Close and distant: Contrasting the metabolism of two closely related subspecies of Scots pine under the effects of folivory and summer drought

Metabolomes, as chemical phenotypes of organisms, are likely not only shaped by the environment but also by common ancestry. If this is the case, we expect that closely related species of pines will tend to reach similar metabolomic solutions to the same environmental stressors. We examined the metabolomes of two sympatric subspecies of Pinus sylvestris in Sierra Nevada (southern Iberian Peninsula), in summer and winter and exposed to folivory by the pine processionary moth. The overall metabolomes differed between the subspecies but both tended to respond more similarly to folivory. The metabolomes of the subspecies were more dissimilar in summer than in winter, and iberica trees had higher concentrations of metabolites directly related to drought stress. Our results are consistent with the notion that certain plant metabolic responses associated with folivory have been phylogenetically conserved. The larger divergence between subspecies metabolomes in summer is likely due to the warmer and drier conditions that the northern iberica subspecies experience in Sierra Nevada. Our results provide crucial insights into how iberica populations would respond to the predicted conditions of climate change under an increased defoliation in the Mediterranean Basin.     

Are the metabolomic responses to folivory of closely related plant species linked to macroevolutionary and plant–folivore coevolutionary processes?

The debate whether the coevolution of plants and insects or macroevolutionary processes (phylogeny) is the main driver determining the arsenal of molecular defensive compounds of plants remains unresolved. Attacks by herbivorous insects affect not only the composition of defensive compounds in plants but also the entire metabolome. Metabolomes are the final products of genotypes and are constrained by macroevolutionary processes, so closely related species should have similar metabolomic compositions and may respond in similar ways to attacks by folivores. We analyzed the elemental compositions and metabolomes of needles from three closely related Pinus species with distant coevolutionary histories with the caterpillar of the processionary moth respond similarly to its attack. All pines had different metabolomes and metabolic responses to herbivorous attack. The metabolomic variation among the species and the responses to folivory reflected their macroevolutionary relationships, with P. pinaster having the most divergent metabolome. The concentrations of terpenes were in the attacked trees supporting the hypothesis that herbivores avoid plant individuals with higher concentrations. Our results suggest that macroevolutionary history plays important roles in the metabolomic responses of these pine species to folivory, but plant–insect coevolution probably constrains those responses. Combinations of different evolutionary factors and trade‐offs are likely responsible for the different responses of each species to folivory, which is not necessarily exclusively linked to plant–insect coevolution.     

Plant ontogeny and chemical defence: older seedlings are better defended

Although patterns of seedling selection by herbivores are strongly influenced by plant age and the expression of anti-herbivore defence, it is unclear how these characteristics interact to influence seedling susceptibility to herbivory. We tracked ontogenetic changes in a range of secondary metabolites (total phenolics, alkaloids and cyanogenic glycosides) commonly associated with seedling defence for nine sympatric British grassland species. Although there was marked variation in concentrations of secondary metabolites between different species, we found a consistent increase in the deployment of phenolics, alkaloids and cyanogenics with seedling age for six of the seven dicotyledonous species examined. The two grass species by contrast exhibited low levels of secondary metabolites across all developmental stages, possibly due to an investment in structural (silica phytoliths) defence. Our results corroborate species-specific patterns of seedling herbivory observed in field studies, and offer some explanation for the relatively high sensitivity to herbivore attack frequently observed for relatively young seedlings compared with their older conspecifics. Our results also support predictions made by the growth–differentiation balance hypothesis regarding ontogenetic changes in resource allocation to anti-herbivore defence for a range of potential chemical defences and across a range of sympatric plant species presumably subject to broadly similar selective pressures at the regeneration stage.     

Anatomical defences against bark beetles relate to degree of historical exposure between species and are allocated independently of chemical defences within trees

Conifers possess chemical and anatomical defences against tree‐killing bark beetles that feed in their phloem. Resins accumulating at attack sites can delay and entomb beetles while toxins reach lethal levels. Trees with high concentrations of metabolites active against bark beetle‐microbial complexes, and more extensive resin ducts, achieve greater survival. It is unknown if and how conifers integrate chemical and anatomical components of defence or how these capabilities vary with historical exposure. We compared linkages between phloem chemistry and tree ring anatomy of two mountain pine beetle hosts. Lodgepole pine, a mid‐elevation species, has had extensive, continual contact with this herbivore, whereas high‐elevation whitebark pines have historically had intermittent exposure that is increasing with warming climate. Lodgepole pine had more and larger resin ducts. In both species, anatomical defences were positively related to tree growth and nutrients. Within‐tree constitutive and induced concentrations of compounds bioactive against bark beetles and symbionts were largely unrelated to resin duct abundance and size. Fewer anatomical defences in the semi‐naïve compared with the continually exposed host concurs with directional differences in chemical defences. Partially uncoupling chemical and morphological antiherbivore traits may enable trees to confront beetles with more diverse defence permutations that interact to resist attack.     

Direct and indirect chemical defence of pine against folivorous insects

The chemical defence of pine against herbivorous insects has been intensively studied with respect to its effects on the performance and behaviour of the herbivores as well as on the natural enemies of pine herbivores. The huge variety of terpenoid pine components play a major role in mediating numerous specific food web interactions. The constitutive terpenoid pattern can be adjusted to herbivore attack by changes induced by insect feeding or oviposition activity. Recent studies on folivorous pine sawflies have highlighted the role of induced pine responses in herbivore attack and have demonstrated the importance of analysing the variability of pine defence and its finely tuned specificity with respect to the herbivore attacker in a multitrophic context.     

Decomposers and root feeders interactively affect plant defence in <Emphasis Type="Italic">Sinapis alba</Emphasis>

Aboveground herbivory is well known to change plant growth and defence. In contrast, effects of soil organisms, acting alone or in concert, on allocation patterns are less well understood. We investigated separate and combined effects of the endogeic earthworm species Aporrectodea caliginosa and the root feeding nematode species Pratylenchus penetrans and Meloidogyne incognita on plant responses including growth and defence metabolite concentrations in leaves of white mustard, Sinapis alba. Soil biota had a strong impact on plant traits, with the intensity varying due to species combinations. Nematode infestation reduced shoot biomass and nitrogen concentration but only in the absence of earthworms. Earthworms likely counteracted the negative effects of nematodes. Infestation with the migratory lesion-nematode P. penetrans combined with earthworms led to increased root length. Earthworm biomass increased in the presence of this species, indicating that these nematodes increased the food resources of earthworms—presumably dead and decaying roots. Nitrogen-based defence compounds, i.e. glucosinolates, did not correlate with nitrogen levels. In the presence of earthworms, concentrations of aromatic glucosinolates in leaves were significantly increased. In contrast, infection with P. penetrans strongly decreased concentrations of glucosinolates (up to 81%). Infestation with the sedentary nematode M. incognita induced aromatic glucosinolates by more than 50% but only when earthworms were also present. Myrosinase activities, glucosinolate-hydrolysing enzymes, were unaffected by nematodes but reduced in the presence of earthworms. Our results document that root-feeding nematodes elicit systemic plant responses in defence metabolites, with the responses varying drastically with nematode species of different functional groups. Furthermore, systemic plant responses are also altered by decomposer animals, such as earthworms, challenging the assumption that induction of plant responses including defence traits is restricted to herbivores. Soil animals even interact and modulate the individual effects on plant growth and plant defence, thereby likely also influencing shoot herbivore attack.     

Temporal consistency in herbivore responses to glucosinolate polymorphism in populations of wild cabbage (Brassica oleracea)

Natural populations of wild cabbage (Brassica oleracea) show significant qualitative diversity in heritable aliphatic glucosinolates, a class of secondary metabolites involved in defence against herbivore attack. One candidate mechanism for the maintenance of this diversity is that differential responses among herbivore species result in a net fitness balance across plant chemotypes. Such top-down differential selection would be promoted by consistent responses of herbivores to glucosinolates, temporal variation in herbivore abundance, and fitness impacts of herbivore attack on plants varying in glucosinolate profile. A 1-year survey across 12 wild cabbage populations demonstrated differential responses of herbivores to glucosinolates. We extended this survey to investigate the temporal consistency of these responses, and the extent of variation in abundance of key herbivores. Within plant populations, the aphid Brevicoryne brassicae consistently preferred plants producing the glucosinolate progoitrin. Among populations, increasing frequencies of sinigrin production correlated positively with herbivory by whitefly Aleyrodes proletella and negatively with herbivory by snails. Two Pieris butterfly species showed no consistent response to glucosinolates among years. Rates of herbivory varied significantly among years within populations, but the frequency of herbivory at the population scale varied only for B. brassicae. B. brassicae emerges as a strong candidate herbivore to impose differential selection on glucosinolates, as it satisfies the key assumptions of consistent preferences and heterogeneity in abundance. We show that variation in plant secondary metabolites structures the local herbivore community and that, for some key species, this structuring is consistent over time. We discuss the implications of these patterns for the maintenance of diversity in plant defence chemistry.     

Herbivory-induced signalling in plants: perception and action

Plants and herbivores have been interacting for millions of years. Over time, plants have evolved mechanisms to defend against herbivore attacks. Herbivore-challenged plants reconfigure their metabolism to produce compounds that are toxic, repellant or anti-digestive for the herbivores. Some compounds are volatile signals that attract the predators of herbivores. All these responses are tightly regulated by a signalling network triggered by the plant's perception machinery. Several compounds that specifically elicit herbivory-induced responses in plants have been isolated from herbivore oral secretions and oviposition fluids. Elicitor perception is rapidly followed by cell membrane depolarization, calcium influx and mitogen-activated protein kinase (MAPK) activation; plants also elevate the concentrations of reactive oxygen and nitrogen species, and modulate phytohormone levels accordingly. In addition to these reactions in the herbivore-attacked regions of a leaf, defence responses are also mounted in unattacked parts of the attacked leaf and as well in unattacked leaves. In this review, we summarize recent progress in understanding how plants recognize herbivory, the involvement of several important signalling pathways that mediate the responses to herbivore attack and the signals that transduce local into systemic responses.     

Variations in leaf traits and susceptibility to insect herbivory within a Salix miyabeana population under field conditions

Variations in leaf traits (toughness, total nitrogen and total phenolic concentrations) and susceptibility to herbivory in Salix miyabeana were studied among individual trees within a population under field conditions. Leaf quality clearly decreased as season progressed, i.e. increases in leaf toughness and total phenolics and decrease in leaf nitrogen. Seasonal pattern and extent of herbivore attack were similar between years. Significant correlation between leaf traits and susceptibility to herbivore attack was detected, while effects of sex and plant size on leaf traits and herbivory were less clear. There was a negative correlation between total nitrogen and total phenolics, and a positive correlation between leaf toughness and total phenolics among trees. Trees with high quality leaves tended to suffer from frequent herbivore attack and leaf damage. Such a clear relationship between leaf traits and susceptibility to herbivory may be related with a life-history strategy of willows, which are rapid-growing pioneer species and generally respond to herbivorous damage not by induced resistance but by compensative growth. This revised version was published online in August 2006 with corrections to the Cover Date.     

Role of castor plant phenolics on performance of its two herbivores and their impact on egg parasitoid behaviour

Plants are reported to reconfigure their metabolism and generate secondary metabolites as a defence response towards herbivore attack. In this study we aimed to evaluate the anti-nutritive and defensive properties of phenolic compounds that were enhanced in Castor, Ricinus communis L. plant due to herbivore damage. Through HPLC studies it was observed that damage caused by two pests, Achaea janata L. and Spodoptera litura F. on the castor plant resulted in quantitative enhancement of syringic, coumaric, cinnamic and vanillic acids. These phenolic compounds showed considerable impact on insect’s feeding and their toxic nature towards the herbivores was determined by estimating three detoxification mid-gut enzymes, glutathione-s-transferase, carboxyl esterase and β-glucosidase. In the ovipositional and orientation bioassays with the egg parasitoid Trichogramma chilonis Ishii, syringic and coumaric acids had shown good kairomonal activity, thus defining a new role of these chemicals in indirect plant defence. Thus, this study demonstrates a quantitative association between plant phenolic accumulation and the elevated reproductive performance of the herbivore natural enemies, by illustrating the role of plant phenolics in both the direct and indirect induced plant defences.     

Priming by airborne signals boosts direct and indirect resistance in maize

Plants counteract attack by herbivorous insects using a variety of inducible defence mechanisms. The production of toxic proteins and metabolites that instantly affect the herbivore's development are examples of direct induced defence. In addition, plants may release mixtures of volatile organic compounds (VOCs) that indirectly protect the plant by attracting natural enemies of the herbivore. Recent studies suggest that these VOCs can also prime nearby plants for enhanced induction of defence upon future insect attack. However, evidence that this defence priming causes reduced vulnerability to insects is sparse. Here we present molecular, chemical and behavioural evidence that VOC-induced priming leads to improved direct and indirect resistance in maize. A differential hybridization screen for inducible genes upon attack by Spodoptera littoralis caterpillars identified 10 defence-related genes that are responsive to wounding, jasmonic acid (JA), or caterpillar regurgitant. Exposure to VOCs from caterpillar-infested plants did not activate these genes directly, but primed a subset of them for earlier and/or stronger induction upon subsequent defence elicitation. This priming for defence-related gene expression correlated with reduced caterpillar feeding and development. Furthermore, exposure to caterpillar-induced VOCs primed for enhanced emissions of aromatic and terpenoid compounds. At the peak of this VOC emission, primed plants were significantly more attractive to parasitic Cotesia marginiventris waSPS. This study shows that VOC-induced priming targets a specific subset of JA-inducible genes, and links these responses at the molecular level to enhanced levels of direct and indirect resistance against insect attack.     

Delayed induced responses of birch glandular trichomes and leaf surface lipophilic compounds to mechanical defoliation and simulated winter browsing

Changes in morphology and chemistry of leaf surface in response to herbivore damage may increase plant resistance to subsequent herbivore attack; however, there is lack of studies on induced responses of glandular trichomes and their exudates in woody plants and on effects of these changes on herbivores. We studied delayed induced responses in leaf surface traits of five clones of silver birch (Betula pendula Roth) subjected to various types of mechanical defoliation and simulated winter browsing. Glandular trichome density and concentrations of the majority of surface lipophilic compounds increased in trees defoliated during the previous summer. This induced response was systemic, since control branches in branch defoliated trees responded to the treatments similarly to defoliated branches, but differently from control trees. In contrast to defoliation treatments, simulated winter browsing reduced glandular trichome density on the following summer and had fewer effects on individual surface lipophilic compounds. Moreover, constitutive density of glandular trichomes was negatively correlated with induced total amount of lipophilic compounds per trichome, indicating a trade-off between constitutive and induced resistance in silver birch. Induced changes in leaf surface traits had no significant effect on leaf damage by chewers, miners and gall mites, but increased susceptibility of birch trees to aphids. However, leaf damage by chewers, miners and gall mites in defoliated (but not in control) trees was correlated with concentrations of some fatty acids and triterpenoids, although the direction of relationships varied among herbivore species. This indicates that induction of surface lipophilic compounds may influence birch resistance to herbivores. Our study thus demonstrated both specificity of elicitation of induced responses of birch leaf surface traits by different types of damage and specificity of the effects of these responses on different types of herbivores.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Latitudinal Patterns of Herbivory in Mangrove Forests: Consequences of Nutrient Over-Enrichment

Ecosystems in the tropics are predicted to have stronger responses to nutrient enrichment, greater diversity, and more intense biotic interactions than in temperate areas. Mangrove forests, which occur across a broad biogeographic range from warm temperate to tropical, provide a unique opportunity to test these hypotheses by investigating the responses of herbivores to nutrient enrichment in temperate versus tropical latitudes. Mangroves are complex intertidal ecosystems with spatial differences in structure and diversity along tidal gradients and are threatened globally by human activities including nutrient over-enrichment. In this study, we used long-term fertilization experiments at the Indian River Lagoon, FL; Twin Cays, Belize; and Bocas del Toro, Panamá to determine how increased nutrients impact herbivore abundance and herbivory of Rhizophora mangle at the tree, forest, and regional scales. At these locations, which span approximately 2185 km and 18.4º of latitude, we fertilized individual trees with one of three treatments (Control, +N, +P) in two zones (fringe, scrub) along transects perpendicular to the shoreline and measured their responses for 4 years. Herbivory was measured as folivory, loss of yield, and tissue mining. Although nutrient enrichment altered plant growth, leaf traits, and nutrient dynamics, these variables had little effect on folivory at any location. Our results did not support the prediction that herbivory and per capita consumption are greatest at the most tropical location. Instead, folivory was highest at the most temperate location and lowest at the intermediate location. Folivory was generally higher in the fringe than in the scrub zone, but the pattern varied by location, herbivore, and nutrient treatment. Folivory by a dominant herbivore, Aratus pisonii, decreased from the highest to the lowest latitude. Our data suggest that factors controlling population dynamics of A. pisonii cascade to the mangrove canopy, linking herbivory to crab densities.     

Roots drive oligogalacturonide‐induced systemic immunity in tomato

Oligogalacturonides (OGs) are fragments of pectin released from the plant cell wall during insect or pathogen attack. They can be perceived by the plant as damage signals, triggering local and systemic defence responses. Here, we analyse the dynamics of local and systemic responses to OG perception in tomato roots or shoots, exploring their impact across the plant and their relevance in pathogen resistance. Targeted and untargeted metabolomics and gene expression analysis in plants treated with purified OGs revealed that local responses were transient, while distal responses were stronger and more sustained. Remarkably, changes were more conspicuous in roots, even upon foliar application of the OGs. The treatments differentially activated the synthesis of defence‐related hormones and secondary metabolites including flavonoids, alkaloids and lignans, some of them exclusively synthetized in roots. Finally, the biological relevance of the systemic defence responses activated upon OG perception was confirmed, as the treatment induced systemic resistance to Botrytis cinerea. Overall, this study shows the differential regulation of tomato defences upon OGs perception in roots and shoots and reveals the key role of roots in the coordination of the plant responses to damage sensing.     

How do plants “notice” attack by herbivorous arthropods?

Precise and deep comprehension of plant responses to herbivorous arthropods requires detailed knowledge of how a plant “notices” the attack. Herbivore attack is not restricted to plant wounding by feeding, but instead different phases of attack that elicit a plant response need to be distinguished: touch, oviposition and feeding. Touch, secretions released with eggs and regurgitate delivered during feeding may act in concert as elicitors of plant defence. Here, we discuss the current knowledge of what a plant “notices” during the different phases of herbivore attack and how it responds at the molecular, physiological and ecological level. Understanding the mechanisms of plant responses to the different phases of herbivore attack will be a key challenge in unravelling the complex communication pathways between plants and herbivores.     

Rethinking the role of many plant phenolics – protection from photodamage not herbivores?

Phenolics have been considered classic defence compounds for protecting plants from herbivores, ever since plant secondary metabolites were suggested to have evolved for that reason. The resource availability and carbon-nutrient balance hypotheses proposed that variation in phenolic levels between and within plant species reflects environmental availability of nutrients and light, and represents a trade-off in allocation by plants to growth and defence against herbivores. In contrast to these concepts, we suggest that (1) the main role of many plant phenolics may be to protect leaves from photodamage, not herbivores; (2) they can achieve this by acting as antioxidants; and (3) their levels may vary with environmental conditions in order to counteract this potential photodamage. We therefore suggest that patterns in phenolic levels, often used to support the concept of trade-off between growth and herbivore defence in relation to resource availability, may actually reflect different risks of photodamage. We suggest that the level of many phenolics is low under some environmental conditions, not because resources to produce them are limited, but simply because the risk of photodamage is low and they are not required. If our photodamage hypothesis is correct, a reassessment of the ecological and evolutionary role of many phenolics in plant defence theory is required.     

Methyl jasmonate does not induce changes in Eucalyptus grandis leaves that alter the effect of constitutive defences on larvae of a specialist herbivore

The up-regulation of secondary metabolic pathways following herbivore attack and the subsequent reduction in herbivore performance have been identified in numerous woody plant species. Eucalypts constitutively express many secondary metabolites in the leaves, including terpenes and formylated phloroglucinol compounds (FPCs). We used clonal ramets from six clones of Eucalyptus grandis and two clones of E. grandis x camaldulensis to determine if methyl jasmonate (MeJA) treatment could induce changes in the foliar concentrations of either of these groups of compounds. We also used bioassays to determine if any changes in the performance of larvae of Paropsis atomaria, a chrysomelid leaf beetle, could be detected in treated ramets versus the untreated controls, thus indicating whether MeJA induced the up-regulation of defences other than terpenes or FPCs. We found no significant effects of MeJA treatment on either the foliar concentrations of terpenes and FPCs or on herbivore performance. We did, however, detect dramatic differences in larval performance between Eucalyptus clones, thereby demonstrating large variations in the levels of constitutive defence. Larval feeding on clones resistant to P. atomaria resulted in high first instar mortality and disruption of normal gregarious feeding behaviour in surviving larvae. Histological examination of larvae feeding on a resistant clone revealed damage to the midgut consistent with the action of a toxin. These findings concur with mounting evidence that most evergreen perennial plants lack foliar-induced defences and suggest that constitutively expressed secondary metabolites other than those commonly examined in studies of interactions between insect herbivores and Eucalyptus may be important in plant defence.     

Can genetically based clines in plant defence explain greater herbivory at higher latitudes?

Greater plant defence is predicted to evolve at lower latitudes in response to increased herbivore pressure. However, recent studies question the generality of this pattern. In this study, we tested for genetically based latitudinal clines in resistance to herbivores and underlying defence traits of Oenothera biennis. We grew plants from 137 populations from across the entire native range of O. biennis. Populations from lower latitudes showed greater resistance to multiple specialist and generalist herbivores. These patterns were associated with an increase in total phenolics at lower latitudes. A significant proportion of the phenolics were driven by the concentrations of two major ellagitannins, which exhibited opposing latitudinal clines. Our analyses suggest that these findings are unlikely to be explained by local adaptation of herbivore populations or genetic variation in phenology. Rather greater herbivory at high latitudes can be explained by latitudinal clines in the evolution of plant defences.     

Plant structural traits and their role in anti-herbivore defence

We consider the role that key structural traits, such as spinescence, pubescence, sclerophylly and raphides, play in protecting plants from herbivore attack. Despite the likelihood that many of these morphological characteristics may have evolved as responses to other environmental stimuli, we show that each provides an important defence against herbivore attack in both terrestrial and aquatic ecosystems. We conclude that leaf-mass–area is a robust index of sclerophylly as a surrogate for more rigorous mechanical properties used in herbivory studies. We also examine herbivore counter-adaptations to plant structural defence and illustrate how herbivore attack can induce the deployment of intensified defensive measures. Although there have been few studies detailing how plant defences vary with age, we show that allocation to structural defences is related to plant ontogeny. Age-related changes in the deployment of structural defences plus a paucity of appropriate studies are two reasons why relationships with other plant fitness characteristics may be obscured, although we describe studies where trade-offs between structural defence and plant growth, reproduction, and chemical defences have been demonstrated. We also show how resource availability influences the expression of structural defences and demonstrate how poorly our understanding of plant structural defence fits into contemporary plant defence theory. Finally, we suggest how a better understanding of plant structural defence, particularly within the context of plant defence syndromes, would not only improve our understanding of plant defence theory, but enable us to predict how plant morphological responses to climate change might influence interactions at the individual (plant growth trade-offs), species (competition), and ecosystem (pollination and herbivory) levels.