1 Running head: Guard cells regulate sesquiterpene emission.

Accepted Article

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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

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Accepted Article

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Abstract

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Maize seedlings emit sesquiterpenes during the day in response to insect herbivory.

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Parasitoids and predators use induced volatile blends to find their hosts or prey. To

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investigate the diurnal regulation of biosynthesis and emission of induced sesquiterpenes

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we applied linolenoyl-L-glutamine (LG) to maize seedlings in the morning or evening

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using a cut-stem assay and tracked farnesene emission, in-planta accumulation, as well as

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transcript levels of Farnesyl Pyrophosphate Synthase3 (ZmFPPS3) and Terpene

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Synthase10 (ZmTPS10) throughout the following day. Independent of time of day of LG

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treatment, maximum transcript levels of ZmFPPS3 and ZmTPS10 occurred within 3-4

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hours after elicitor application. The similarity between the patterns of farnesene emission

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and in-planta accumulation in light-exposed seedlings in both time courses suggested

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unobstructed emission in the light. After evening induction, farnesene biosynthesis

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increased dramatically during early morning hours. Contrary to light-exposed seedlings

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dark-kept seedlings retained the majority of the synthesized farnesene. Two treatments to

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reduce stomatal aperture, dark exposure at midday, and ABA treatment before daybreak,

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resulted in significantly reduced amounts of emitted and significantly increased amounts

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of in-planta accumulating farnesene. Our results suggest that stomata not only play an

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important role in gas exchange for primary metabolism but also for indirect plant

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defenses.

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Keywords: Zea mays, herbivore-induced plant volatiles, farnesene, linolenoyl-l-

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glutamine, stomata, nocturnal emission, diurnal emission, ZmFPPS3, ZmTPS10, GC-

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FID, quantitative Realtime PCR.

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This article is protected by copyright. All rights reserved.

3

Introduction

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Plants defend themselves against pathogens and herbivores directly by producing either

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constitutive or inducible barriers, toxins, and deterrents, and indirectly by attracting

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enemies, i.e. predators or parasites of the attackers. Terpenoids have been shown to

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perform all these defense roles in numerous representatives of the plant kingdom

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(Gershenzon & Dudareva 2007). The antimicrobial properties of terpenoids have been

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demonstrated in members of monocots and dicots, herbaceous and woody plants, (Raffa

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et al. 1985; Huffaker et al. 2011; Huang et al. 2012). In various species in the pine family

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terpenoids function as barriers, toxins and deterrents against insect attackers (Alfaro

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1995; Werner 1995; Martin et al. 2002). And finally, in both monocots and dicots volatile

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terpenoids act above and below ground as attractants for predators and parasites of

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different life stages of diverse insect herbivores (Turlings, Tumlinson & Lewis 1990;

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Turlings et al. 1991; Turlings 2000; Kessler & Baldwin 2001; Hoballah & Turlings 2005;

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Rasmann et al. 2005; Tamiru et al. 2011).

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The biosynthesis pathways for terpenes are well documented (Ashour, Wink &

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Gershenzon 2010). Therefore we will only give a brief summary here; all terpenoids are

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synthesized from the same basic building blocks, isopentenyl pyrophosphate (IPP) and

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dimethylallyl pyrophosphate (DMAPP), which are produced in the cytosol from acetyl-

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CoA via the mevalonate (MAV pathway) and in plastids from pyruvate, thiamine

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pyrophosphate, and glyceraldehyde 3-phosphate via methylerthyritol 4-phosphate (MEP

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pathway). As a general rule, sesquiterpenes are synthesized by terpene synthases (TPS) in

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the cytosol from farnesyl pyrophosphate (FPP), which in turn is made by FPP synthase

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(FPPS) from IPP and DMAPP produced via the MVA pathway. Mono- and diterpenes

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are made in plastids from geranyldiphosphate and geranylgeranyl diphosphate

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respectively, produced from MEP pathway derived IPP and DMAPP, but exceptions have

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been reported (Dudareva et al. 2005; Nagegowda, 2010).

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Maize is a model system to study induced volatile emissions (Degenhardt 2009); the

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volatile terpenoid bouquet is not only emitted in response to lepidopteran larvae feeding,

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but also to treatments with larval regurgitants, and fatty acid amino acid conjugate (FAC)

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elicitors like linolenoyl-L-glutamine (LG) (Turlings et al. 1990; Schmelz, Alborn &

Accepted Article

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4 Tumlinson 2001; Yoshinaga et al. 2010). Furthermore, FPP synthases and several terpene

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synthases are well characterized. Recently, Richter et al. (submitted) characterized the

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only insect herbivore-inducible farnesyl pyrophosphate synthase (ZmFPPS3) in maize.

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Transcript levels of ZmFPPS3 increase within 1 hour after caterpillar feeding, and

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caterpillar feeding mimics like wounding and applications of the FAC elicitors volicitin

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and LG to the wounding site (Richter et al. submitted). FPP is the substrate for the

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terpene synthases ZmTPS23 and ZmTPS10, whose major products are caryophyllene

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(Koellner et al. 2008), and bergamotene and farnesene (Koellner, Gershenzon &

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Degenhardt 2009), respectively. Just as for ZmFPPS3, transcript levels of these two

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terpene synthases increase within hours of insect feeding or FAC elicitor treatments

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(Koellner et al. 2008; 2009). While the components of the terpenoid blend vary in

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different cultivars of maize, the sesquiterpenes caryophyllene, bergamotene and

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farnesene are common in most volatile profiles (Gouinguene, Degen & Turlings 2001;

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Degen et al. 2004).

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Terpene synthases are thought to be key regulators of terpene biosynthesis, because

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developmental (Dudareva, Pichersky & Gershenzon 2004) as well as induced terpene

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emissions (Koellner et al. 2008; 2009) are correlated with terpene synthase expression.

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Furthermore, upon having been fed on by beet armyworm caterpillars, cotton plants

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emitted de novo synthesized terpenes, which were different from the constitutive volatiles

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released from damaged glands (Pare & Tumlinson 1997). Yet, it is not clear, how well

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terpene synthase transcripts are correlated with terpene synthase activity, and whether

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whatever is synthesized is emitted at a similar rate without any regulatory step between

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synthesis and emission.

Accepted Article

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Induced volatile emissions in general follow a diurnal pattern (Loughrin et al. 1994; De

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Moraes, Mescher & Tumlinson 2001). Yet, it is unclear whether, and if so how, light

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and/or the circadian clock regulate biosynthesis, or emission, or both. In order to identify

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points of regulation of volatile emissions, both biosynthesis and the mechanics of

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emission have to be well understood. While the biosynthetic pathway of terpenoids has

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been studied extensively, studies of the physical path volatiles take within and through

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plant tissues are mostly studied for isoprenes and monoterpenes (Kesselmeier & Staudt

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1999). Isoprenes are clearly emitted from stomata in oak and aspen leaves but

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5 surprisingly emission rates are not affected by aperture of stomata (Fall & Monson 1992).

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The authors explain these seemingly contradictory findings by postulating that the high

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internal build-up of isoprenes in leaves compensates quickly for the decreased diffusion

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rate due to decreased stomatal aperture. This compensation happens somewhat slower in

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Pinus pinea for oxygenated monoterpenes (Niinemets et al. 2002).

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Maize stomata, in particular, respond within minutes to a change in light conditions

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(Raschke & Fellows 1971; Pallaghy 1971). Flaccid guard cells close stomatal aperture in

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the dark (Humble & Hsiao 1969; Humble & Hasiao 1970; Humble & Raschke 1971). In

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preliminary experiments LG-induced sesquiterpenes accumulated during the dark period

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in the tissue of maize seedlings suggesting that closed stomata restrict emission of

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sesquiterpenes for several hours. We investigated this phenomenon through detailed time

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course experiments, monitoring transcript levels of ZmFPPS3 and ZmTPS10, as well as

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emitted and in-planta accumulated farnesene amounts. More specifically, we tested the

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following hypotheses (1) the patterns of induced transcript levels of ZmFPPS3 and

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ZmTPS10 depend on the time of day when the elicitor was applied and the subsequent

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light availability; (2) gene expression patterns of ZmFPPS3 and ZmTPS10 predict

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farnesene biosynthesis and emission; (3) farnesene biosynthesis is light-dependent, and

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finally; (4) stomata play a role in the observed emission patterns for farnesene.

Accepted Article

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Materials and methods

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Plant growth conditions

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Delprim seeds (Delley Switzerland) were treated with FLINT fungicide (Bayer

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CropScience Kansas City, MO) according to the manufacturer’s recommendations.

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Fungicide treated seeds were planted directly into autoclaved moistened Sunshine MVP

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(Sun Grow Horticulture, Agwam MA) and a quarter teaspoon of slow release fertilizer,

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Osmocote Plus 15-9-12 (Scotts®) was sprinkled on the surface of the soil. Seedlings

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were grown in growth chambers under a 17/7 day/night cycle, with lights on between

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4:30 AM and 9:30 PM. Temperature from 6:30 AM until 9:30 PM was set at 25°C, and

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from 9:30 PM to 6:30 AM was set at 23°C. All light conditions were produced using an

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equal number of metal halide and high pressure sodium lamps with an output of 400W

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each. One hour of dawn between 4:30 AM and 5:30 AM was simulated at a

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6 photosynthetic photon flux of 130 mol m-2 s-1. Full daylight between 5:30 AM and 6:30

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PM was simulated with 270 mol m-2 s-1. A prolonged dusk period between 6:30 PM and

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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

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Time course experiments

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All day and night time courses were conducted similarly. Since transcript level and in-

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planta terpenoids measurements are destructive, seedlings were harvested at the

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respective time points and gene expression and accumulated farnesene amounts were

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determined for each seedling. To measure emissions a separate experiment was

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conducted where emissions from the same set of seedlings were measured in 90 minute

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intervals throughout the course of the experiment.

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Morning induction: At 9:00 AM, 14-day old maize seedlings, stage v3, were cut off 1 cm

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above the first leaf, and the cut end of the stem was immersed in 250 L of either elicitor

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solution (25 mM phosphate buffer pH7.8, 4 L of LG at 100 ng L-1 in 100 mM

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phosphate buffer pH7.8) or 25 mM phosphate buffer, pH7.8. At 9:30 AM, after the

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seedlings had taken up the treatment solution, seedlings were transferred to ddH2O. For a

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total of seven time points, four buffer treated and five LG-treated seedlings were

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harvested every 90 min between 10:30 AM and 6 PM into liquid N2 for subsequent

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extraction of RNA and in-planta sesquiterpenes. Similarly, for base line establishment

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three buffer treated and three LG-treated seedlings were harvested at 9:30 AM into liquid

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N2.

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Emitted volatiles: At 9 AM, six seedlings each were induced with LG and buffer as

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described above. After having taken up all the treatment solution the stem of each

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individual seedling was inserted through the lid into 10 mL glass vials containing ddH2O.

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At 9:30 AM these seedlings were then inserted into volatile collection chambers, which

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were placed at a 15° angle from the horizontal. Charcoal filtered clean air entering the

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chambers at 10 psi was pulled over the seedlings and volatiles were drawn onto SuperQ

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filter traps at a flow of 0.5 L min-1. Filter traps were replaced every 90 min and eluted

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with two aliquots of 50 L 1:1 dichloromethane: hexane containing 4 ng L-1

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nonylacetate, internal standard. At 6 PM all seedlings were weighed.

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7 Evening induction: Since it took seedlings more time to take up the elicitor solution the

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evening induction experiment was started already at 8:30 PM, 1 hour before dark, to

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insure that by 9:30 PM all seedlings were induced. Otherwise the induction procedure

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was conducted in the same manner as in the morning induction experiment. 14-day old

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maize seedlings, stage v3, were cut off 1 cm above the first leaf, and the cut end of the

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stem was immersed in either elicitor solution or phosphate buffer. They were transferred

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to 25 mL beakers with ddH2O after they had taken up the elicitor solution (around 9:30

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PM) and placed into the dark growth chamber. One third of the LG-treated seedlings

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were placed under plexiglass tubes covered with aluminum foil, to keep these seedlings

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in the environmental conditions of the growth chamber, but in the dark, when the lights

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came on the following morning (LG+DARK). A diagram of the treatments is shown in

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Supplemental Figure 1. We verified that temperature reads inside these tubes did not

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differ from growth chamber temperature and that no light entered the tubes.

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Every 90 min three seedlings from each treatment were harvested, corresponding to six

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nocturnal (9:30 PM, 10:30 PM, midnight, 1:30 AM, 3 AM, 4:30 AM) and six diurnal

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samplings (6 AM, 7:30 AM, 9 AM, 10:30 AM, noon, 1:30 PM) for subsequent extraction

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of RNA and internal sesquiterpenes. During the night three buffer treated and three LG-

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treated seedlings were harvested per time point, while during the daytime hours three

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buffer-treated seedlings and three seedlings of both the LG treatment and the LG+Dark

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treatment were harvested. Seedlings were harvested directly into liquid N2 immediately

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after cutting off an additional centimeter from the base to eliminate the tissue that had

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been in immediate contact with the elicitor solution. To establish a baseline three

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seedlings per treatment were harvested immediately after the treatment solution had been

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taken up at 9:30 PM.

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Emitted volatiles: At 8:30 PM 12 seedlings were induced with LG and six seedlings with

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buffer as described above. After having taken up all the treatment solution the stem of

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each individual seedling was inserted through slits in the lid into 10 mL glass vials

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containing ddH2O. At 9:30 PM these seedlings were then inserted into volatile collection

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chambers, which were placed at a 15° angle from the horizontal. The LG-treated

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seedlings designated to remain in the dark were placed into collection chambers wrapped

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with aluminum foil. Emitted VOC were entrained in clean air and trapped on SuperQ

Accepted Article

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8 filters as before. Filters were changed every 90 min and eluted as before. At 1:30 PM all

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seedlings were weighed.

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Tests of stomatal involvement in volatile release

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Midday dark treatment: Eleven seedlings were treated with LG at 9 AM. At 9:30 AM all

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seedlings had taken up the LG solution and had been transferred to individual vials

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containing ddH20. Subsequently seedlings were placed into individual volatile collection

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chambers as described above. At 1:30 PM the chambers of five of the LG-treated

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seedlings were wrapped with aluminum foil to block the light. Filters were exchanged

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every 90 min from 10:30 AM through 6 PM. At 6 PM all seedlings were weighed and

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harvested into liquid N2 for subsequent extraction and measurements of in-planta

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terpenoids.

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ABA treatment: Eighteen 14 day-old Delprim seedlings were induced with LG at 8:30

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PM outside of the growth chamber as described above for the night time course

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experiment. After elicitor solution was taken up, seedlings were transferred to individual

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vials containing ddH2O, and inserted into the tubular volatile collection chambers. Six

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collection chambers were wrapped in aluminum foil to block the light. At 3 AM the

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ddH20 of six other seedlings was exchanged for vials containing 250 M ABA in ddH2O.

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Volatiles were collected on SuperQ filters as described above. Filters were exchanged at

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3 AM, 4:30 AM (just prior to the lights coming on), 6 AM, 7:30 AM, 9 AM and 10:30

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AM. At 10:30 AM seedlings were harvested into liquid nitrogen for in-planta terpenoids

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measurements.

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Internal terpene concentration measurements

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Terpene extraction from ground plant tissue was adapted from a published protocol

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(Koellner et al. 2004): Frozen samples were ground in a Genogrinder with three metal

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balls ( 6mm) for 2 min at 1200 strokes per min. About 500 mg of frozen tissue powder

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was suspended in 2 mL of pentane containing 400 ng humulene as internal standard.

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Glass vials were shaken in the refrigerator at 10

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centrifugation at 10

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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

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9 evaporated under a stream of nitrogen to dryness. Afterwards a slit was cut into the

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septum with a clean razor blade. Through this slid a SuperQ filter attached to vacuum and

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a thin metal tube attached to a nitrogen manifold were inserted. By heating vials for 7

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min at 80

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0.4 L min-1. Volatile organic compounds were eluted from the SuperQ filters with two

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aliquots of 50 L 1:1 dichloromethane: hexane each containing 4 ng L-1 nonylacetate a

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second internal standard. One L of the eluent was analyzed on an Equity-5 column (30

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m x 0.2 mm x 0.2 μm film thickness; Supelco, Bellefonte, PA) by GC-FID. Terpenoids

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were quantified relative to the square root of the product of both internal standards.

Accepted Article

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volatiles were drawn under nitrogen gas onto SuperQ filters at a flow rate of

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Chromatographic and spectroscopic analysis and identification

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Components of volatile blends emitted or extracted from plants were identified by

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comparison of GC retention times and mass spectra with those of authentic synthetic

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compounds, and by matching spectra to those from the NIST 02 mass spectral libraries.

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Samples were analyzed in an Agilent 6890 gas chromatograph-flame ionization detector

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system (GC-FID) equipped with an Equity-5 column (30 m x 0.2 mm x 0.2 μm film

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thickness; Supelco, Bellefonte, PA). Helium was used as carrier gas at an average linear

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velocity of 26 cm s-1. Samples, 1 μL, were injected in the splitless mode and the injector

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was changed to split mode after 0.75 min. The initial oven temperature was held at 40°C

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for 1 min, then programmed to increase at 8°C min-1 to 180°C; followed by ramping at

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30°C min-1 to 300°C, and holding at 300°C for 5 min. The injector and the detector

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temperatures were set to 280°C and 300°C, respectively. Selected samples were analyzed

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in a GC-MS system consisting of an Agilent 6890N gas chromatograph interfaced with

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an Agilent 5973N mass selective detector. The capillary column and GC conditions were

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equivalent to those used in the GC-FID. The MS was used in electron impact (EI)

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ionization mode with the default temperature settings (ion source: 230°C, and

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quadrupole: 150°C).

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Reverse transcriptase quantitative PCR (RT-qPCR)

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RNA extraction and cDNA synthesis: Total RNA was extracted from 100 mg ground

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tissue according to the manufacturer’s protocol with the RNeasy Plant mini kit (Qiagen)

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10 including the shredder step. Genomic DNA in 3 g total RNA was digested in a 50 L

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reaction with the Turbo DNA-free™ kit (Applied Biosystems) according to the

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manufacturer’s protocol. 550 ng of DNase treated total RNA was reverse transcribed in a

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20 L reaction with SMART™ MMLV Reverse Transcriptase (Clontech) according to

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the manufacturer’s protocol using a mix of anchored 18mer polydT and random 8mer

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oligo primers (Genomics Core Facility PennState University) at a final concentration of

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3.75 M.

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Quantitative PCR: cDNA was diluted 1:10 and used in 5 L aliquots as template in 20 l

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PCRs. Final primer concentration was 0.5 M. All other components necessary for qPCR

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were contained in the SsoFast™ EvaGreen® Supermix (BioRad). All PCRs were run in

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triplicate. A water control and a standard curve were run on each of the 96-well plates.

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The standard curve was generated using five three-fold serial dilutions of pooled cDNA

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from highly expressing samples as template. The highest concentrated template for the

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standard curves (3x) was obtained by diluting 1.5 L undiluted pooled cDNA into 3.5 L

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H2O and scaled up correspondingly so that on all plates standard curves were generated

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from the same templates. Similarly, enough cDNA of individual samples was diluted so

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that all reactions on all plates were from the same template pool thus guaranteeing that

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the same amount of template was used in all reactions. Standard curves were constructed

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assigning 81 arbitrary units as the starting amount of the transcript of interest when the 3x

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pooled cDNA was used as template. To control for the amount of cDNA in each of the

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samples adenine phosphate transferase 1 (ZmAPT1) was used as reference gene. Its

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expression is invariant during our experimental conditions. All transcript levels of the

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genes of interest were expressed as the ratio of their and ZmAPT1 starting quantities

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based on the respective standard curves.

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Primer sequences: ZmTPS10 F363: AGGGAACTTCGTGGTGGATGATAC, ZmTPS10

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R476: TGGCGTCTGGTGAAGGTAATGG; ZmFPPS3 F1044:

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CCTGGCTAGTTGTGCAAGCT, ZmFPPS3 R1262: GAAAACAGTTTGGACTGCCT;

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ZmAPT1 F380: AGGCGTTCCGTGACACCATC, ZmAPT1 R541:

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CTGGCAACTTCTTCGGCTTCC.

Accepted Article

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Statistical analysis

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11 All statistical analyses were conducted in SAS ver. 9.3. General and generalized linear

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mixed models were used to model the effects of treatment, harvest time, and their

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interaction. Based on the Bayesian information criterion (BIC) and the Akaikie

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information criterion (AIC), an unstructured covariance structure was selected as the best

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structure within in the repeated measures analyses. Bonferroni adjusted p-values for

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multiple comparisons were used based on a priori comparisons of interest; the respective

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adjusted level of significance for each analysis are listed in Table I. We tested two

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separate hypotheses for each time course: (Hypothesis1) mean treatment effects at any

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particular time point do not differ significantly, and (Hypothesis 2) mean treatment

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effects for a particular treatment do not differ between time points.

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Analyses of emission measurements from the morning induction were modeled with a

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repeated measures general linear model. None of the other emission measurements nor

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in-tissue measurements (gene expression and tissue-extracted farnesene measurements)

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satisfied the requirements of normality or equal variance, even after transformations.

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Therefore the data from these experiments were analyzed using a generalized linear

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mixed model assuming a lognormal distribution, with the main factors treatment and

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harvest time and a linear interaction term of treatment and harvest time. All pairwise

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interactions were significant except for extracted farnesene after morning induction and

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during the dark period after evening induction. After testing for significant interaction of

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treatment and harvest time within light/dark period we split the analysis of evening

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induction time courses into separate analyses for the light and dark period.

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The percent emitted farnesene was analyzed with a generalized linear model assuming a

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binomial distribution.

Accepted Article

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Results

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Time course after morning induction

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After morning induction transcript levels of ZmFPPS3 (Figure 1A) and ZmTPS10 (Figure

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1B) increased rapidly in response to wounding with both buffer (wounding control) and

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LG treatment. Within 1-2 hours of treatment transcript levels of ZmFPPS3 and ZmTPS10

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increased at least eight-fold compared to background levels at 9:30 AM. About 3-4 hours

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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

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PM. Buffer treatment had a similar effect on ZmFPPS3 and ZmTPS10 transcript levels,

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with maximum amounts at noon, about 50% of LG-induced maximum levels, and a more

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rapid decline to background levels (Figure 1 A, B).

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After morning induction the dynamics of farnesene accumulations within the tissue

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(Figure 1C) and emissions (Figure 1D) were similar: both increased steadily until they

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reached a plateau during the measurement interval between 1:30 PM and 3 PM. Extracted

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and emitted amounts were of the same order of magnitude at all time points; maximum

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in-planta accumulation of farnesene was 1 g per g freshweight (g g-1) in LG-induced

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and 0.4g g-1 in buffer-treated seedlings (Figure 1C), compared to maximum emission of

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1.3g g-1 hr-1 by LG-induced and 0.75 g g-1 hr-1 by buffer-treated seedlings (Figure 1D).

Accepted Article

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335 336

Time course after evening induction

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When seedlings were treated with LG in the evening during the last hour of the light

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cycle (between 8:30 PM and 9:30 PM), the dynamics of transcript accumulation (Figure

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1E, F), despite happening in the dark, were similar to the morning induction: a rapid

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increase until midnight (3 to 4 hours after induction) and for ZmFPPS3 a slow and steady

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decline continuing into the following morning (Figure 1E). ZmTPS10 transcript levels

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appeared to reach a plateau around midnight (Figure 1F). This plateau persisted until 7:30

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AM when transcript levels started to decline rapidly in light-exposed seedlings (Figure

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1F). Interestingly, after evening induction transcript levels of ZmTPS10 in the seedlings

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that were kept in the dark during the following light period (dark-kept) were higher than

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transcript levels of seedlings exposed to light (Figure 1F).

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Buffer-induced transcript levels of ZmFPPS3 declined more rapidly than LG-induced

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transcript levels (Figure 1E); buffer-induced relative to LG-induced transcript levels of

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ZmTPS10 never reached levels as high as after morning induction. (Figure 1B, F).

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Transcript levels of ZmFPPS3 were more than twice as high for corresponding time

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points after evening induction (i.e. midnight) compared to plants induced in the morning

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(i.e. noon) (Figure 1A, E). This comparison is legitimate because the same template was

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used to generate the standard curves for both qPCR assays.

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13 Although transcript levels of ZmFPPS3 declined steadily after midnight (Figure 1E),

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farnesene amounts extracted from the tissue (Figure 1G) began increasing at 1:30 AM.

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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 2.3g 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 3.17g 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 0.12g 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 0.3g 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 6.62g 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

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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

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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

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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

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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

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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

Emission of herbivore elicitor-induced sesquiterpenes is regulated by stomatal aperture in maize (Zea mays) seedlings.

Maize seedlings emit sesquiterpenes during the day in response to insect herbivory. Parasitoids and predators use induced volatile blends to find thei...
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