Metabolomics reveals herbivore‐induced metabolites of resistance and susceptibility in maize leaves and roots

Plants respond to herbivory by reprogramming their metabolism. Most research in this context has focused on locally induced compounds that function as toxins or feeding deterrents. We developed an ultra‐high‐pressure liquid chromatography time‐of‐flight mass spectrometry (UHPLC‐TOF‐MS)‐based metabolomics approach to evaluate local and systemic herbivore‐induced changes in maize leaves, sap, roots and root exudates without any prior assumptions about their function. Thirty‐two differentially regulated compounds were identified from Spodoptera littoralis‐infested maize seedlings and isolated for structure assignment by microflow nuclear magnetic resonance (CapNMR). Nine compounds were quantified by a high throughput direct nano‐infusion tandem mass spectrometry/mass spectrometry (MS/MS) method. Leaf infestation led to a marked local increase of 1,3‐benzoxazin‐4‐ones, phospholipids, N‐hydroxycinnamoyltyramines, azealic acid and tryptophan. Only few changes were found in the root metabolome, but 1,3‐benzoxazin‐4‐ones increased in the vascular sap and root exudates. The role of N‐hydroxycinnamoyltyramines in plant–herbivore interactions is unknown, and we therefore tested the effect of the dominating p‐coumaroyltyramine on S. littoralis. Unexpectedly, p‐coumaroyltyramine was metabolized by the larvae and increased larval growth, possibly by providing additional nitrogen to the insect. Taken together, this study illustrates that herbivore attack leads to the induction of metabolites that can have contrasting effects on herbivore resistance in the leaves and roots.     

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.     

Fighting on two fronts: Elevated insect resistance in flooded maize

To grow and thrive plants must be able to adapt to both adverse environmental conditions and attack by a variety of pests. Elucidating the sophisticated mechanisms plants have developed to achieve this has been the focus of many studies. What is less well understood is how plants respond when faced with multiple stressors simultaneously. In this study, we assess the response of Zea mays (maize) to the combinatorial stress of flooding and infestation with the insect pest Spodoptera frugiperda (fall armyworm). This combined stress leads to elevated production of the defence hormone salicylic acid, which does not occur in the individual stresses, and the resultant salicylic acid-dependent increase in S. frugiperda resistance. Remodelling of phenylpropanoid pathways also occurs in response to this combinatorial stress leading to increased production of the anti-insect C-glycosyl flavones (maysins) and the herbivore-induced volatile phenolics, benzyl acetate, and phenethyl acetate. Furthermore, changes in cellular redox status also occur, as indicated by reductions in peroxidase and polyphenol oxidase activity. These data suggest that metabolite changes important for flooding tolerance and anti-insect defence may act both additively and synergistically to provide extra protection to the plant.     

Herbivore‐induced plant responses in Brassica oleracea prevail over effects of constitutive resistance and result in enhanced herbivore attack

1. Plant responses to herbivore attack may have community‐wide effects on the composition of the plant‐associated insect community. Thereby, plant responses to an early‐season herbivore may have profound consequences for the amount and type of future attack. 2. Here we studied the effect of early‐season herbivory by caterpillars of Pieris rapae on the composition of the insect herbivore community on domesticated Brassica oleracea plants. We compared the effect of herbivory on two cultivars that differ in the degree of susceptibility to herbivores to analyse whether induced plant responses supersede differences caused by constitutive resistance. 3. Early‐season herbivory affected the herbivore community, having contrasting effects on different herbivore species, while these effects were similar on the two cultivars. Generalist insect herbivores avoided plants that had been induced, whereas these plants were colonised preferentially by specialist herbivores belonging to both leaf‐chewing and sap‐sucking guilds. 4. Our results show that community‐wide effects of early‐season herbivory may prevail over effects of constitutive plant resistance. Induced responses triggered by prior herbivory may lead to an increase in susceptibility to the dominant specialists in the herbivorous insect community. The outcome of the balance between contrasting responses of herbivorous community members to induced plants therefore determines whether induced plant responses result in enhanced plant resistance.     

Root hair formation and elongation of primary maize roots

In maize ( Zea mays L. cv. LG 11) roots cultured in humid air, the presence of hairs was not related to root growth. However, maximum hair length and length of the hair zone could be correlated to the elongation rate of the primary root. Under the growth conditions used, the emergence of root hairs always took place in the extending zone. In more basal regions, rhizodermal cells could not give rise to root hairs. Results were similar for roots preincubated in a buffer solution.     

Effect of decreased irradiance on N and C metabolism in leaves and roots of maize

The effects of decreased irradiance on fresh and dry weight, root respiration, levels of carbohydrates and N-compounds, and extractable activities of enzymes involved in C and N metabolism were evaluated in maize ( Zea mays L. cv. Plauto) seedlings during the 7 days following transfer from 450 to 200 μmol m⁻² s⁻¹ PAR. The fresh weight of roots and stems, the initiation of new leaves, root respiration rate, and the accumulation of dry matter, soluble sugars, starch, malate and amino acids in both leaves and roots were strongly reduced at low irradiance. In contrast, the level of nitrate was increased in leaves and only marginally affected in roots. Leaf phosphoenolpyruvate carboxylase (EC 4.1.1.31) activity started to decrease after 24–34 h, whereas ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) activity and chlorophyll content were unaffected or only slightly reduced. In both leaves and roots, the adjustment of N metabolism to low irradiance occurred through a relatively rapid (30% after 10 h) and large (60% after 3 days) decrease of nitrate reductase (NR; EC 1.6.6.1) activity, followed by slower and smaller changes in the activity of nitrite reductase (EC 1.7.7.1), glutamine synthetase (EC 6.3.1.2) and NAD-dependent glutamate dehydrogenase (EC 1.4.1.2). We suggest that the preferential decrease of NR activity relative to other N-assimilating enzymes may be important for preventing the accumulation of toxic N-compounds like ammonia in both leaf and root tissues.     

Appearance of bulges on maize roots as affected by 6-benzylaminopurine and α-naphthylacetic acid

The thickening that appeared on maize roots under the influence of 6-benzylaminopurine and α-naphthylacetic acid (concentration 10⁻⁵, 10⁻⁶, 10⁻⁷ and 10⁻⁸ M) were analysed. The changes in length and width of maize roots at the edge of elongation zone after 24,48 and 72 h of treatment were studied. The growth in length of cells at the edge of elongation zone stopped abruptly but the growth in width slowly continued. So, the growth of cells in length and width under the influence of growth regulators was not simultaneous. They had distinct time limits.     

Silicon as a plant defence against insect herbivory: response to Massey, Ennos and Hartley

1. Silicon (Si) has received increased attention as a nutrient capable of providing some measure of defence for plants against fungal pathogens, and insect and mammalian herbivores. 2. On the basis of a study including two generalist insect folivores and a phloem feeder, Massey, Ennos & Hartley (2006; Journal of Animal Ecology, 75, 595-603) have drawn attention to a putative distinction between the effects of plant Si in defending against folivorous and phloem-feeding insects. On the basis of their results they imply that phloem feeders are less likely to be adversely affected by increased plant Si than folivores. 3. However, in making this suggestion, Massey et al. have ignored many previous studies demonstrating a clear effect of plant Si on a range of phloem-feeding and some xylem-feeding insects, and that this effect stems not only from leaf mechanical properties based on opaline silica, but also from induced chemical defences seemingly mediated by soluble Si. 4. Furthermore, Massey et al. cannot claim that their study was the first demonstration of a direct effect of Si on insect herbivore preference and performance; there have been numerous earlier studies demonstrating this from folivores, stem borers, and phloem and xylem feeders. 5. We contend that current evidence indicates that Si is likely to be involved to a similar extent in enhancing resistance to all four insect feeding guilds and that any conclusion to the contrary is, at this stage, premature.     

A specialist root herbivore exploits defensive metabolites to locate nutritious tissues

The most valuable organs of plants are often particularly rich in essential elements, but also very well defended. This creates a dilemma for herbivores that need to maximise energy intake while minimising intoxication. We investigated how the specialist root herbivore Diabrotica virgifera solves this conundrum when feeding on wild and cultivated maize plants. We found that crown roots of maize seedlings were vital for plant development and, in accordance, were rich in nutritious primary metabolites and contained higher amounts of the insecticidal 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and the phenolic compound chlorogenic acid. The generalist herbivores Diabrotica balteata and Spodoptera littoralis were deterred from feeding on crown roots, whereas the specialist D. virgifera preferred and grew best on these tissues. Using a 1,4-benzoxazin-3-one-deficient maize mutant, we found that D. virgifera is resistant to DIMBOA and other 1,4-benzoxazin-3-ones and that it even hijacks these compounds to optimally forage for nutritious roots.     

Plant behavioural ecology: dynamic plasticity in secondary metabolites

Behaviour is in part the ability to respond rapidly and reversibly in response to environmental stimuli during the lifetime of an individual. Plants and animals both exhibit behaviour, but plant behaviour is most often examined in the context of morphologically plastic growth. Rapid and reversible secondary metabolite production and release is also a key mechanism by which plants behave. Here, we review plant biochemical plasticity as plant behaviour, and explicitly focus on evidence for responses that display rapid induction, reversibility and ecological relevance. Rapid induction and attenuation of plant secondary metabolites occur as chemically mediated root foraging, plant defence, allelochemistry and to regulate mutualistic relationships. We describe a wealth of information on the induction of various plant biochemical responses to environmental stimuli but found a limited body of literature on the reversibility of induced biochemical responses. Understanding the full cycle of dynamic plasticity in secondary metabolites is an important niche for future research. Biochemical behaviours extend beyond the plant kingdom; however, they clearly illustrate the capacity for plants to behave in ways that closely mirror the classic definitions and research approaches applied to behaviour in animals.     

Field resistance in maize cultivars to stem borer Chilo partellus

The Kenyan maize varieties, Hybrid 511, Hybrid 622, Pwani hybrid, Katumani Composite B, and local populations were evaluated along with the varieties of Multiple Insect-Resistant Tropical (MIRT) pool and experimental varieties from The Maize and Wheat Improvement Centre (CIMMYT), Mexico for resistance to the stem borer, Chilo partellus (Swinhoe). The artificial infestation by first-generation C. partellus on maize during the early whorl stage revealed that the leaf feeding damage, the dead heart formation, and the stem damage on the Kenyan hybrids and populations were significantly greater than on the MIRT varieties. The experimental varieties of CIMMYT were also susceptible to C. partellus. When the maize was infested at anthesis, the Kenyan varieties displayed a moderate level of resistance in terms of stem tunnelling and grain damage by C. partellus. The varieties of Multiple Insect Resistance Tropical pool can be effectively used by the breeders to enhance the resistance of agronomically good, but susceptible, varieties of Kenya.     

Gone with the wind: trembling leaves may deter herbivory

Plants employ various defensive tactics against herbivores but are rarely considered to use rapid movements to resist predation. However, the aboveground parts of plants are often forcefully moved by wind and rain. This passive movement has been overlooked as an anti‐herbivore trait. The leaves of many plant species, such as aspens, Indian sacred fig, bamboos, and palms, tremble even in a slight breeze. Leaves that are easily moved by gentle winds can sometimes resist strong winds and may have other benefits as well. In the present study, it is proposed that the movement of such plant leaves physically deters arthropod herbivory and pathogen infection by repelling colonization and oviposition by herbivorous insects. This leads to herbivores and pathogens being dislodged from the plants, and the ensuing death of the herbivores on the ground or at least their recolonization to other plants, as well as the interruption of feeding, intraspecific communication and the mating behaviour of herbivores, thus lowering their performance on the plant or increasing enemy attack of the herbivores. In addition, passive leaf movements may undermine herbivore camouflage and expose them to predation, and may also allow plant volatiles to diffuse efficiently to repel herbivores and attract natural enemies. Thus, the mechanistic properties of these leaves may have anti‐herbivore effects in the wind and rain. This hypothesis can also be applied to aquatic plants that tremble in gentle water currents. In addition, genetic manipulation of the tendency for leaf movement may be beneficial for the management of pest insects and pathogens with reduced pesticides in forestry and agriculture. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 738–747.     

Anion uptake in maize roots: Interactions between chlorate and nitrate

Effects of nitrate, chloride and chlorate ions upon nitrate and chlorate uptake by roots of maize ( Zea mays L., cv. B73) seedlings were examined. Net nitrate uptake, ³⁶ClO₃⁻ influx and ³⁶Cl⁻ influx (the latter two in a background of 0.5 m M KNO₃) displayed similar pH profiles with optima at pH 5.5 and below. External, non-labeled chloride had little effect on the accumulation of ³⁶ClO₃⁻ (both in 5 h and 20 min uptake assays), while nitrate and chlorate had almost identical, marked inhibitory effects. Nitrate pretreatment caused an apparent induction of both ³⁶ClO₃⁻ and ¹⁵NO₃⁻ uptake activities. After 5 h of treatment in nitrate, the uptake activities of chloride- and chlorate-pretreated plants increased to that of nitrate-pretreated plants. During 6 h exposure to chlorate, ³⁶ClO₃⁻ uptake activity of nitrate-pretreated plants decreased to that of chlorate- and chloride-pretreated plants. The results support the existence of a shared nitrate/chlorate transport system in maize roots which is not inhibited by external chloride, and which is induced by nitrate, but not by chlorate or chloride. The suggestion is made that selection of chlorate-resistant mutants of maize can identify nitrate uptake as well as nitrate reductase mutants.     

Host‐searching responses to herbivory‐associated chemical information and patch use depend on mating status of female solitary parasitoid wasps

1. In a tritrophic interaction system consisting of plants, herbivores, and their parasitoids, chemicals released from plants after herbivory are known to play important roles for many female parasitoids to find their hosts efficiently. On the plant side, chemical information associated with herbivory can act as an indirect defence by attracting the natural enemies of the host herbivores. 2. However, mated and virgin females of haplodiploid parasitoids might not necessarily respond to such chemical cues in the same way. Since virgin females can produce only sons, they might refrain from searching for hosts to invest eggs until copulation, in order to produce both sexes. 3. Here, we investigated differential host‐searching behaviours shown by mated and virgin females in the solitary parasitoid wasp, Cotesia vestalis, in response to herbivory‐associated chemical information from cruciferous plants infested by their host larvae, Plutella xylostella. 4. Mated females showed a significantly higher flight preference for host‐infested plants over intact plants, while no preference was observed with virgin females. Mated females also showed more intensive antennal searching and ovipositor probing behaviours to leaf squares with wounds caused by hosts than did virgin females. Furthermore, mated females stayed longer in host patches with higher parasitism rates than virgin females. 5. These results indicate that mating status of C. vestalis females clearly influences their host‐searching behaviour in response to herbivory‐associated chemical information and patch exploitation. Female parasitoids seem to forage for hosts depending on their own physiological condition in a tritrophic system.     

Specific herbivore-induced volatiles defend plants and determine insect community composition in the field

In response to insect attack, plants release complex blends of volatile compounds. These volatiles serve as foraging cues for herbivores, predators and parasitoids, leading to plant-mediated interactions within and between trophic levels. Hence, plant volatiles may be important determinants of insect community composition. To test this, we created rice lines that are impaired in the emission of two major signals, S-linalool and (E)-β-caryophyllene. We found that inducible S-linalool attracted predators and parasitoids as well as chewing herbivores, but repelled the rice brown planthopper Nilaparvata lugens, a major pest. The constitutively produced (E)-β-caryophyllene on the other hand attracted both parasitoids and planthoppers, resulting in an increased herbivore load. Thus, silencing either signal resulted in specific insect assemblages in the field, highlighting the importance of plant volatiles in determining insect community structures. Moreover, the results imply that the manipulation of volatile emissions in crops has great potential for the control of pest populations.     

Influence of silicon pretreatment on aluminium toxicity in maize roots

The influence of Si pretreatment on Al toxicity in an Al sensitive maize variety (Zea mays L. var. BR 201 F) was investigated using root elongation rates (RER) and hematoxylin staining as stress indicators. Plants pretreated with 1 mt M Si (+ Si) and then exposed for 24 h to Al in nutrient solution without concurrent Si supply in the rooting medium exhibited higher RER than plants that were not pretreated with Si (-Si). The ameliorative effect of Si was due to lower Al uptake and to the exclusion of Al from the root tips in + Si plants. Lower Al uptake in + Si plants was not a consequence of decreased Al availability in the bulk solution. The possible mechanisms of Si-induced increase of Al resistance are discussed     

A six-arm olfactometer permitting simultaneous observation of insect attraction and odour trapping

Abstract.  Behavioural assays to study insect attraction to specific odours are tedious, time consuming and often require large numbers of replications. Olfactometer and flight tunnel tests can usually only be conducted with one or two odour sources at a time. Moreover, chemical information on the odour sources has to be obtained in separate analytical studies. An olfactometer was developed in which six odours can be tested simultaneously for their relative attractiveness while during the assays, part of each test odour can be trapped for further analyses. The effectiveness of this six-arm olfactometer was tested by observing the responses of the solitary endoparasitoid Cotesia marginiventris (Cresson) to host-induced odours from young maize plants. For statistical analyses, we used log-linear models were adapted to account for overdispersion and possible positional biases. Female wasps responded extremely well in tests where they were offered a single odour source, as well as in tests with multiple choices. The responses of wasps released in groups were the same as those released individually and it was found that females did not attract or repel each other, but males preferred arms in which females had been released. Dose–response tests with varying numbers of plants or host larvae on plants revealed that the wasps responded in a dose-related manner, thus showing that the system is well suited to measure relative preference. The clear choices of the insects amongst six possibilities provided substantial statistical power. Gas chromatographic analyses of sampled air revealed clean and effective odour trapping, which largely facilitates the comparison of results from behavioural assays with the actual blends of volatiles that were emitted by the various odour sources. Advantages and disadvantages compared to other methods are discussed.