Priming and filtering of antiherbivore defences among Nicotiana attenuata plants connected by mycorrhizal networks

Arbuscular mycorrhizal fungi (AMF) establish symbiotic associations with a majority of terrestrial plants to form underground common mycorrhizal networks (CMNs) that connect neighbouring plants. Because Nicotiana attenuata plants do not respond to herbivory‐elicited volatiles from neighbours, we used this ecological model system to evaluate if CMNs function in interplant transmission of herbivory‐elicited responses. A mesocosm system was designed to establish and remove CMNs linking N. attenuata plants to examine the herbivory‐elicited metabolic and hormone responses in CMNs‐connected “receiver” plants after the elicitation of “donor” plants by wounding (W) treated with Manduca sexta larval oral secretions (OS). AMF colonization increased constitutive jasmonate (JA and JA‐Ile) levels in N. attenuata roots but did not affect well‐characterized JAs‐regulated defensive metabolites in systemic leaves. Interestingly, larger JAs bursts, and higher levels of several amino acids and particular sectors of hydroxygeranyllinalool diterpene glycoside metabolism were elevated in the leaves of W + OS‐elicited “receivers” with CMN connections with “donors” that had been W + OS‐elicited 6 hr previously. Our results demonstrate that AMF colonization alone does not enhance systemic defence responses but that sectors of systemic responses in leaves can be primed by CMNs, suggesting that CMNs can transmit and even filter defence signalling among connected plants.     

Compensation to simulated insect leaf herbivory in wild cotton (Gossypium hirsutum): responses to multiple levels of damage and associated traits

• Identifying the mechanisms of compensation to insect herbivory remains a major challenge in plant biology and evolutionary ecology. Most previous studies have addressed plant compensatory responses to one or two levels of insect herbivory, and the underlying traits mediating such responses remain elusive in many cases.
• We evaluated responses associated with compensation to multiple intensities of leaf damage (0% control, 10%, 25%, 50%, 75% of leaf area removed) by means of mechanical removal of foliar tissue and application of a caterpillar (Spodoptera exigua) oral secretions in 3‐month‐old wild cotton plants (Gossypium hirsutum). Four weeks post‐treatment, we measured plant growth and multiple traits associated with compensation, namely: changes in above‐ and belowground, biomass and the concentration of nutrients (nitrogen and phosphorus) and non‐structural carbon reserves (starch and soluble sugars) in roots, stems and leaves.
• We found that wild cotton fully compensated in terms of growth and biomass allocation when leaf damage was low (10%), whereas moderate (25%) to high leaf damage in some cases led to under‐compensation. Nonetheless, high levels of leaf removal (50% and 75%) in most cases did not cause further reductions in height and allocation to leaf and stem biomass relative to low and moderate damage. There were significant positive effects of leaf damage on P concentration in leaves and stems, but not roots, as well as a negative effect on soluble sugars in roots.
• These results indicate that wild cotton fully compensated for a low level of leaf damage but under‐compensated under moderate to high leaf damage, but can nonetheless sustain growth despite increasing losses to herbivory. Such responses were possibly mediated by a re‐allocation of carbohydrate reserves from roots to shoots.

     

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.     

What happens in the pith stays in the pith: tissue‐localized defense responses facilitate chemical niche differentiation between two spatially separated herbivores

Herbivore attack is known to elicit systemic defense responses that spread throughout the host plant and influence the performance of other herbivores. While these plant‐mediated indirect competitive interactions are well described, and the co‐existence of herbivores from different feeding guilds is common, the mechanisms of co‐existence are poorly understood. In both field and glasshouse experiments with a native tobacco, Nicotiana attenuata, we found no evidence of negative interactions when plants were simultaneously attacked by two spatially separated herbivores: a leaf chewer Manduca sexta and a stem borer Trichobaris mucorea. T. mucorea attack elicited jasmonic acid (JA) and jasmonoyl‐l ‐isoleucine bursts in the pith of attacked stems similar to those that occur in leaves when M. sexta attacks N. attenuata leaves. Pith chlorogenic acid (CGA) levels increased 1000‐fold to levels 6‐fold higher than leaf levels after T. mucorea attack; these increases in pith CGA levels, which did not occur in M. sexta‐attacked leaves, required JA signaling. With plants silenced in CGA biosynthesis (irHQT plants), CGA, as well as other caffeic acid conjugates, was demonstrated in both glasshouse and field experiments to function as a direct defense protecting piths against T. mucorea attack, but not against leaf chewers or sucking insects. T. mucorea attack does not systemically activate JA signaling in leaves, while M. sexta leaf‐attack transiently induces detectable but minor pith JA levels that are dwarfed by local responses. We conclude that tissue‐localized defense responses allow tissue‐specialized herbivores to share the same host and occupy different chemical defense niches in the same hostplant.     

Leaf herbivory counteracts nematode-triggered repression of jasmonate-related defenses in tomato roots

Shoot herbivores may influence the communities of herbivores associated with the roots via inducible defenses. However, the molecular mechanisms and hormonal signaling underpinning the systemic impact of leaf herbivory on root-induced responses against nematodes remain poorly understood. By using tomato (Solanum lycopersicum) as a model plant, we explored the impact of leaf herbivory by Manduca sexta on the performance of the root knot nematode Meloidogyne incognita. By performing glasshouse bioassays, we found that leaf herbivory reduced M. incognita performance in the roots. By analyzing the root expression profile of a set of oxylipin-related marker genes and jasmonate root content, we show that leaf herbivory systemically activates the 13-Lipoxigenase (LOX) and 9-LOX branches of the oxylipin pathway in roots and counteracts the M. incognita-triggered repression of the 13-LOX branch. By using untargeted metabolomics, we also found that leaf herbivory counteracts the M. incognita-mediated repression of putative root chemical defenses. To explore the signaling involved in this shoot-to-root interaction, we performed glasshouse bioassays with grafted plants compromised in jasmonate synthesis or perception, specifically in their shoots. We demonstrated the importance of an intact shoot jasmonate perception, whereas having an intact jasmonate biosynthesis pathway was not essential for this shoot-to-root interaction. Our results highlight the impact of leaf herbivory on the ability of M. incognita to manipulate root defenses and point to an important role for the jasmonate signaling pathway in shoot-to-root signaling.

Leaf herbivory counteracts the repression of jasmonate-related defenses triggered by a root knot nematode in tomato roots impairing the nematode performance via shoot-to-root jasmonate signaling     

Changes in cytokinins are sufficient to alter developmental patterns of defense metabolites in Nicotiana attenuata

Plant defense metabolites are well known to be regulated developmentally. The optimal defense (OD) theory posits that a tssue's fitness values and probability of attack should determine defense metabolite allocations. Young leaves are expected to provide a larger fitness value to the plant, and therefore their defense allocations should be higher when compared with older leaves. The mechanisms that coordinate development with defense remain unknown and frequently confound tests of the OD theory predictions. Here we demonstrate that cytokinins (CKs) modulate ontogeny‐dependent defenses in Nicotiana attenuata. We found that leaf CK levels highly correlate with inducible defense expressions with high levels in young and low levels in older leaves. We genetically manipulated the developmental patterns of two different CK classes by using senescence‐ and chemically inducible expression of CK biosynthesis genes. Genetically modifying the levels of different CKs in leaves was sufficient to alter ontogenic patterns of defense metabolites. We conclude that the developmental regulation of growth hormones that include CKs plays central roles in connecting development with defense and therefore in establishing optimal patterns of defense allocation in plants.     

Changes in nutrient concentrations of leaves and roots in response to global change factors

Global change impacts on biogeochemical cycles have been widely studied, but our understanding of whether the responses of plant elemental composition to global change drivers differ between above‐ and belowground plant organs remains incomplete. We conducted a meta‐analysis of 201 reports including 1,687 observations of studies that have analyzed simultaneously N and P concentrations changes in leaves and roots in the same plants in response to drought, elevated [CO₂], and N and P fertilization around the world, and contrasted the results within those obtained with a general database (838 reports and 14,772 observations) that analyzed the changes in N and P concentrations in leaves and/or roots of plants submitted to the commented global change drivers. At global level, elevated [CO₂] decreased N concentrations in leaves and roots and decreased N:P ratio in roots but no in leaves, but was not related to P concentration changes. However, the response differed among vegetation types. In temperate forests, elevated [CO₂] was related with lower N concentrations in leaves but not in roots, whereas in crops, the contrary patterns were observed. Elevated [CO₂] decreased N concentrations in leaves and roots in tundra plants, whereas not clear relationships were observed in temperate grasslands. However, when elevated [CO₂] and N fertilization coincided, leaves had lower N concentrations, whereas root had higher N concentrations suggesting that more nutrients will be allocated to roots to improve uptake of the soil resources not directly provided by the global change drivers. N fertilization and drought increased foliar and root N concentrations while the effects on P concentrations were less clear. The changes in N and P allocation to leaves and root, especially those occurring in opposite direction between them have the capacity to differentially affect above‐ and belowground ecosystem functions, such as litter mineralization and above‐ and belowground food webs.     

Root damage to apple plants by cockchafer larvae induces a change in volatile signals below‐ and above‐ground

Volatile organic compounds (VOCs) mediate communication between plants and insects. Plants under insect herbivore attack release VOCs either at the site of attack or systemically, indicating within‐plant communication. Some of these VOCs, which may be induced only upon herbivore attack, recruit parasitoids and predatory insects to feed on the attacking insects. Moreover, some plants are able to ‘eavesdrop’ on herbivore‐induced plant volatiles (HIPVs) to prime themselves against impending attack; such eavesdropping exemplifies plant–plant communication. In apple orchards, the beetle Melolontha melolontha L. (Coleoptera: Scarabaeidae) is an important insect pest whose larvae live and feed on roots for about 4 years. In this study, we investigated whether the feeding activity of M. melolontha larvae (1) alters the volatile profile of apple roots, (2) induces the release of HIPVs systemically in the leaves, and (3) whether infested plants communicate to neighbouring non‐infested conspecifics through HIPVs. To answer these questions, we collected constitutive VOCs from intact M9 roots as well as M. melolontha larvae‐damaged roots using a newly designed ‘rhizobox’, to collect root‐released volatiles in situ, without damaging the plant root system. We also collected VOCs from the leaf‐bearing shoots of M9 whose roots were under attack by M. melolontha larvae and from shoots of neighbouring non‐infested conspecifics. Gas chromatography‐mass spectrometry analysis showed that feeding activity of M. melolontha larvae induces the release of specific HIPVs; for instance, camphor was found in the roots only after larvae caused root damage. Melolontha melolontha also induced the systemic release of methyl salicylate and (E,E)‐α‐farnesene from the leaf‐bearing shoots. Methyl salicylate and (E,E)‐α‐farnesene were also released by the shoots of non‐infested neighbouring conspecifics. These phenomena indicate the induction of specific VOCs below‐ and above‐ground upon M. melolontha larvae feeding on apple roots as well as plant–plant communication in apple plants.     

The Nicotiana attenuata GLA1 lipase controls the accumulation of Phytophthora parasitica‐induced oxylipins and defensive secondary metabolites

Nicotiana attenuata plants silenced in the expression of GLYCEROLIPASE A1 (ir‐gla1 plants) are compromised in the herbivore‐ and wound‐induced accumulation of jasmonic acid (JA). However, these plants accumulate wild‐type (WT) levels of JA and divinyl‐ethers during Phytophthora parasitica infection. By profiling oxylipin‐enriched fractions with targeted and untargeted liquid chromatography‐tandem time‐of‐flight mass spectrometry approaches, we demonstrate that the accumulation of 9‐hydroxy‐10E,12Z‐octadecadienoic acid (9‐OH‐18:2) and additional C₁₈ and C₁₉ oxylipins is reduced by ca. 20‐fold in P. parasitica‐infected ir‐gla1 leaves compared with WT. This reduced accumulation of oxylipins was accompanied by a reduced accumulation of unsaturated free fatty acids and specific lysolipid species. Untargeted metabolic profiling of total leaf extracts showed that 87 metabolites accumulated differentially in leaves of P. parasitica‐infected ir‐gla1 plants with glycerolipids, hydroxylated‐diterpene glycosides and phenylpropanoid derivatives accounting together for ca. 20% of these 87 metabolites. Thus, P. parasitica‐induced oxylipins may participate in the regulation of metabolic changes during infection. Together, the results demonstrate that GLA1 plays a distinct role in the production of oxylipins during biotic stress responses, supplying substrates for 9‐OH‐18:2 and additional C₁₈ and C₁₉ oxylipin formation during P. parasitica infection, whereas supplying substrates for the biogenesis of JA during herbivory and mechanical wounding.     

Palatability, decomposition and insect herbivory: patterns in a successional old-field plant community

We tested the hypothesis that selective feeding by insect herbivores in an old‐field plant community induces a shift of community structure towards less palatable plant species with lower leaf and litter tissue quality and may therefore affect nutrient cycling. Leaf palatability of 20 herbaceous plant species which are common during the early successional stages of an old‐field plant community was assayed using the generalist herbivores Deroceras reticulatum (Mollusca: Agriolomacidae) and Acheta domesticus (Ensifera: Gryllidae). Palatability was positively correlated with nitrogen content, specific leaf area and water content of leaves and negatively correlated with leaf carbon content and leaf C/N‐ratio. Specific decomposition rates were assessed in a litter bag experiment. Decomposition was positively correlated with nitrogen content of litter, specific leaf area and water content of living leaves and negatively correlated with leaf C/N‐ratio. When using phylogentically independent contrasts the correlations between palatability and decomposition versus leaf and litter traits remained significant (except for specific leaf area) and may therefore reflect functional relationships. As palatability and decomposition show similar correlations to leaf and litter traits, the correlation between leaf palatability and litter decomposition rate was also significant, and this held even in a phylogenetically controlled analysis. This correlation highlights the possible effects of invertebrate herbivory on resource dynamics. In a two‐year experiment we reduced the density of above‐ground and below‐ground insect herbivores in an early successional old‐field community in a two‐factorial design by insecticide application. The palatability ranking of plants showed no relationship with the specific change of cover abundance of plants due to the reduction of above‐ or below‐ground herbivory. Thus, changes in the dominance structure as well as potentially associated changes in the resource dynamics are not the result of differences in palatability between plant species. This highlights fundamental differences between the effects of insect herbivory on ecosystems and published results from vertebrate‐grazing systems.     

Counteractive biomass allocation responses to drought and damage in the perennial herb Convolvulus demissus

Herbivory and water shortage are key ecological factors affecting plant performance. While plant compensatory responses to herbivory include reallocation of biomass from below‐ground to above‐ground structures, plant responses to reduced soil moisture involve increased biomass allocation to roots and a reduction in the number and size of leaves. In a greenhouse study we evaluated the effects of experimental drought and leaf damage on biomass allocation in Convolvulus demissus (Convolvulaceae), a perennial herb distributed in central Chile, where it experiences summer drought typical of Mediterranean ecosystems and defoliation by leaf beetles and livestock. The number of leaves and internode length were unaffected by the experimental treatments. The rest of plant traits showed interaction of effects. We detected that drought counteracted some plant responses to damage. Thus, only in the control watering environment was it observed that damaged plants produced more stems, even after correcting for main stem length (index of architecture). In the cases of shoot : root ratio, relative shoot biomass and relative root biomass we found that the damage treatment counteracted plant responses to drought. Thus, while undamaged plants under water shortage showed a significant increase in root relative biomass and a significant reduction in both shoot : root ratio and relative shoot biomass, none of these responses to drought was observed in damaged plants. Total plant biomass increased in response to simulated herbivory, apparently due to greater shoot size, and in response to drought, presumably due to greater root size. However, damaged plants under experimental drought had the same total biomass as control plants. Overall, our results showed counteractive biomass allocation responses to drought and damage in C. demissus. Further research must address the fitness consequences under field conditions of the patterns found. This would be of particular importance because both current and expected climatic trends for central Chile indicate increased aridity.