant volatile terpenoid metabolism: biosynthetic genes, transcriptional regulation and subcellular compartmentation. FEBS Lett, 584, 2965-2973. Niinemets U., Loreto F. & Reichstein M. (2004) Physiological and physicochemical controls on foliar volatile organic compound emissions. Trends in Plant Science, 9, 180-186. Niinemets U. & Reichstein M. (2003a) Controls on the emission of plant volatiles through stomata: Differential sensitivity of emission rates to stomatal closure explained. Journal of Geophysical Research-Atmospheres, 108, ACH 2-1-17. Niinemets U. & Reichstein M. (2003b) Controls on the emission of plant volatiles through stomata: A sensitivity analysis. Journal of Geophysical ResearchAtmospheres, 108, ACH 3-1-10. Niinemets U., Reichstein M., Staudt M., Seufert G. & Tenhunen J.D. (2002) Stomatal constraints may affect emission of oxygenated monoterpenoids from the foliage of Pinus pinea. Plant Physiol, 130, 1371-1385. Pallaghy C.K. (1971) Stomatal Movement and Potassium Transport in Epidermal Strips of ZeaMays - Effect of Co2. Planta, 101, 287-295. Pandey R., Chacko P.M., Choudhary M.L., Prasad K.V. & Pal M. (2007) Higher than optimum temperature under CO2 enrichment influences stomata anatomical characters in rose (Rosa hybrida). Scientia Horticulturae, 113, 74-81. Pare P.W. & Tumlinson J.H. (1997) De novo biosynthesis of volatiles induced by insect herbivory in cotton plants. Plant Physiology, 114, 1161-1167. Parent A.C., Belanger M.C., Parent L.E., Santerre R., Viau A.A., Anctil F., et al. (2008) Soil properties and landscape factors affecting maize yield under wet spring conditions in eastern Canada. Biosystems Engineering, 99, 134-144. Raffa K.F., Berryman A.A., Simasko J., Teal W. & Wong B.L. (1985) Effects of Grand Fir Monoterpenes on the Fir Engraver, Scolytus-Ventralis (Coleoptera, Scolytidae), and Its Symbiotic Fungus. Environmental Entomology, 14, 552-556. Raschke K. & Fellows M.P. (1971) Stomatal Movement in Zea-Mays - Shuttle of Potassium and Chloride between Guard Cells and Subsidiary Cells. Planta, 101, 296-313. Raschke K. & Hedrich R. (1985) Simultaneous and Independent Effects of Abscisic-Acid on Stomata and the Photosynthetic Apparatus in Whole Leaves. Planta, 163, 105-118. Rasmann S., Koellner T.G., Degenhardt J., Hiltpold I., Toepfer S., Kuhlmann U., et al. (2005) Recruitment of entomopathogenic nematodes by insect-damaged maize roots. Nature, 434, 732-737. Richter A., Seidl-Adams I., Koellner T. & Degenhardt J. (2014) A small, differentially regulated family of farnesyl diphosphate synthases in maize (Zea mays) provides farnesyl diphosphate for the biosynthesis of herbivore-induced sesquiterpenes. (submitted) Rodriguez-Concepcion M., Fores O., Martinez-Garcia J.F., Gonzalez V., Phillips M.A., Ferrer
Accepted Article
703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748
This article is protected by copyright. All rights reserved.
25 A., et al. (2004) Distinct light-mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development. Plant Cell, 16, 144-156. Schmelz E.A., Alborn H.T. & Tumlinson J.H. (2001) The influence of intact-plant and excisedleaf bioassay designs on volicitin- and jasmonic acid-induced sesquiterpene volatile release in Zea mays. Planta, 214, 171-179. Shah S.P.J. & Rogers L.J. (1969) Compartmentation of Terpenoid Biosynthesis in Green Plants a Proposed Route of Acetyl-Coenzyme a Synthesis in Maize Chloroplasts. Biochemical Journal, 114, 395-&. Sharkey T.D. & Yeh S.S. (2001) Isoprene emission from plants. Annual Review of Plant Physiology and Plant Molecular Biology, 52, 407-436. Siekmann G., Keller M.A. & Tenhumberg B. (2004) The sweet tooth of adult parasitoid Cotesia rubecula: Ignoring hosts for nectar? Journal of Insect Behavior, 17, 459-476. Stecker T. (2012) Drought-tolerant corn efforts show positive early results. Scientific America. Tallman G. (2004) Are diurnal patterns of stomatal movement the result of alternating metabolism of endogenous guard cell ABA and accumulation of ABA delivered to the apoplast around guard cells by transpiration? Journal of Experimental Botany, 55, 19631976. Tamiru A., Bruce T.J., Woodcock C.M., Caulfield J.C., Midega C.A., Ogol C.K., et al. (2011) Maize landraces recruit egg and larval parasitoids in response to egg deposition by a herbivore. Ecol Lett, 14, 1075-1083. Turlings T.C.J., Alborn H.T., Loughrin J.H. & Tumlinson J.H. (2000) Volicitin, an elicitor of maize volatiles in oral secretion of Spodoptera exigua: Isolation and bioactivity. Journal of Chemical Ecology, 26, 189-202. Turlings T.C.J., Loughrin J.H., McCall P.J., Rose U.S.R., Lewis W.J. & Tumlinson J.H. (1995) How caterpillar-damaged plants protect themselves by attracting parasitic wasps. Proceedings of the National Academy of Sciences of the United States of America, 92, 41694174. Turlings T.C.J., Tumlinson J.H., Heath R.R., Proveaux A.T. & Doolittle R.E. (1991) Isolation and identification of allelochemicals that attract the larval parasitoid, Cotesia marginiventris (cresson), to the microhabitat of one of its hosts. Journal of Chemical Ecology, 17, 22352251. Turlings T.C.J., Tumlinson J.H. & Lewis W.J. (1990) Exploitation of herbivore-induced plant odors by host-seeking parasitic wasps. Science, 250, 1251-1253. Underwood W., Melotto M. & He S.Y. (2007) Role of plant stomata in bacterial invasion. Cellular Microbiology, 9, 1621-1629. van Gardingen P.R., Jeffree C.E. & Grace J. (1989) Variation in Stomatal Aperture in Leaves of Avena-Fatua L Observed by Low-Temperature Scanning Electron-Microscopy. Plant Cell and Environment, 12, 887-897. Voegeli U. & Chappell J. (1988) Induction of Sesquiterpene Cyclase and Suppression of Squalene Synthetase Activities in Plant-Cell Cultures Treated with Fungal Elicitor. Plant Physiol, 88, 1291-1296. Werner R.A. (1995) Toxicity and Repellency of 4-Allylanisole and Monoterpenes from White Spruce and Tamarack to the Spruce Beetle and Eastern Larch Beetle (Coleoptera, Scolytidae). Environmental Entomology, 24, 372-379. Willmer C.M., Don R. & Parker W. (1978) Levels of Short-Chain Fatty-Acids and of AbscisicAcid in Water-Stressed and Non-Stressed Leaves and their Effects on Stomata in Epidermal
Accepted Article
749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794
This article is protected by copyright. All rights reserved.
26 Strips and Excised Leaves. Planta, 139, 281-287. Yoshinaga N., Alborn H.T., Nakanishi T., Suckling D.M., Nishida R., Tumlinson J.H., et al. (2010) Fatty acid-amino acid conjugates diversification in Lepidopteran caterpillars. Journal of Chemical Ecology, 36, 319-325.
Accepted Article
795 796 797 798 799
This article is protected by copyright. All rights reserved.
27
Table I. Bonferroni corrected cut-off values for multiple comparisons. Cut-off values
802
were calculated as 0.05/# of comparisons.
Accepted Article
800 801
Experiment
Hypothesis1
Hypothesis2
# of Comparisons Hypothesis1/2
Morning induction emission
= 0.0083
= 0.0033
Morning induction in-planta
= 0.0071
= 0.0024
Evening induction light period emission
= 0.0028
= 0.0033
Evening induction light period in-
= 0.0028
= 0.0033
Evening induction dark period emission
= 0.01
= 0.005
Evening induction dark period in-planta
= 0.0083
= 0.0042
ABA treatment
= 0.0028
18
Morning induction with darkening
= 0.01
5
planta
Percent emitted farnesene for ABA experiment
= 0.017
Percent emitted farnesene for darkening experiment
= 0.05
803 804
This article is protected by copyright. All rights reserved.
Evening Induction Buffer treated in normal light cycle LG treated in normal light cycle LG+Dark
A
1.5
*
1.0
*
0.5
*
*
*
0.0
B * *
0.5
*
*
* b
* *
b
b
b
b
b a
ab
a
a
b
c ab
c ab
b
F *
*
*
*
b
b
b
b b
a
a
a
a
c
b
b ab
a
aa
c
Extracted
C
7.5
5.0
c c
Extracted
G
c
5.0
b b
H
Emitted
b
ab
b b b
b
a
a
b
bb
a
b
a
a
a
a
a
a
1:30 PM
a
noon
*
6:00 PM
*
4:30 PM
*
3:00 PM
1:30 PM
noon
10:30 AM
9:30 AM
*
5 4 3 2 1 0
a
a
10:30 AM
Emitted
b
b a
a
9:00 AM
D
b a
7:30 AM
0.0
b
6:00 AM
0.0
4:30 AM
2.5
3:00 AM
2.5
1:30 AM
Farnesene [µg g-1]
0.0
Farnesene [µg g-1 hr-1]
1.5 1.0 0.5 0.0 2.0
1.0
5 4 3 2 1 0
*
b
1.5
7.5
E
midnight
TPS10/APT1
2.0
1.5 1.0 0.5 0.0 2.0
10:30 PM
FPPS3/APT1
2.0
Morning Induction
9:30 PM
Accepted Article
Figure 1
Farnesene emitted [µg g-1 hr-1]
A 0.75
0.50
0.25
0.00
B 3 Total Farnesene [µg g-1]
Accepted Article
Normal light exposure Dark exposure after 1:30PM
Figure 2
*
* *
1:30PM 3PM 4:30PM 6PM
noon
Total emissions until 6PM Internal pool at 6PM
2 94%
1
*
0 Ln-L-Gln light
64%
** Ln-L-Gln darkened
ABA, normal light cycle Normal light cycle Continued darkness
Emitted Farnesene [µg g-1 hr-1]
A 7.5
b
5
2.5
a
a
b b a
c
a
a
a
ba
0
3AM 4:30AM 6AM 7:30AM 9AM 10:30AM
B
Total emission until 10:30AM Internal pool at 10:30AM
20
Total Farnesene [µg g-1]
Accepted Article
Figure 3
b 3%
15 10 5 0
*** a
a
76%
* Dark
29%
**
ABA + light
light