Green leaf volatile production by plants: a meta-analysis.

666 I. Introduction 667 II. Biosynthesis 667 III. Meta-analysis 669 IV. The type of stress influences the total amount of GLVs released 669 V. Herbivores can modulate the wound-induced release of GLVs 669 VI. Fungal infection greatly induces GLV production 672 VII. Monocots and eudicots respond differentially to different types of stress 673 VIII. The type of stress does not influence the proportion of GLVs per chemical class 673 IX. The type of stress does influence the isomeric ratio within each chemical class 674 X. GLVs: from signal perception to signal transduction 676 XI. GLVs influence the C/N metabolism 677 XII. Interaction with plant hormones 678 XIII. General conclusions and unanswered questions 678 Acknowledgements 679 References 679 SUMMARY: Plants respond to stress by releasing biogenic volatile organic compounds (BVOCs). Green leaf volatiles (GLVs), which are abundantly produced across the plant kingdom, comprise an important group within the BVOCs. They can repel or attract herbivores and their natural enemies; and they can induce plant defences or prime plants for enhanced defence against herbivores and pathogens and can have direct toxic effects on bacteria and fungi. Unlike other volatiles, GLVs are released almost instantly upon mechanical damage and (a)biotic stress and could thus function as an immediate and informative signal for many organisms in the plant's environment. We used a meta-analysis approach in which data from the literature on GLV production during biotic stress responses were compiled and interpreted. We identified that different types of attackers and feeding styles add a degree of complexity to the amount of emitted GLVs, compared with wounding alone. This meta-analysis illustrates that there is less variation in the GLV profile than we presumed, that pathogens induce more GLVs than insects and wounding, and that there are clear differences in GLV emission between monocots and dicots. Besides the meta-analysis, this review provides an update on recent insights into the perception and signalling of GLVs in plants.

Green Leaf Volatiles in Plant Signaling and Response.

Most 'green' plants form green leaf volatiles (GLVs). GLVs are a familiar plant secondary metabolite, but knowledge of their physiological and ecological functions is limited. GLV formation is tightly suppressed when plant tissues are intact, but upon mechanical wounding, herbivore attack, or abiotic stresses, GLVs are formed rapidly, within seconds or minutes. Thus, this may be an important system for defense responses, allowing plants to protect themselves from damage as soon as possible. Because GLV formation in the natural environment is roughly related to the degree of stress in the plant life, sensing the amount of GLVs in the atmosphere might allow plants to recognize their surroundings. Because some plants respond to GLVs, they may communicate with GLVs. GLVs that contain α,ß-unsaturated carbonyl groups might activate signaling systems regulated under the redox state of plant cells. Plasma membranes would also be targets of interactions with GLVs. Additionally, the metabolism of GLVs in plant cells after absorption from the atmosphere could also be classified as a plant-plant interaction.

Physiological and metabolomic analysis of Punica granatum (L.) under drought stress.

MAIN CONCLUSION: Punica granatum has a noticeable adaptation to drought stress. The levels of the green leaf volatile trans-2-hexenal increased in response to drought stress suggesting a possible role of this compound in drought stress response in pomegranate. Punica granatum (L.) is a highly valued fruit crop for its health-promoting effects and it is mainly cultivated in semi-arid areas. Thus, understanding the response mechanisms to drought stress is of great importance. In the present research, a metabolomics analysis was performed to evaluate the effects of drought stress on volatile organic compounds extracted from the leaves of pomegranate plants grown under water shortage conditions. The time course experiment (7 days of water deprivation and 24-h recovery) consisted of three treatments (control, drought stress, and rehydration of drought-stressed plants). Plant weights were recorded and control plants were irrigated daily at pot capacity to provide the lost water. Fraction of transpirable soil water has been evaluated as indicator of soil water availability in stressed plants. The levels of proline, hydrogen peroxide and lipid peroxidation as well as of the photosynthetic parameters such as photosynthesis rate (A), stomatal conductance (g s), photosynthetic efficiency of photosystem II, and photochemical quenching were monitored after the imposition of drought stress and recovery as markers of plant stress. Constitutive carbon volatile components were analyzed in the leaf of control and drought-stressed leaves using Head Space Solid Phase Micro Extraction sampling coupled with Gas Chromatography Mass Spectrometry. A total of 12 volatile compounds were found in pomegranate leaf profiles, mainly aldehydes, alcohols, and organic acids. Among them, the trans-2-hexenal showed a significant increase in water-stressed and recovered leaves respect to the well-watered ones. These data evidence a possible role of the oxylipin pathway in the response to water stress in pomegranate plants.

Factors Influencing Male Plutella xylostella (Lepidoptera: Plutellidae) Capture Rates in Sex Pheromone-Baited Traps on Canola in Western Canada.

Optimization of male moth trapping rates in sex pheromone-baited traps plays a key role in managing Plutella xylostella (L.). We investigated various ways to increase the attractiveness of pheromone-baited traps to P. xylostella in canola agroecosystems in AB, Canada. Factors tested included pheromone blend and dose, addition of a green leaf volatile to the pheromone at different times during the season, lure type, trap color, and height. The industry standard dose of 100 µg of pheromone (four-component blend) per lure (ConTech Enterprises Inc., Delta, British Columbia [BC], Canada) captured the most moths in the two lure types tested. Traps baited with pheromone released from gray rubber septa captured more males than those baited with red rubber septa. Traps baited with lures in which Z11-16: Ac is the main component attracted significantly more moths than those in which Z11-16: Ald is the main component. The addition of the green leaf volatile, (Z)-3-hexenyl acetate, to pheromone at a range of doses, did not increase moth capture at any point during the canola growing season. Unpainted white traps captured significantly more male moths than pheromone-baited traps that were painted yellow. Trap height had no significant effect on moth capture. Recommendations for monitoring P. xylostella in canola agroecosystems of western Canada include using a pheromone blend with Z11-16: Ac as the main component released from gray rubber septa at a dose of 100 µg.

Caffeoylputrescine-hexenal-mediated nonhost resistance against leafhoppers.

Despite its critical role in repelling damaging insects, our understanding of nonhost resistance against herbivores remains very limited. Recently, Bai et al. identified a novel caffeoylputrescine-green leaf volatile (GLV) compound in wild tobacco plants that confers nonhost resistance to Empoasca leafhoppers through high-throughput multi-omics analyses.

Green Leaf Volatile Confers Management of Late Blight Disease: A Green Vaccination in Potato.

Yield losses of crops due to plant pathogens are a major threat in all agricultural systems. In view of environmental issues and legislative limitations for chemical crop protection products, the need to design new environmentally friendly disease management strategies has gained interest. Despite the unique capability of green leaf volatiles (GLVs) to suppress a broad spectrum of plant pathogens, their capacity to control the potato late-blight-causing agent Phytophthora infestans has not been well studied. This study addresses the potential role of the GLV Z-3-hexenyl acetate (Z-3-HAC) in decreasing the severity of late blight and the underlying gene-based evidence leading to this effect. Nine-week-old potato plants (Solanum tuberosum L.) were exposed to Z-3-HAC before they were inoculated with P. infestans genotypes at different time points. These pre-exposed potato plants exhibited slower disease development after infection with the highly pathogenic genotype of P. infestans (EU-13-A2) over time. Qualitative assessment showed that the exposed, infected plants possessed significantly lower sporulation intensity and disease severity compared to the control plants. Hypersensitive response (HR)-like symptoms were observed on the treated leaves when inoculated with different pathogen genotypes. No HR-like lesions were detected on the untreated leaves after infection. It was shown that the transcript levels of several defense-related genes, especially those that are involved in reactive oxygen species (ROS) production pathways were significantly expressed in plants at 48 and 72 h postexposure to the Z-3-HAC. The current work provides evidence on the role of Z-3-HAC in the increased protection of potato plants against late blight through plant immunity and offers new opportunities for the sustainable control of potato diseases.

The Synthesis of Pentyl Leaf Volatiles and Their Role in Resistance to Anthracnose Leaf Blight.

Volatiles are important airborne chemical messengers that facilitate plant adaptation to a variety of environmental challenges. Lipoxygenases (LOXs) produce a bouquet of non-volatile and volatile oxylipins, including C6 green leaf volatiles (GLVs), which are involved in a litany of plant physiological processes. GLVs are emitted by a diverse array of plant species, and are the best-known group of LOX-derived volatiles. Five-carbon pentyl leaf volatiles (PLVs) represent another widely emitted group of LOX-derived volatiles that share structural similarity to GLVs, however, relatively little is known about their biosynthesis or biological activity. In this study, we utilized PLV-deficient mutants of maize and Arabidopsis and exogenous PLV applications to elucidate the biosynthetic order of individual PLVs. We further measured PLVs and GLVs after tissue disruption of leaves by two popular methods of volatile elicitation, wounding and freeze-thawing. Freeze-thawing distorted the volatile metabolism of both GLVs and PLVs relative to wounding, though this distortion differed between the two groups of volatiles. These results suggest that despite the structural similarity of these two volatile groups, they are differentially metabolized. Collectively, these results have allowed us to produce the most robust PLV pathway to date. To better elucidate the biological activity of PLVs, we show that PLVs induce maize resistance to the anthracnose pathogen, Colletotrichum graminicola, the effect opposite to that conferred by GLVs. Further analysis of PLV-treated and infected maize leaves revealed that PLV-mediated resistance is associated with early increases of oxylipin α- and γ-ketols, and later increases of oxylipin ketotrienes, hydroxytrienes, and trihydroxydienes. Ultimately, this study has produced the most up-to-date pathway for PLV synthesis, and reveals that PLVs can facilitate pathogen resistance through induction of select oxylipins.

Metabolomics Reveal Induction of ROS Production and Glycosylation Events in Wheat Upon Exposure to the Green Leaf Volatile Z-3-Hexenyl Acetate.

The activation and priming of plant defense upon perception of green leaf volatiles (GLVs) have often been reported. However, information as to which metabolic pathways in plants are affected by GLVs remains elusive. We report the production of reactive oxygen species in the tip of young wheat leaves followed by activation of antioxidant-related enzyme activity. In this study, we aimed to uncover metabolic signatures upon exposure to the GLV Z-3-hexenyl acetate (Z-3-HAC). By using an untargeted metabolomics approach, we observed changes in the phenylpropanoid pathways which yield metabolites that are involved in many anti-oxidative processes. Furthermore, exposure to GLV, followed by infection with Fusarium graminearum (Fg), induced significantly greater changes in the phenylpropanoid pathway compared to a sole Z-3-HAC treatment. Fragmentation of a selection of metabolites, which are significantly more upregulated in the Z-3-HAC + Fg treatment, showed D-glucose to be present as a substructure. This suggests that Z-3-HAC induces early glycosylation processes in plants. Additionally, we identified the presence of hexenyl diglycosides, which indicates that aerial Z-3-HAC is metabolized in the leaves by glycosyltransferases. Together these data indicate that GLV Z-3-HAC is taken up by leaves and incites oxidative stress. This subsequently results in the modulation of the phenylpropanoid pathway and an induction of glycosylation processes.

Variation Between Three Eragrostis tef Accessions in Defense Responses to Rhopalosiphum padi Aphid Infestation.

Tef (Eragrostis tef), a staple crop that originated in the Horn of Africa, has been introduced to multiple countries over the last several decades. Crop cultivation in new geographic regions raises questions regarding the molecular basis for biotic stress responses. In this study, we aimed to classify the insect abundance on tef crop in Israel, and to elucidate its chemical and physical defense mechanisms in response to insect feeding. To discover the main pests of tef in the Mediterranean climate, we conducted an insect field survey on three selected accessions named RTC-144, RTC-405, and RTC-406, and discovered that the most abundant insect order is Hemiptera. We compared the differences in Rhopalosiphum padi (Hemiptera; Aphididae) aphid performance, preference, and feeding behavior between the three accessions. While the number of aphid progeny was lower on RTC-406 than on the other two, the aphid olfactory assay indicated that the aphids tended to be repelled from the RTC-144 accession. To highlight the variation in defense responses, we investigated the physical and chemical mechanisms. As a physical barrier, the density of non-granular trichomes was evaluated, in which a higher number of trichomes on the RTC-406 than on the other accessions was observed. This was negatively correlated with aphid performance. To determine chemical responses, the volatile and central metabolite profiles were measured upon aphid attack for 4 days. The volatile analysis exposed a rich and dynamic metabolic profile, and the central metabolism profile indicated that tef plants adjust their sugars and organic and amino acid levels. Overall, we found that the tef plants possess similar defense responses as other Poaceae family species, while the non-volatile deterrent compounds are yet to be characterized. A transcriptomic time-series analysis of a selected accession RTC-144 infested with aphids revealed a massive alteration of genes related to specialized metabolism that potentially synthesize non-volatile toxic compounds. This is the first report to reveal the variation in the defense mechanisms of tef plants. These findings can facilitate the discovery of insect-resistance genes leading to enhanced yield in tef and other cereal crops.

Green leaf volatiles and jasmonic acid enhance susceptibility to anthracnose diseases caused by Colletotrichum graminicola in maize.

Colletotrichum graminicola is a hemibiotrophic fungus that causes anthracnose leaf blight (ALB) and anthracnose stalk rot (ASR) in maize. Despite substantial economic losses caused by these diseases, the defence mechanisms against this pathogen remain poorly understood. Several hormones are suggested to aid in defence against C. graminicola, such as jasmonic acid (JA) and salicylic acid (SA), but supporting genetic evidence was not reported. Green leaf volatiles (GLVs) are a group of well-characterized volatiles that induce JA biosynthesis in maize and are known to function in defence against necrotrophic pathogens. Information regarding the role of GLVs and JA in interactions with (hemi)biotrophic pathogens remains limited. To functionally elucidate GLVs and JA in defence against a hemibiotrophic pathogen, we tested GLV- and JA-deficient mutants, lox10 and opr7 opr8, respectively, for resistance to ASR and ALB and profiled jasmonates and SA in their stalks and leaves throughout infection. Both mutants were resistant and generally displayed elevated levels of SA and low amounts of jasmonates, especially at early stages of infection. Pretreatment with GLVs restored susceptibility of lox10 mutants, but not opr7 opr8 mutants, which coincided with complete rescue of JA levels. Exogenous methyl jasmonate restored susceptibility in both mutants when applied before inoculation, whereas methyl salicylate did not induce further resistance in either of the mutants, but did induce mutant-like resistance in the wild type. Collectively, this study reveals that GLVs and JA contribute to maize susceptibility to C. graminicola due to suppression of SA-related defences.

Herbivorous Caterpillars Can Utilize Three Mechanisms to Alter Green Leaf Volatile Emission.

Green plants emit green leaf volatiles (GLVs) as a general damage response. These compounds act as signals for the emitter plant, neighboring plants, and even for insects in the ecosystem. However, when oral secretions from certain caterpillars are applied to wounded leaves, GLV emissions are significantly decreased or modified. We examined four caterpillar species representing two lepidopteran families for their capacity to decrease GLV emissions from Zea mays leaf tissue. We also investigated the source of the GLV modifying components in the alimentary tract of the various caterpillars. In Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae), Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), and Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae), we found three distinct mechanisms to modify GLV emission: a heat-stable compound in the gut, a heat-labile enzyme in salivary gland homogenate (previously described in Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae), and an isomerase in the salivary gland homogenate, which catalyzes the conversion of (Z)-3-hexenal to (E)-2-hexenal (previously described in M. sexta). These mechanisms employed by caterpillars to suppress or modify GLV emission suggest a counteraction against the induced indirect volatile defenses of a plant and provides further insights into the ecological functions of GLVs.

Use of density sorting for the selection of aromatic grape berries with different volatile profile.

The aim of the study was to investigate the application of berry density sorting as a tool for the selection of grapes with different volatile and precursor profiles. The study was carried out on Moscato giallo, Malvasia di Schierano, Malvasia nera lunga, and Brachetto aromatic grape varieties. Free and glycosidically-bound terpene compounds including linalool, geraniol, nerol, citronellol, and terpineol, as well as lipoxygenases activity-derived compounds, were evaluated using head space-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) in density sorted berries (1075-1119 kg m-3). Total free terpenes changed with the berry density, while no significant changes were found in total glycosylated compounds, except for Malvasia nera lunga grapes where nerol, linalool, and geraniol contributed strongly to the increase of total contents with increasing berry density. Given that these variations were strongly variety-dependent, the possible use of density sorting equipment in winery for this aim may be less effective.

Transcriptome analysis of Arabidopsis thaliana treated with green leaf volatiles: possible role of green leaf volatiles as self-made damage-associated molecular patterns.

Green leaf volatiles (GLVs), which include C6 aldehydes, alcohols, and their esters, are emitted by damaged plants and are, therefore, thought to be involved in stress responses. However, the effects of GLVs on gene expression are not fully understood. Thus, the aim of the present study was to analyze the early transcriptional responses of Arabidopsis to the major GLVs-(Z)-3-hexenal, (Z)-3-hexenol, (E)-2-hexenal, and (Z)-3-hexenyl acetate-using comprehensive microarray gene expression analysis. All of the GLVs induced changes in gene expression, and (Z)-3-hexenal, (Z)-3-hexenol, and (Z)-3-hexenyl acetate commonly triggered the expression of defense-related genes, whereas (E)-2-hexenal mainly induced genes responsible for responding to abiotic stress, such as heat and oxidative stress. These results suggest that GLVs can function as airborne infochemicals that regulate the rapid expression of defense response-related genes and that GLVs might play a physiological role as self-made damage-associated molecular patterns (DAMPs) in damaged leaves.

An oriental melon 9-lipoxygenase gene CmLOX09 response to stresses, hormones, and signal substances.

In plants, lipoxygenases (LOXs) play a crucial role in biotic and abiotic stresses. In our previous study, five 13-LOX genes of oriental melon were regulated by abiotic stress but it is unclear whether the 9-LOX is involved in biotic and abiotic stresses. The promoter analysis revealed that CmLOX09 (type of 9-LOX) has hormone elements, signal substances, and stress elements. We analyzed the expression of CmLOX09 and its downstream genes-CmHPL and CmAOS-in the leaves of four-leaf stage seedlings of the oriental melon cultivar "Yumeiren" under wound, hormone, and signal substances. CmLOX09, CmHPL, and CmAOS were all induced by wounding. CmLOX09 was induced by auxin (indole acetic acid, IAA) and gibberellins (GA3); however, CmHPL and CmAOS showed differential responses to IAA and GA3. CmLOX09, CmHPL, and CmAOS were all induced by hydrogen peroxide (H2O2) and methyl jasmonate (MeJA), while being inhibited by abscisic acid (ABA) and salicylic acid (SA). CmLOX09, CmHPL, and CmAOS were all induced by the powdery mildew pathogen Podosphaera xanthii. The content of 2-hexynol and 2-hexenal in leaves after MeJA treatment was significantly higher than that in the control. After infection with P. xanthii, the diseased leaves of the oriental melon were divided into four levels-levels 1, 2, 3, and 4. The content of jasmonic acid (JA) in the leaves of levels 1 and 3 was significantly higher than that in the level 0 leaves. In summary, the results suggested that CmLOX09 might play a positive role in the response to MeJA through the hydroperoxide lyase (HPL) pathway to produce C6 alcohols and aldehydes, and in the response to P. xanthii through the allene oxide synthase (AOS) pathway to form JA.

CYP74B24 is the 13-hydroperoxide lyase involved in biosynthesis of green leaf volatiles in tea (Camellia sinensis).

Green leaf volatiles (GLVs) are C6-aliphatic aldehydes/alcohols/acetates, and biosynthesized from the central precursor fatty acid 13-hydroperoxides by 13-hydroperoxide lyases (HPLs) in various plant species. While GLVs have been implicated as defense compounds in plants, GLVs give characteristic grassy note to a bouquet of aroma in green tea, which is manufactured from young leaves of Camellia sinensis. Here we identify three HPL-related genes from C. sinensis via RNA-Sequencing (RNA-Seq) in silico, and functionally characterized a candidate gene, CYP74B24, as a gene encoding tea HPL. Recombinant CYP74B24 protein heterologously expressed in Escherichia coli specifically produced (Z)-3-hexenal from 13-HPOT with the optimal pH 6.0 in vitro. CYP74B24 gene was expressed throughout the aerial organs in a rather constitutive manner and further induced by mechanical wounding. Constitutive expression of CYP74B24 gene in intact tea leaves might account for low but substantial and constitutive formation of a subset of GLVs, some of which are stored as glycosides. Our results not only provide novel insights into the biological roles that GLVs play in tea plants, but also serve as basis for the improvement of aroma quality in tea manufacturing processes.

Conversion of volatile alcohols into their glucosides in Arabidopsis.

Exposure of tomato plants to volatile chemicals emitted from common cutworm (Spodoptera litura)-infested conspecifics led to accumulation of the glycoside, (Z)-3-hexenyl vicianoside. Accumulation of (Z)-3-hexenyl vicianoside in the exposed plants has adverse impacts on the performance of the common cutworms. The aglycon of (Z)-3-hexenyl vicianoside is derived from airborne (Z)-3-hexenol emitted from infested plants. The ability to incorporate and convert (Z)-3-hexenol to its corresponding glycoside is widely conserved in an array of plant species. However, the specificity of this ability to discriminate between the chemical structures of different volatile alcohols remains unknown. In this study, we investigated glycosylation of several volatile alcohols in Arabidopsis (Arabidopsis thaliana). The exposure of Arabidopsis to a variety of volatile alcohols, (Z)-2-pentenol, (Z)-3-hexenol, (Z)-3-heptenol, (Z)-3-octenol, (Z)-3-nonenol, cyclohexanol, benzyl alcohol, verbenol, perillyl alcohol, myrtenol, geraniol, or linalool　led to the detection of the putative corresponding glucosides. These results suggest that Arabidopsis might convert a broad range of volatile alcohols into the corresponding glucosides.

An oriental melon 9-lipoxygenase gene CmLOX09 response to stresses, hormones, and signal substances / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

In plants, lipoxygenases (LOXs) play a crucial role in biotic and abiotic stresses. In our previous study, five 13-LOX genes of oriental melon were regulated by abiotic stress but it is unclear whether the 9-LOX is involved in biotic and abiotic stresses. The promoter analysis revealed that CmLOX09 (type of 9-LOX) has hormone elements, signal substances, and stress elements. We analyzed the expression of CmLOX09 and its downstream genes-CmHPL and CmAOS-in the leaves of four-leaf stage seedlings of the oriental melon cultivar "Yumeiren" under wound, hormone, and signal substances. CmLOX09, CmHPL, and CmAOS were all induced by wounding. CmLOX09 was induced by auxin (indole acetic acid, IAA) and gibberellins (GA3); however, CmHPL and CmAOS showed differential responses to IAA and GA3. CmLOX09, CmHPL, and CmAOS were all induced by hydrogen peroxide (H2O2) and methyl jasmonate (MeJA), while being inhibited by abscisic acid (ABA) and salicylic acid (SA). CmLOX09, CmHPL, and CmAOS were all induced by the powdery mildew pathogen Podosphaera xanthii. The content of 2-hexynol and 2-hexenal in leaves after MeJA treatment was significantly higher than that in the control. After infection with P. xanthii, the diseased leaves of the oriental melon were divided into four levels-levels 1, 2, 3, and 4. The content of jasmonic acid (JA) in the leaves of levels 1 and 3 was significantly higher than that in the level 0 leaves. In summary, the results suggested that CmLOX09 might play a positive role in the response to MeJA through the hydroperoxide lyase (HPL) pathway to produce C6 alcohols and aldehydes, and in the response to P. xanthii through the allene oxide synthase (AOS) pathway to form JA.

Herbivory-induced volatiles function as defenses increasing fitness of the native plant Nicotiana attenuata in nature.

From an herbivore's first bite, plants release herbivory-induced plant volatiles (HIPVs) which can attract enemies of herbivores. However, other animals and competing plants can intercept HIPVs for their own use, and it remains unclear whether HIPVs serve as an indirect defense by increasing fitness for the emitting plant. In a 2-year field study, HIPV-emitting N. attenuata plants produced twice as many buds and flowers as HIPV-silenced plants, but only when native Geocoris spp. predators reduced herbivore loads (by 50%) on HIPV-emitters. In concert with HIPVs, plants also employ antidigestive trypsin protease inhibitors (TPIs), but TPI-producing plants were not fitter than TPI-silenced plants. TPIs weakened a specialist herbivore's behavioral evasive responses to simulated Geocoris spp. attack, indicating that TPIs function against specialists by enhancing indirect defense.DOI:http://dx.doi.org/10.7554/eLife.00007.001.

Indirect routes to reproductive success.

By comparing wild-type and transgenic tobacco plants in a natural ecosystem, researchers have confirmed that the indirect defence mechanisms employed by plants to fend off herbivorous insects can increase Darwinian fitness.