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Little peaks with big effects: Establishing the role of minor plant

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volatiles in plant-insect interactions1

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Andrea Clavijo McCormick, Jonathan Gershenzon and Sybille B. Unsicker*

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Max Planck Institute for Chemical Ecology, Department of Biochemistry

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*corresponding author: Sybille B. Unsicker, Hans-Knöll-Straße 8, 07745 Jena,

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Germany, [email protected]

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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.12357 This article is protected by copyright. All rights reserved.

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Abstract

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Plants emit complex mixtures of volatile organic compounds from floral and

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vegetative tissue, especially after herbivore damage, so it is difficult to associate

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individual compounds with activity towards pollinators, herbivores or herbivore

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enemies. Attention has usually focused on the biological activity of the most

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abundant compounds, but here we detail a number of reports implicating minor

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volatiles in attractant or deterrent roles. This is not surprising given the exquisite

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sensitivity of insect olfactory systems for certain substances. In this context, it is

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worth reconsidering the methods involved in sampling volatile compounds from

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plants, measuring their abundance, and determining their biological activity to be sure

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that minor compounds are not overlooked. Here we describe various experimental

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approaches and chemical and statistical methods that should increase the chance of

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detecting minor compounds with major biological activities.

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Keywords: herbivore-induced volatile emission, gas chromatography, herbivory,

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pollination, indirect plant defense, multivariate statistics

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This article is protected by copyright. All rights reserved.

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Introduction

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Plant volatile blends typically consist of a complex mixture of metabolites with a few

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compounds being highly abundant, but most being emitted in low amounts. For

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instance, banana fruits emit a few major volatile compounds when ripe, including 2-

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methylpropyl acetate, 3-methylbutan-1-ol, 3-methybutyl-3-methylbutanoate, 2-pentyl

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acetate, and eugenol, at a high release rate (Pino & Febles 2013). However, at least

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140 other volatiles in the bouquet are emitted at much lower rates of which 31 are

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considered important sensory cues for humans. It is easy to wonder why such an

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enormous variety of compounds are released from banana fruits and other plant

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parts. Do all of these constituents have biological roles, even the minor compounds,

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or are some just biosynthetic intermediates or catabolites without any further

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function?

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The functional importance of diverse volatile mixtures has already been

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demonstrated in other groups of organisms. Insects, for example, release complex

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blends of volatiles as pheromones. Like plant volatile blends, lepidopteran

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pheromones typically have one or two components present in high amounts with a

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number of other components present in low quantities. The minor compounds of

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pheromones have been shown to contribute to the specificity of mate attraction by

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providing cues for individuals of the same species while inhibiting the attraction of

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non-target sympatric species (Dunkelblum & Mazor 1993; Groot et al. 2010; Groot et

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al. 2008; Landolt & Heath 1987; Lofstedt & Vanderpers 1985; Mazor & Dunkelblum

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1992).

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It has long been theorized that pheromone mixtures may be a way to achieve

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specificity in the face of limitations in the capacity of biosynthetic machinery. This article is protected by copyright. All rights reserved.

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Lepidopteran females produce a mixture of long-chain fatty acid derivatives as

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pheromones which are 12-18 carbons in length and are modified with 1-3 double

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bonds of E or Z geometry and various oxygenated functional groups. There are not

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enough possible structures in this class to give every species a single unique

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component as a pheromone, but the use of mixtures and different ratios among the

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components of mixtures provides the necessary species specificity (Ando et al. 2004;

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Roelofs & Wolf 1988).

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In the case of plant volatiles, much less is known about their ecological roles.

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Although they clearly do not function solely to attract other organisms (Clavijo

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McCormick et al. 2012; Mumm & Dicke 2010), the occurrence of major compounds

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that are widespread among plant species together with complex blends of minor

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constituents also suggests limitations in the ability to produce unique compounds

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coupled with a drive for specificity in the blend as a whole. Unique blends may help in

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the attraction of pollinators to flowers and herbivore enemies to herbivore-damaged

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foliage (Bruce et al. 2005; Clavijo McCormick et al. 2012). In the latter case, the

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blend of minor compounds could inform herbivore enemies about the identity,

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quantity and properties of their herbivore prey or hosts (Clavijo McCormick et al.

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2012).

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In this review, we will survey previous reports about the role of minor plant

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volatiles in the attraction of insect herbivores, pollinators and herbivore enemies, and

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suggest methods for better detecting their importance in future work. The chemical

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identity of plant minor volatiles depends on their context. For example, the blend of

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volatiles released from herbivore-damaged foliage is typically dominated by terpenes

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and green leaf volatiles with aromatics, nitrogen-containing compounds and other

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fatty acid derivatives being minor constituents (Dudareva et al. 2004; Mumm & Dicke

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2010). In contrast, floral bends often have aromatics as their main components with This article is protected by copyright. All rights reserved.

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terpenes being minor volatiles. Examples of each major class of plant volatiles

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mentioned in the text are given in Fig. 1.

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Minor plant volatiles in interactions with herbivores

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Plant-produced volatiles are well known to facilitate host plant recognition by insect

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herbivores at a distance. Recognition may occur either by the detection of specific

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volatiles restricted to a particular host plant or a group of related plant species or by

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assessment of specific blends and ratios among widespread volatiles. Thus

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recognition is dependent upon the ability of the insect’s olfactory system to perceive

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these volatiles and how sensory input is processed (Bruce et al. 2005).

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Electrophysiological studies on various phytophagous insects have revealed that

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they can perceive both specific and widespread volatiles, some of which may be

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present in only minor amounts (Bruce et al. 2005).

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One widely documented example of attractive host plant volatiles found at low

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levels is the isothiocyanates. These volatile compounds, produced by the

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myrosinase-mediated hydrolysis of glucosinolates (Halkier & Gershenzon 2006), are

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characteristic of plants of the Brassicales and typically present in only minute

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amounts in the headspace of undamaged plants. While isothiocyanates are highly

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toxic to generalist herbivores, specialized insect herbivores commonly use them as

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host finding cues (Hopkins et al. 2009). A study comparing the volatile profiles of

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intact plants of five Brassicaceae species: Brassica napus, B. campestris, B. juncea,

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B. nigra and Sinapis alba showed that monoterpenes and sesquiterpenes (such as

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E,E,-α-farnesene) and aromatic compounds (such as benzaldehyde) were the most

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dominant compounds reaching values as high as 80 % of the total mixture whereas

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isothiocyanates were present in very low quantities ranging from 0.1% to 5% of the

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total blend (Tollsten & Bergstrom 1988). In another study, the volatile bouquet of This article is protected by copyright. All rights reserved.

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macerated oilseed rape (Brassica napus ssp. oleifera) leaves was found to be

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dominated by two widespread green leaf volatiles, (Z)-3-hexenol and (Z)-3-hexenyl

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acetate (Fig. 1), accounting together for almost 90% of the composition of the odor,

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whereas 3-butenyl isothiocyanate, sec-butyl isothiocyanate and 2-phenylethyl

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isothiocyanate (Fig. 2) accounted for less than 1% each. Despite the low amounts of

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isothiocyanates, these characteristic volatiles elicited much higher antennal

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responses than most green leaf volatiles and terpenoids tested in the antennae of the

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cabbage seed weevil Ceutorhynchus assimilis (Evans & Allenwilliams 1992), it was

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later shown that this insect has specialized olfactory receptor neurons tuned to the

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detection of isothiocyanates (Blight et al. 1995). The presence of an isothiocyanate-

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specialized olfactory receptor neuron has also been demonstrated for the cabbage

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aphid Brevicoryne brassicae which is behaviorally attracted to isothiocyanates and

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uses them as host-derived cues. Interestingly, some insects which do not feed on

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Brassica species and are behaviorally repelled by isothiocyanates (such as the black

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bean aphid, Aphis fabae) also possess isothiocyanate-specialized olfactory receptor

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neurons, indicating that these minor volatiles are also important in the avoidance of

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potentially toxic non-host plants (Nottingham et al. 1991).

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Minor plant volatiles in interactions with herbivore enemies

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Herbivory and oviposition induce qualitative and quantitative changes in the volatile

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emission from vegetative plant parts (Arimura et al. 2009; Clavijo McCormick et al.

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2012; Mumm & Dicke 2010), which leads to the attraction of a broad range of

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herbivore enemies in over 150 tri-trophic systems studied to date (Clavijo McCormick

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et al. 2012; Mumm & Dicke 2010; Turlings & Wackers 2004; Unsicker et al. 2009).

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However, despite the many reports on this subject, the precise volatile constituents This article is protected by copyright. All rights reserved.

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mediating attraction remain unknown in most cases (but see Kappers et al. 2005;

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Schnee et al. 2006).

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One of the main difficulties in identifying herbivore enemy attractants is the

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lack of knowledge on how herbivore enemies perceive and process olfactory

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information (Clavijo McCormick et al. 2012). It is possible that natural enemies of

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herbivores perceive plant odors in the same way as herbivores, either by recognition

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of specific-odors or recognition of blends and ratios. On the other hand, it has been

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recently suggested that plant odors are perceived by natural enemies as a block

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rather than as individual attractants (van Wijk et al. 2011). The presence of

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background odors also plays an important role in recognition of herbivore-induced

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volatiles by natural enemies since they provide the “context” in which odors are

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perceived. For this reason individual compounds may only be attractive against an

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appropriate background which could include minor compounds (Beyaert et al. 2010;

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Dicke & Baldwin 2010; Mumm & Hilker 2005).

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Two major classes of herbivore-induced volatiles, the green leaf volatiles and

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terpenoids, have often been associated with herbivore enemy recruitment because

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they are the most abundant compounds emitted from plants after herbivore attack

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(Arimura et al. 2009; Gershenzon & Dudareva 2007; Mumm & Dicke 2010), stimulate

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strong physiological responses by insect antennae (Gouinguene et al. 2005; Ngumbi

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et al. 2010; Smid et al. 2002) and can be associatively learned by herbivore enemies

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(Costa et al. 2010; De Boer et al. 2005; Glinwood et al. 2011). Nevertheless,

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compounds present in minor amounts could also be important for attraction as

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suggested earlier by Dicke (Dicke, 1999), especially considering the extreme

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sensitivity of olfactory receptor neurons in insect antennae (Angioy et al. 2003). The

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significant role of minor constituents is also highlighted by studies showing that the

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major compounds of the herbivore induced blend are sometimes unattractive or even This article is protected by copyright. All rights reserved.

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repellent to herbivore enemies when presented alone, or that the absence of major

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compounds does not decrease enemy attraction (Beyaert et al. 2010; Clavijo

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McCormick et al. 2014; Michereff et al. 2013; Mumm & Hilker 2005; van Wijk et al.

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2011).

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One of the best studied examples of an herbivore enemy being attracted to

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volatiles emitted from herbivore-damaged plants is the orientation of the braconid

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wasp, Cotesia marginiventris, to maize plants damaged by one of its lepidopteran

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hosts, such as Spodoptera littoralis (Turlings & Wackers 2004). The maize blend

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consists of many possible candidate compounds for attraction. Curiously,

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investigations on C. marginiventris with gas chromatography-electroantennography

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(GC-EAG) systems showed high antennal responses to compounds below the GC

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detection threshold (Gouinguene et al. 2005). Meanwhile, sesquiterpenes which are

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major constituents of the blend produced by S. littoralis feeding on maize did not

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seem to be significant for host location by C. marginiventris (D'Alessandro & Turlings

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2005). When the blend was filtered through different solid adsorbent filters, the

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parasitic wasps were still attracted to blends from which most sesquiterpenes were

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removed, but not attracted to a blend from which highly polar volatiles (nearly all

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minor components) were removed. Further fractionation employing chemical solvents

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and preparative GC confirmed C. marginiventris attraction to polar volatiles, many of

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which are emitted in very small amounts (D'Alessandro et al. 2009). Interestingly,

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another species of parasitic wasp (Microplitis rufiventris) was still attracted to a maize

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blend from which polar volatiles had been removed indicating that, although

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attraction of herbivore enemies by plant volatiles is a widespread phenomenon,

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volatile cues for host location are likely species-specific (D'Alessandro & Turlings

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2005).

This article is protected by copyright. All rights reserved.

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Recently we found that several major compounds of the volatile blend of black

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poplar (Populus nigra) foliage released after gypsy moth (Lymantria dispar) caterpillar

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herbivory, including green leaf volatiles and terpenes, were unattractive to the

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braconid parasitoid Glyptapanteles liparidis when offered individually, and two

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widespread compounds (Z)-3-hexenol and (E)-β-ocimene were even repellent

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(Clavijo McCormick et al. 2014). On the other hand, two minor nitrogen-containing

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compounds, 2- and 3-methyl butyraldoxime (Fig. 2), were highly attractive to G.

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liparidis when tested alone and showed stronger physiological responses than most

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sesquiterpenes and green leaf volatiles when tested on their antennae. These

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aldoximes were also attractive to natural parasitoids under field conditions suggesting

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that several different herbivore enemies might use these minor compounds to locate

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their hosts on poplar foliage. Although the aldoximes are minor compounds, we

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found that they are emitted only at the site of damage and not from adjacent

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undamaged foliage as many herbivore-induced volatiles are. In addition, aldoximes

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are produced in higher amounts after attack of early instar L. dispar larvae

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(McCormick and Unsicker, unpublished results), which are often preferred by

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koinobiont parasitoids, indicating that these compounds might provide specific

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information to herbivore enemies regarding the exact location of their hosts at the

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preferred physiological stage.

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Sometimes the effect of minor plant volatiles cannot be attributed to individual

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compounds, but just to the blend itself. For instance, (E)-β-farnesene is the only

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compound produced in higher amounts after oviposition by the sawfly Diprion pini

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feeding on the Scots pine Pinus sylvestris. While the egg parasitoid Chrysonotomyia

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ruforum is attracted to egg laden pine twigs over ones without sawfly eggs, (E)-β-

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farnesene is not attractive by itself (Mumm et al. 2003). However, when (E)-β-

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farnesene was offered in combination with a mixture of volatiles emitted by This article is protected by copyright. All rights reserved.

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undamaged plants the parasitoid was attracted (Mumm & Hilker 2005). Similar

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situations have been described for bark beetle parasitoids in spruce and predatory

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mites of spider mites feeding on lima bean plants where major herbivore-induced

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compounds elicit no attraction of natural enemies when presented alone but only

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against a background odor (De Boer & Dicke 2004; Pettersson et al. 2001;

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Pettersson et al., 2000). These examples point to the critical role of context in

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determining odor-guided behavior. Minor compounds might be of great relevance in

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establishing the proper context (Hilker & Meiners 2006).

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Minor plant volatiles in interactions with pollinators

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Pollinator attraction to floral volatiles also offers examples of the importance of minor

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compounds.

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(Asparagaceae) are either pollinated by carrion flies or wasps of the Pompilidae, but

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neither the color nor the composition of the nectar seemed to be responsible for the

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differences between fly and wasp-pollinated species pointing to volatiles as the major

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factor (Shuttleworth & Johnson 2009; Shuttleworth & Johnson 2010). The main

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components in the odor bouquet of these species are the monoterpene linalool and

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the benzenoid 3,5-dimethoxytoluene (Fig. 1), accounting for as much as 50% of the

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total odour. However, numerous minor volatiles differ between the individual species

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making it a hard task to establish which compounds mediate the differences in

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pollination systems. A non-metric multidimensional scaling based on the Bray-Curtis

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similarity index followed by analysis of similarities was performed for the compounds

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in the odor bouquet of four Eucomis species: two wasp pollinated species, E.

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autumnalis and E. comosa, and two fly pollinated species, E. bicolor and E. humilis.

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This statistical approach revealed that the minor sulfur-containing volatiles, dimethyl

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disulfide (DMDS) and dimethyl trisulfide (DMTS) (Fig. 2) contributed significantly to

Flowers

of

closely

related

This article is protected by copyright. All rights reserved.

species

of

the

genus

Eucomis

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the average similarity in both fly-pollinated species (Shuttleworth & Johnson 2010).

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These compounds, which typically derive from protein decomposition, had been

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previously reported as blowfly attractants involved in the pollination of Saponia and

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Helicodiceros flowers (Jürgens et al. 2003; Stensmyr et al. 2002). Experimental

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supplementation of the wasp-pollinated E. autumnalis and E. comosa with a blend of

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dimethyl disulfide and dimethyl trisulfide caused a highly significant increase in the

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visitation of these flowers by flies suggesting that these minor volatiles could be

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responsible for fly attraction. However, vials containing these sulphur compounds

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alone were not attractive to the flies indicating that visual cues and other odors are

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also necessary in addition for pollinator attraction. Altogether these examples

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illustrate the importance of minor compounds in mediating plant-insect interactions

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suggersting that efforts should be made to include them in volatile collection and

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analysis.

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Methods for identifying minor volatiles with biological activity

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The significance of minor plant volatiles may have been overlooked in the past

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because of limitations in experimental methodology. Here we discuss some of the

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procedures commonly used for chemically analyzing volatiles, assessing their relative

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abundance and determining biological activity, and make suggestions that might

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increase the ability to identify minor active compounds.

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Volatile collection and analysis

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As a first consideration, it is important to collect and analyze volatile mixtures in a

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way that maximizes the recovery of all classes of compounds, gives adequate

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separation of peaks on gas chromatography and allows for the identification of as

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many substances as possible. Standard methods for headspace sampling and GCThis article is protected by copyright. All rights reserved.

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MS analysis (Bicchi & Maffei 2012; Qualley & Dudareva 2009; Qualley & Dudareva

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2014; Tholl et al. 2006) can identify hundreds of peaks in typical plant volatile blends

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at nanogram or picogram levels per injection, although they may not adequately

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detect molecules of higher mass and polarity that are sometimes referred to as

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“semivolatiles” (Vaughan et al. 2013). More detailed recommendations for the

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collection and analysis of minor volatile compounds are given in Box 1.

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Comparison of volatile composition

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To determine the plant volatiles responsible for specific biological activities,

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researchers commonly compare the composition of active vs. inactive mixtures. For

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example, to determine which compounds attract an herbivore enemy, the blend of

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volatiles from an attractive herbivore-damaged plant might be compared to that of an

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unattractive, undamaged control. But, many other blends can be compared too. The

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emission of herbivore-induced volatiles varies substantially among populations or

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genotypes of some plant species (Eller et al. 2012; Hare 2011; Kariyat et al. 2012;

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Schuman et al. 2009) and with plant age and development (Köllner et al. 2004;

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Shiojiri & Karban 2006). The nature of the herbivore species (Pierre et al. 2011), its

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abundance (Cai et al. 2012; Girling et al. 2011) and developmental stage

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(Gouinguene et al. 2003) affect emission too. So can other biotic influences, such as

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pathogens (Ponzio et al. 2013), mycorrhizal and endophytic fungi (Fontana et al.

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2009; Jallow et al. 2008), competing plants (Kigathi et al. 2013) and abiotic factors

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including light and nutrient availability (Gouinguene & Turlings 2002; Sampedro et al.

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2010), photoperiod (Cai et al. 2012) and temperature (Gouinguene & Turlings 2002;

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Kigathi et al. 2009). Blends collected under all of these conditions, whether attractive

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or unattractive to herbivore enemies, can be compared to narrow down the actual

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compounds responsible for the attraction. This article is protected by copyright. All rights reserved.

285 286

Statistical comparison of volatile collections

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The composition of volatile blends cannot always be compared by simple inspection

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of chromatographic peak areas. Several authors have realized the necessity of using

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appropriate statistical methods for analyzing plant volatile datasets to reveal the

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compounds differing significantly among experimental treatments (Hare 2011; van

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Dam & Poppy 2008). Factor reduction analyses such as principal component

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analysis (PCA) or non-metric multidimensional scaling (NMDS) as well as

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classification algorithms such as canonical discriminant analysis (CDA) have been

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applied in recent studies on herbivore induced plant volatile emission (Dong et al.

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2011; Girling et al. 2011; Jürgens et al. 2006). These and other multivariate statistical

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analyses can be carried out with free online software, such as R (R Development

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Core

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(http://www.metaboanalyst.ca). PCA has been particularly favored in volatile

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analyses since factor reduction allows generating a few principal components that

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explain most of the variation in the data, which can then be used as explanatory

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variables in univariate statistical testing (e.g. Pareja et al. 2009).

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However, complex volatile datasets often do not meet the assumptions for PCA and

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similar statistical approaches such as having normal distribution of data. Therefore,

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random forest analysis (Breiman 2001) was recently suggested as a mutltivariate

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statistical technique to classify large plant volatile datasets and identify relevant

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compounds for biological assays (Ranganathan & Borges 2010). Random forest

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analysis is a computer-learning algorithm that constructs multiple decision trees using

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bootstrapping. It selects a variable set of attributes at each node of the decision tree,

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leading to the selection of a minimum set of predictor variables. The algorithm

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calculates two numerical values that help to ascertain the importance of a given

Team,

http://www.R-project.org)

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or

MetaboAnalyst

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variable: the mean decrease accuracy (MDA) is a hierarchical value of the relative

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importance of a given variable to the classification, and the out of bag error (OOB)

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indicates the probability of the variable being incorrectly classified (Breiman 2001).

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The advantages of the random forest analysis over other statistical analysis methods

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include that it is not biased towards abundant compounds and thus especially useful

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in finding minor compounds that characterize different volatile blends. In addition, it is

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well suited to analyze data sets with more variables than samples and variables of an

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auto-correlated nature, such as plants volatiles with common biosynthetic pathways.

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Finally, it is robust to correlation and interdependence as well as to other interactions

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among variables and has good classification efficiency even for variables with low or

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zero values (Ranganathan & Borges 2010).

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In our search for herbivore-induced black poplar volatiles responsible for attraction of

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the parasitoid G. liparidis, we used random forest analysis to identify volatile

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compounds characteristic of the blend from gypsy moth-damaged foliage (attractive)

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vs. the blend of adjacent undamaged foliage (unattractive) (Clavijo McCormick et al.

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2014). As stated above, the most abundant compounds from the infested poplar

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headspace were the terpenoids (E)-DMNT, (E)-β-ocimene and (E)-β-caryophyllene.

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However, random forest analysis revealed that three nitrogen containing compounds

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(benzyl cyanide, 2-methylbutyraldoxime and 3-methylbutyraldoxime), two of which

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were only present in minor amounts, were the most characteristic components of the

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herbivore-induced blend. Both groups of compounds (the highly abundant and those

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suggested by random forest analysis) were subjected individually to behavioral and

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electrophysiological experiments to investigate their attractive potential, which led to

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the discovery of the biological importance of minor nitrogenous compounds for

335

parasitoid wasps.

This article is protected by copyright. All rights reserved.

336 337

Other methods for assessing biological activity

338

Another approach to reveal the biological role of individual volatiles from a complex

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blend is to fractionate the blend and then test the single fractions in bioassays with

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parasitoids or herbivores. Repeated fractionation could lead to the identification of

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individual active compounds. Previous work from Ted Turlings’ group showed that

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volatile mixtures could be readily fractionated using preparative GC and by different

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types of adsorbent filters coupled with desorption by different solvents (D'Alessandro

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et al. 2009; D'Alessandro & Turlings 2005).

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Other tools to simplify volatile profiles and allow better assessment of the roles

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of specific compounds include metabolic inhibitors. For example, treating plant tissue

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with inhibitors of specific biosynthetic pathways can eliminate or reduce many

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components of the blend. Two inhibitors of terpene biosynthetic pathways which

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block the mevalonate (mevinolin) and methylerythritol phosphate (fosmidomycin)

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pathways have been employed to reduce emission of terpenes in studies on floral

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(e.g. Junker et al. 2011) and vegetative (Mumm et al. 2008) volatiles.

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Mutant lines or plant lines over-expressing volatiles can also be valuable tools

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to test the role of individual volatiles or groups of volatiles in attracting herbivores,

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herbivore enemies and pollinators (Cheng et al. 2007; Fontana et al. 2011;

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Halitschke et al. 2008; Schnee et al. 2006; Schuman et al. 2012). For certain well-

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studied model plant species, such as Zea mays, Arabidopsis thaliana and Nicotiana

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attenuata, the availability of large collections of mutants coupled with the ability to

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carry out genetic transformation can allow researchers to create custom volatile

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profiles for testing.

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The biological relevance of individual compounds from volatile blends can also

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be inferred from investigations on insect olfaction. Studies employing combined gas This article is protected by copyright. All rights reserved.

362

chromatography-electroantennograms (GC-EAG) or a gas chromatograph combined

363

with single cell recording can highlight the importance of otherwise minor compounds

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which can then be tested in behavioral bioassays (Ulland et al. 2008). Unfortunately,

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the minor components of volatile blends may not be widely available from commercial

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suppliers, and once identified it may be necessary to isolate them from natural

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sources or carry out chemical synthesis to obtain enough for testing.

368 369

Conclusions

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Gas chromatograms of plant volatile samples commonly contain many minor peaks

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that have become much easier to separate and identify in recent years thanks to

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advances in gas chromatograph and mass spectrometer instrumentation. However,

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in searching for the compounds responsible for activity in biotic interactions,

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researchers usually focus on the most abundant compounds. This is a judicious

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approach since abundant compounds are usually readily available for testing. But,

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we have described a number of examples in this review where minor rather than

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major members of volatile blends were shown to be responsible for their biological

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activity. Hence, methods should be employed to insure that volatile collection,

379

chemical analysis, data handling and biological testing do not exclude the minor

380

components of volatile mixtures. Given the ability of insects to perceive minute

381

amounts of many plant volatiles, minor constituents should always be given as much

382

consideration as possible.

383 384

BOX 1. RECOMMENDATIONS FOR COLLECTING AND ANALYZING MINOR

385

PLANT VOLATILES

386

There are some excellent reviews that give a good overview of volatile collection

387

techniques, gas chromatographic analysis and identification (Qualley and Dudareva, This article is protected by copyright. All rights reserved.

388

2009; Tholl et al. 2006; Tholl & Röse 2006). Here we list some suggestions to

389

improve detection of minor volatile compounds.

390

First of all it is important to avoid contamination during volatile collection since

391

this will increase the signal to noise ratio and make it harder to detect the

392

components of the sample. The usage of materials which are easy to clean and emit

393

only low levels of volatiles, such as Teflon, glass or polyethylene terephthalate (PET,

394

a plastic polymer) is recommended. In dynamic headspace collection, it is important

395

to filter the incoming air with activated charcoal to avoid aerial contamination. The

396

use of collection techniques such as solid-phase microextraction (SPME) fibers or

397

thermal desorption cartridges which do not require solvent elution prevents small

398

compounds from being diluted and increases the chances of detection. However, for

399

both solid-phase microextraction and thermal desorption, the samples cannot be

400

recovered for further analysis and quantification is more challenging. When trapping

401

on solid adsorbents, the combination of polymer-based (e.g. SuperQ®, Porapak Q®,

402

Tenax GC®, Tenax TA®) with carbon-based adsorbents (e.g. Carboxen™,

403

Carbosieve ™, Supelco, Carbotrap) can be helpful to recover minor compounds

404

since different materials have different selectivities.

405

During gas chromatography, peaks of minor volatile compounds might be overlaid by

406

peaks of major ones. Therefore, it might be advisable to use at least two different

407

types of GC columns for the analysis (e.g. a non-polar column such as DB-5 and a

408

polar column, such as DB-WAX). In addition, columns of 60 m in length instead of the

409

common 30 m columns should improve separation. An example of how using

410

different columns might aid in compound separation is given in Fig. 3. At the detector

411

level, the sensitivity of GC-MS analysis to minor compounds can be increased by

412

using the selected ion monitoring mode (SIM) when the base peak is known or

413

suspected, In general gas chromatography, nitrogen-phosphorus detectors can This article is protected by copyright. All rights reserved.

414

increase the sensitivity to compounds containing atoms of these elements, but are

415

insensitive to most other compounds (Tholl & Röse 2006).

416 417

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683 684 685

Figure legends

686

Figure 1: Examples of major classes of plant volatiles

687

Figure 2: Minor plant volatiles with significant biological activity

688

Figure 3: Separation of minor volatiles by chromatography on two different GC

689

columns. Two GC-MS chromatograms are depicted for the same sample run on two

690

different columns. Sample contains an artificial mixture of nitrogen-containing

691

volatiles emitted by poplar trees after herbivory. The chromatogram in the upper

692

panel was run on a non-polar DB-5 column ((5% phenyl)-methylpolysiloxane), and

693

that in the lower panel on a polar DB-Wax column (polyethylene glycol). In the DB-5

694

column, compounds 1 and 2 overlap and minor compounds 9 and 10 are not visible.

695

By contrast, in the DB-Wax column compounds 1 and 2 are separated and This article is protected by copyright. All rights reserved.

696

compounds 9 and 10 appear, but compounds 6 and 7 are not clearly separated any

697

longer. It is sometimes necessary to run samples on more than one type of column to

698

adequately detect minor volatiles. Compound identity: IS = internal standard (nonyl

699

acetate), 1. (E)-2-methylbutyraldoxime, 2. (E)-3-methylbutyraldoxime, 3. (Z)-2-

700

methylbutyraldoxime, 4. (Z)-3-methylbutyraldoxime, 5. benzyl cyanide, 6. (E)-

701

phenylacetaldoxime, 7. (Z)-phenylacetaldoxime, 8) 2-phenylnitroethane, 9) 3-

702

methylbutane nitrile, 10) 2-methylbutane nitrile.

703 704

This article is protected by copyright. All rights reserved.

705 706

Figure 1:

707 708 This article is protected by copyright. All rights reserved.

709

Figure 2:

710 711 712 713 714 715 716

This article is protected by copyright. All rights reserved.

717 718

Figure 3:

719 720 721 722 723 724

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Figure 1. Examples of major classes of plant volatiles

Green leaf volatiles (Z)-3-Hexenol

Monoterpenes (C10) (E)-β-Ocimene (R)-(-)-Linalool

Terpenoids Hemiterpene (C5) Isoprene

(E)-2-Hexenal Sesquiterpenes (C15) (Z)-3-Hexenyl acetate

(E,E)-α-Farnesene

Homoterpenes (+1 C) DMNT TMTT

Aromatic compounds Salicylaldehyde

Nitrogen and sulphur containing compounds Amines

Oximes

Methyl salicylate

Isothiocyanates

Nitriles

Figure 2. Minor plant volatiles with significant biological activity. Image of Pollinator fly on Eucomis bicolor courtesy of Prof Steven Johnson, School of Life Sciences, University of KwaZulu-Natal, South Africa. Image of Ceutorhynchus assimilis on Brassica napus courtesy of Astrid Oldenburg.

Pollinator attraction

Herbivore attraction

Parasitoid attraction

Dimethyl disulfide

3-Butenyl isothiocyanate

3-Methylbutyraldoxime

Dimethyl trisulfide

2-Phenylethyl isothiocyanate

2-Methylbutyraldoxime

Ceutorhynchus assimilis on Brassica napus

Glyptapanteles liparidis and Lymantria dispar induced Populus nigra

Pollinator fly on Eucomis bicolor

Figure 3. Separation of different compounds by using different GC columns and running times