Green Leaf Volatiles-The Forefront of Plant Responses Against Biotic Attack.

Green leaf volatiles (GLVs) are six-carbon volatile oxylipins ubiquitous in vascular plants. GLVs are produced from acyl groups in the biological membranes via oxygenation by a pathway-specific lipoxygenase (LOX) and a subsequent cleavage reaction by hydroperoxide lyase. Because of the universal distribution and ability to form GLVs, they have been anticipated to play a common role in vascular plants. While resting levels in intact plant tissues are low, GLVs are immediately synthesized de novo in response to stresses, such as insect herbivory, that disrupt the cell structure. This rapid GLV burst is one of the fastest responses of plants to cell-damaging stresses; therefore, GLVs are the first plant-derived compounds encountered by organisms that interact with plants irrespective of whether the interaction is competitive or friendly. GLVs should therefore be considered important mediators between plants and organisms that interact with them. GLVs can have direct effects by deterring herbivores and pathogens as well as indirect effects by attracting predators of herbivores, while other plants can recruit them to prepare their defenses in a process called priming. While the beneficial effects provided to plants by GLVs are often less dramatic and even complementary, the buildup of these tiny effects due to the multiple functions of GLVs can amass to levels that become substantially beneficial to plants. This review summarizes the current understanding of the spatiotemporal resolution of GLV biosynthesis and GLV functions and outlines how GLVs support the basic health of plants.

Herbivorous Caterpillars and the Green Leaf Volatile (GLV) Quandary.

Several herbivorous caterpillars contain effectors in their oral secretions that alter the emission of green leaf volatiles (GLVs) produced by the plants upon which the caterpillars are feeding. These effectors include an isomerase, a fatty acid dehydratase (FHD), and a heat-stable hexenal trapping (HALT) molecule. GLVs serve as signaling compounds in plant-insect interactions and inter-and intra-plant communication. However, it is not known whether these GLV-altering effectors are common among herbivorous caterpillars, or the evolutionary context of these effectors in relation to GLV emission by host plants in response to feeding damage. Here, we examined the distribution and activity of the isomerase, FHD, and HALT effectors across 10 species spanning 7 lepidopteran families. Six of the 10 species possessed all three effectors in their oral secretions. Activity from the HALT and FHD effectors was observed in all examined caterpillar species, while activity from the isomerase effector varied in some species and was absent in others. There was no discernable pattern in effector activity based on evolutionary divergence, since individual species within a family did not possess similar mechanisms to alter GLV emission. These data, demonstrating the GLV-altering effectors acting at different steps in the GLV biosynthetic pathway and present in the examined caterpillar species at different combinations with different activities, highlight the importance of these effectors in changing the emission of these compounds during caterpillar herbivory. Understanding the prevalence and roles of GLV-altering effectors and GLV emission itself will open new research areas in the dynamics of plant-insect interactions.

Green leaf volatiles protect maize (Zea mays) seedlings against damage from cold stress.

Although considerable evidence has accumulated on the defensive activity of plant volatile organic compounds against pathogens and insect herbivores, less is known about the significance of volatile organic compounds emitted by plants under abiotic stress. Here, we report that green leaf volatiles (GLVs), which were previously shown to prime plant defences against insect herbivore attack, also protect plants against cold stress (4 °C). We show that the expression levels of several cold stress-related genes are significantly up-regulated in maize (Zea mays) seedlings treated with physiological concentrations of the GLV, (Z)-3-hexen-1-yl acetate (Z-3-HAC), and that seedlings primed with Z-3-HAC exhibit increased growth and reduced damage after cold stress relative to unprimed seedlings. Together, these data demonstrate the protective and priming effect of GLVs against cold stress and suggest an activity of GLVs beyond the activation of typical plant defence responses against herbivores and pathogens.

Disruption of OPR7 and OPR8 reveals the versatile functions of jasmonic acid in maize development and defense.

Here, multiple functions of jasmonic acid (JA) in maize (Zea mays) are revealed by comprehensive analyses of JA-deficient mutants of the two oxo-phytodienoate reductase genes, OPR7 and OPR8. Single mutants produce wild-type levels of JA in most tissues, but the double mutant opr7 opr8 has dramatically reduced JA in all organs tested. opr7 opr8 displayed strong developmental defects, including formation of a feminized tassel, initiation of female reproductive buds at each node, and extreme elongation of ear shanks; these defects were rescued by exogenous JA. These data provide evidence that JA is required for male sex determination and suppression of female reproductive organ biogenesis. Moreover, opr7 opr8 exhibited delayed leaf senescence accompanied by reduced ethylene and abscisic acid levels and lack of anthocyanin pigmentation of brace roots. Remarkably, opr7 opr8 is nonviable in nonsterile soil and under field conditions due to extreme susceptibility to a root-rotting oomycete (Pythium spp), demonstrating that these genes are necessary for maize survival in nature. Supporting the importance of JA in insect defense, opr7 opr8 is susceptible to beet armyworm. Overall, this study provides strong genetic evidence for the global roles of JA in maize development and immunity to pathogens and insects.

The maize lipoxygenase, ZmLOX10, mediates green leaf volatile, jasmonate and herbivore-induced plant volatile production for defense against insect attack.

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, major regulatory genes and the signals that modulate these defense metabolites are vastly understudied, especially in important agro-economic monocot species. Here we show that products and signals derived from a single Zea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbivory. We provide genetic evidence that two 13-LOXs, ZmLOX10 and ZmLOX8, specialize in providing substrate for the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the specialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indicating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression of JA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10-derived signaling is required for LOX8-mediated JA. The possible role of GLVs in JA signaling is supported by their ability to partially restore wound-induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produce GLVs and JA led to dramatic reductions in herbivore-induced plant volatiles (HIPVs) and attractiveness to parasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic link to the diurnal regulation of GLVs and HIPVs. GLV-, JA- and HIPV-deficient lox10 mutants display compromised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence that LOX10-dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene to agro-ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.

Phytohormone-based activity mapping of insect herbivore-produced elicitors.

In response to insect attack, many plants exhibit dynamic biochemical changes, resulting in the induced production of direct and indirect defenses. Elicitors present in herbivore oral secretions are believed to positively regulate many inducible plant defenses; however, little is known about the specificity of elicitor recognition in plants. To investigate the phylogenic distribution of elicitor activity, we tested representatives from three different elicitor classes on the time course of defense-related phytohormone production, including ethylene (E), jasmonic acid (JA), and salicylic acid, in a range of plant species spanning angiosperm diversity. All families examined responded to at least one elicitor class with significant increases in E and JA production within 1 to 2 h after treatment, yet elicitation activity among species was highly idiosyncratic. The fatty-acid amino acid conjugate volicitin exhibited the widest range of phytohormone and volatile inducing activity, which spanned maize (Zea mays), soybean (Glycine max), and eggplant (Solanum melongena). In contrast, the activity of inceptin-related peptides, originally described in cowpea (Vigna unguiculata), was limited even within the Fabaceae. Similarly, caeliferin A16:0, a disulfooxy fatty acid from grasshoppers, was the only elicitor with demonstrable activity in Arabidopsis thaliana. Although precise mechanisms remain unknown, the unpredictable nature of elicitor activity between plant species supports the existence of specific receptor-ligand interactions mediating recognition. Despite the lack of an ideal plant model for studying the action of numerous elicitors, E and JA exist as highly conserved and readily quantifiable markers for future discoveries in this field.

Developmental Stages Affect the Capacity to Produce Aldehyde Green Leaf Volatiles in Zea mays and Vigna radiata.

Green leaf volatiles (GLV) are essentially produced by the green parts of plants upon damage. GLV are mainly 6-carbon molecules derived from fatty acids through the hydroperoxide lyase pathway and can serve as airborne signals to other parts of the same plant and to neighboring plants and help to protect them against biotic and abiotic stresses. However, while the biosynthesis is generally well understood, little is known about how plants regulate the production of these important signaling molecules. To better understand how the developmental stage of the plant affects aldehyde GLV production, we selected Zea mays and Vigna radiata to represent mono- and dicot plants for this analysis. We show that the capacity to produce aldehyde GLV strongly depends on the developmental stage of the plant. Major differences in the quantity, and in the quality of these compounds were found, not only in leaves from different developmental stages, but also in different areas within a leaf. The results demonstrate that the capacity to produce GLV varies significantly within a plant and the potential implications of these findings are discussed.

Low concentrations of salicylic acid stimulate insect elicitor responses in Zea mays seedlings.

Salicylic acid (SA) generally is thought to suppress jasmonic acid (JA) related signaling events. However, when we treated the roots of corn seedlings overnight with low physiological concentrations of SA (50 µM), we found a priming effect of this pretreatment on typical insect elicitor (IE)-induced responses in the leaves of these plants. IE-induced JA was more than 2-fold up regulated in SA-pretreated plants. Consequently, IE-induced volatile organic compounds (VOC) release also was significantly increased. In contrast, when corn seedlings were treated with SA overnight and then mechanically damaged, we found no significant differences in JA accumulation. We also found that the application of even lower concentrations of SA (5 µM) had no significant effect on IE-induced responses, while higher concentrations (500 µM) inhibited IE-induced JA accumulation. Likewise, shorter exposure to SA did not affect subsequent JA accumulation induced by IE or mechanical wounding. These results provide evidence for the existence of non-compatible defense priming by signaling molecules that usually are involved in a conflictive defense signaling pathway and suggests common elements in the regulation of priming plant defense responses.

Plants on constant alert: elevated levels of jasmonic acid and jasmonate-induced transcripts in caterpillar-resistant maize.

This study was conducted to determine if constitutive levels of jasmonic acid (JA) and other octadecanoid compounds were elevated prior to herbivory in a maize genotype with documented resistance to fall armyworm (Spodoptera frugiperda) and other lepidopteran pests. The resistant inbred Mp708 had approximately 3-fold higher levels of jasmonic acid (JA) prior to herbivore feeding than the susceptible inbred Tx601. Constitutive levels of cis-12-oxo-phytodienoic acid (OPDA) also were higher in Mp708 than Tx601. In addition, the constitutive expression of JA-inducible genes, including those in the JA biosynthetic pathway, was higher in Mp708 than Tx601. In response to herbivory, Mp708 generated comparatively higher levels of hydrogen peroxide, and had a greater abundance of NADPH oxidase transcripts before and after caterpillar feeding. Before herbivore feeding, low levels of transcripts encoding the maize insect resistance cysteine protease (Mir1-CP) and the Mir1-CP protein were detected consistently. Thus, Mp708 appears to have a portion of its defense pathway primed, which results in constitutive defenses and the ability to mount a stronger defense when caterpillars attack. Although the molecular mechanisms that regulate the constitutive accumulation of JA in Mp708 are unknown, it might account for its enhanced resistance to lepidopteran pests. This genotype could be valuable in studying the signaling pathways that maize uses to response to insect herbivores.

Primed to grow: a new role for green leaf volatiles in plant stress responses.

Green leaf volatiles (GLV) have been well described to prime plants against biotic and abiotic stresses resulting in an accelerated and/or enhanced protective response. Since investments in priming are considered to be minor, it has been assumed that costs for plants using this mechanism are negligible. By analyzing the costs of defense priming by GLV, we found that while initially growth rates of plants were reduced within the first hours after treatment, significantly increased growth rates were found at later time points. This primed growth response in maize seedlings differs from primed defense responses in that it also affects systemic parts of the plant and suggests a metabolic component to be involved in the regulation of this process.

Variability in the Capacity to Produce Damage-Induced Aldehyde Green Leaf Volatiles among Different Plant Species Provides Novel Insights into Biosynthetic Diversity.

Green leaf volatiles (GLVs) are commonly released by plants upon damage, thereby providing volatile signals for other plants to prepare against the major causes of damage, herbivory, pathogen infection, and cold stress. However, while the biosynthesis of these compounds is generally well understood, little is known about the qualities and quantities that are released by different plant species, nor is it known if release patterns can be associated with different clades of plants. Here, we provide a first study describing the damage-induced release of major GLVs by more than 50 plant species. We found major differences in the quantity and quality of those compounds between different plant species ranging from undetectable levels to almost 100 µg per gram fresh weight. We also found major shifts in the composition that correlate directly to the quantity of emitted GLV. However, we did not find any major patterns that would associate specific GLV release with distinct clades of plants.

The Costs of Green Leaf Volatile-Induced Defense Priming: Temporal Diversity in Growth Responses to Mechanical Wounding and Insect Herbivory.

Green leaf volatiles (GLVs) have long been associated with plant defense responses against insect herbivory. Although some of their biological activities appear to directly affect the attacking herbivore, one of the major functions of GLVs seems to be the priming of these defense responses. This priming is generally considered to impose low costs on the plant should no direct attack happen. Here, we demonstrate that priming of maize seedlings with GLVs is costly for the plants as it results in significantly reduced growth. We further demonstrate that priming very selectively affects growth responses after insect elicitor treatment and mechanical wounding depending on the age and/or the developmental stage of the treated plant. The differential growth response of maize seedlings to treatment with GLVs and subsequent herbivory-related damage sheds new light on the biological activity of these important plant volatile compounds and indicates consequences that go beyond defense.

In-Cold Exposure to Z-3-Hexenal Provides Protection Against Ongoing Cold Stress in Zea mays.

Green leaf volatiles (GLVs), which have mostly been described as providers of protection against insect herbivory and necrotrophic pathogen infections, were recently shown to provide significant fortification against cold stress damage. It was further demonstrated that cold-damaged maize seedlings released a significant amount of GLVs, in particular, Z-3-hexenal (Z-3-HAL). Here, we report that the in-cold treatment of maize seedlings with Z-3-HAL significantly improved cold stress resistance. The transcripts for cold-protective genes were also significantly increased in the Z-3-HAL treated maize seedlings over those found in only cold stressed plants. Consequently, the maize seedlings treated with HAL during cold showed a significantly increased structural integrity, significantly less damage, and increased growth after cold stress, relative to the non-HAL treated maize seedlings. Together, these results demonstrate the protective effect of in-cold treatment with HAL against cold damage, and suggest that the perception of these compounds during cold episodes significantly improves resistance against this abiotic stress.

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.

Maize 9-lipoxygenase ZmLOX3 controls development, root-specific expression of defense genes, and resistance to root-knot nematodes.

Root-knot nematodes (RKN) are severe pests of maize. Although lipoxygenase (LOX) pathways and their oxylipin products have been implicated in plant-nematode interactions, prior to this report there was no conclusive genetic evidence for the function of any plant LOX gene in such interactions. We showed that expression of a maize 9-LOX gene, ZmLOX3, increased steadily and peaked at 7 days after inoculation with Meloidogyne incognita RKN. Mu-insertional lox3-4 mutants displayed increased attractiveness to RKN and an increased number of juveniles and eggs. A set of jasmonic acid (JA)- and ethylene (ET)-responsive and biosynthetic genes as well as salicylic acid (SA)-dependent genes were overexpressed specifically in the roots of lox3-4 mutants. Consistent with this, levels of JA, SA, and ET were elevated in lox3-4 mutant roots, but not in leaves. Unlike wild types, in lox3-4 mutant roots, a phenylalanine ammonia lyase (PAL) gene was not RKN-inducible, suggesting a role for PAL-mediated metabolism in nematode resistance. In addition to these alterations in the defense status of roots, lox3-4 knockout mutants displayed precocious senescence and reduced root length and plant height compared with the wild type, suggesting that ZmLOX3 is required for normal plant development. Taken together, our data indicate that the ZmLOX3-mediated pathway may act as a root-specific suppressor of all three major defense signaling pathways to channel plant energy into growth processes, but is required for normal levels of resistance against nematodes.

Insect elicitors and exposure to green leafy volatiles differentially upregulate major octadecanoids and transcripts of 12-oxo phytodienoic acid reductases in Zea mays.

The induction of jasmonic acid (JA) is one of the major signaling events in plants in response to insect herbivore damage and leads to the activation of direct and indirect defensive measures. Green leafy volatiles, which constitute a major portion of volatile organic compounds, often are released in response to insect herbivore attack and have been shown to significantly activate JA production in exposed corn (Zea mays) seedlings, thereby priming these plants specifically against subsequent herbivore attack. To explore the factors determining the specificity of the octadecanoid signaling pathway in corn, we analyzed qualitative and quantitative changes in major octadecanoids. The time course and the amount of induced JA and 12-oxophytodienoic acid levels in corn seedlings were strikingly different after wounding, application of caterpillar regurgitant, or treatment with cis-3-hexenyl acetate (Z-3-6:AC). Exposure to Z-3-6:AC induced accumulation of transcripts encoded by three putative 12-oxophytodienoate10,11-reductase genes (ZmOPR1/2, ZmOPR5, and ZmOPR8). Although changes in ZmOPR5 RNAs were detected only after exposure to Z-3-6:AC, ZmOPR1/2 RNAs and ZmOPR8 RNAs also were abundant after treatment with crude regurgitant elicitor or mechanical damage. The physiological implications of these findings in the context of plant-insect interactions are discussed.

Defense Priming and Jasmonates: A Role for Free Fatty Acids in Insect Elicitor-Induced Long Distance Signaling.

Green leaf volatiles (GLV) prime plants against insect herbivore attack resulting in stronger and faster signaling by jasmonic acid (JA). In maize this response is specifically linked to insect elicitor (IE)-induced signaling processes, which cause JA accumulation not only around the damage site, but also in distant tissues, presumably through the activation of electrical signals. Here, we present additional data further characterizing these distal signaling events in maize. Also, we describe how exposure to GLV increases free fatty acid (fFA) levels in maize seedlings, but also in other plants, and how increased fFA levels affect IE-induced JA accumulation. Increased fFA, in particular α-linolenic acid (LnA), caused a significant increase in JA accumulation after IE treatment, while JA induced by mechanical wounding (MW) alone was not affected. We also identified treatments that significantly decreased certain fFA level including simulated wind and rain. In such treated plants, IE-induced JA accumulation was significantly reduced when compared to un-moved control plants, while MW-induced JA accumulation was not significantly affected. Since only IE-induced JA accumulation was altered by changes in the fFA composition, we conclude that changing levels of fFA affect primarily IE-induced signaling processes rather than serving as a substrate for JA.

Defense priming by non-jasmonate producing fatty acids in maize (Zea mays).

Previously, we described a priming effect of α-linolenic acid (LnA) on anti-herbivore defense response in maize seedlings. 1 We showed that exogenous application of LnA stimulated higher jasmonic acid (JA) accumulation and herbivore-induced plant volatile (HIPV) emission after treatment with insect elicitor (IE). To further investigate the specificity of LnA's priming effect, we incubated maize seedlings in palmitoleic acid (PeicA), γ-linolenic acid (γ LnA) and stearic acid (StA) solutions, and analyzed HIPV emission in response to IE. Seedlings incubated in PeicA and γ LnA had 3 and 1.8 times higher HIPV release when compared to controls. In contrast, treatment with StA did not up-regulate HIPV release. We propose that the elevated level and/or the presence of unsaturated fatty acids sensitize the defense signaling system, which in turn augments the defense response of maize when under insect herbivore attack.

Mechanosensing and signal transduction in tendrils.

The perception of thigmic stimuli is a widespread phenomenon among plants with decisive meaning for the ability to survive. Beside a general sensitivity for mechanical stimuli many plants have evolved specialized organs with highly developed mechanisms to perceive and transduce the applied forces. Tendrils of Bryonia dioica and Pisum sativum have been chosen to study the effects of mechanical stimulation on plant physiology. Both types of tendrils, although exhibiting different morphology, respond to such a stimulus with a rapid coiling response to the dorsal side of the organ within minutes. The actual perception of the stimulus is most likely coupled to the cytoskeleton serving as the mediator between the physical stimulus and the biochemical response. Drugs affecting the status of the cytoskeleton were used to get more insights into this specific process. The results indicate that microtubuli (MT) play the most important role in the perception of thigmic stimuli in tendrils. Colchicine-mediated disruption of MT lead to total inhibition of the response to the thigmic stimulus in tendrils of Pisum and to a reduced response in Bryonia. Alamethicin, an ionophore that can mimic action potentials in membranes, was able to bypass this inhibition suggesting a direct involvement of MT in depolarization of the membranes. Auxin, however, which is also supposed to be involved in the regulation of the coiling response, failed to bypass colchicine-dependent inhibition. Vinblastine, another microtubule depolimerizing agent, did induce tendril coiling in Pisum without further stimulation. Application of taxol and other MT-stabilizing drugs as well as disruption of the actin network did not affect the coiling response of tendrils. In Pisum indole-3-acetic acid (IAA) is induced after mechanical stimulation during the coiling response, but not jasmonic acid. A further consequence of mechanical stimulation is the induction of an oxidative burst and an increase in soluble sugar. A model is presented integrating these results and might serve as a common basis for the understanding of the perception of mechanical stimuli.

Early transcriptome analyses of Z-3-Hexenol-treated zea mays revealed distinct transcriptional networks and anti-herbivore defense potential of green leaf volatiles.

Green leaf volatiles (GLV), which are rapidly emitted by plants in response to insect herbivore damage, are now established as volatile defense signals. Receiving plants utilize these molecules to prime their defenses and respond faster and stronger when actually attacked. To further characterize the biological activity of these compounds we performed a microarray analysis of global gene expression. The focus of this project was to identify early transcriptional events elicited by Z-3-hexenol (Z-3-HOL) as our model GLV in maize (Zea mays) seedlings. The microarray results confirmed previous studies on Z-3-HOL -induced gene expression but also provided novel information about the complexity of Z-3-HOL -induced transcriptional networks. Besides identifying a distinct set of genes involved in direct and indirect defenses we also found significant expression of genes involved in transcriptional regulation, Ca(2+)-and lipid-related signaling, and cell wall reinforcement. By comparing these results with those obtained by treatment of maize seedlings with insect elicitors we found a high degree of correlation between the two expression profiles at this early time point, in particular for those genes related to defense. We further analyzed defense gene expression induced by other volatile defense signals and found Z-3-HOL to be significantly more active than methyl jasmonate, methyl salicylate, and ethylene. The data presented herein provides important information on early genetic networks that are activated by Z-3-HOL and demonstrates the effectiveness of this compound in the regulation of typical plant defenses against insect herbivores in maize.