Mycorrhiza-Tree-Herbivore Interactions: Alterations in Poplar Metabolome and Volatilome.

Plants are continuously interacting with other organisms to optimize their performance in a changing environment. Mycorrhization is known to affect the plant growth and nutrient status, but it also can lead to adjusted plant defense and alter interactions with other trophic levels. Here, we studied the effect of Laccaria bicolor-mycorrhization on the poplar (Populus x canescens) metabolome and volatilome on trees with and without a poplar leaf beetle (Chrysomela populi) infestation. We analyzed the leaf and root metabolomes employing liquid chromatography-mass spectrometry, and the leaf volatilome employing headspace sorptive extraction combined with gas-chromatography-mass spectrometry. Mycorrhization caused distinct metabolic adjustments in roots, young/infested leaves and old/not directly infested leaves. Mycorrhization adjusted the lipid composition, the abundance of peptides and, especially upon herbivory, the level of various phenolic compounds. The greatest change in leaf volatile organic compound (VOC) emissions occurred four to eight days following the beetle infestation. Together, these results prove that mycorrhization affects the whole plant metabolome and may influence poplar aboveground interactions. The herbivores and the mycorrhizal fungi interact with each other indirectly through a common host plant, a result that emphasizes the importance of community approach in chemical ecology.

Mycorrhiza-Triggered Transcriptomic and Metabolomic Networks Impinge on Herbivore Fitness.

Symbioses between plants and mycorrhizal fungi are ubiquitous in ecosystems and strengthen the plants' defense against aboveground herbivores. Here, we studied the underlying regulatory networks and biochemical mechanisms in leaves induced by ectomycorrhizae that modify herbivore interactions. Feeding damage and oviposition by the widespread poplar leaf beetle Chrysomela populi were reduced on the ectomycorrhizal hybrid poplar Populus × canescens Integration of transcriptomics, metabolomics, and volatile emission patterns via mass difference networks demonstrated changes in nitrogen allocation in the leaves of mycorrhizal poplars, down-regulation of phenolic pathways, and up-regulation of defensive systems, including protease inhibitors, chitinases, and aldoxime biosynthesis. Ectomycorrhizae had a systemic influence on jasmonate-related signaling transcripts. Our results suggest that ectomycorrhizae prime wounding responses and shift resources from constitutive phenol-based to specialized protective compounds. Consequently, symbiosis with ectomycorrhizal fungi enabled poplars to respond to leaf beetle feeding with a more effective arsenal of defense mechanisms compared with nonmycorrhizal poplars, thus demonstrating the importance of belowground plant-microbe associations in mitigating aboveground biotic stress.

Characterization of poplar metabotypes via mass difference enrichment analysis.

Instrumentation technology for metabolomics has advanced drastically in recent years in terms of sensitivity and specificity. Despite these technical advances, data analytical strategies are still in their infancy in comparison with other 'omics'. Plants are known to possess an immense diversity of secondary metabolites. Typically, more than 70% of metabolomics data are not amenable to systems biological interpretation because of poor database coverage. Here, we propose a new general strategy for mass-spectrometry-based metabolomics that incorporates all exact mass features with known sum formulas into the evaluation and interpretation of metabolomics studies. We extend the use of mass differences, commonly used for feature annotation, by redefining them as variables that reflect the remaining 'omic' domains. The strategy uses exact mass difference network analyses exemplified for the metabolomic description of two grey poplar (Populus × canescens) genotypes that differ in their capability to emit isoprene. This strategy established a direct connection between the metabotype and the non-isoprene-emitting phenotype, as mass differences pertaining to prenylation reactions were over-represented in non-isoprene-emitting poplars. Not only was the analysis of mass differences able to grasp the known chemical biology of poplar, but it also improved the interpretability of yet unknown biochemical relationships.

Isoprene emission by poplar is not important for the feeding behaviour of poplar leaf beetles.

BACKGROUND: Chrysomela populi (poplar leaf beetle) is a common herbivore in poplar plantations whose infestation causes major economic losses. Because plant volatiles act as infochemicals, we tested whether isoprene, the main volatile organic compound (VOC) produced by poplars (Populus x canescens), affects the performance of C. populi employing isoprene emitting (IE) and transgenic isoprene non-emitting (NE) plants. Our hypothesis was that isoprene is sensed and affects beetle orientation or that the lack of isoprene affects plant VOC profiles and metabolome with consequences for C. populi feeding. RESULTS: Electroantennographic analysis revealed that C. populi can detect higher terpenes, but not isoprene. In accordance to the inability to detect isoprene, C. populi showed no clear preference for IE or NE poplar genotypes in the choice experiments, however, the beetles consumed a little bit less leaf mass and laid fewer eggs on NE poplar trees in field experiments. Slight differences in the profiles of volatile terpenoids between IE and NE genotypes were detected by gas chromatography - mass spectrometry. Non-targeted metabolomics analysis by Fourier Transform Ion Cyclotron Resonance Mass Spectrometer revealed genotype-, time- and herbivore feeding-dependent metabolic changes both in the infested and adjacent undamaged leaves under field conditions. CONCLUSIONS: We show for the first time that C. populi is unable to sense isoprene. The detected minor differences in insect feeding in choice experiments and field bioassays may be related to the revealed changes in leaf volatile emission and metabolite composition between the IE and NE poplars. Overall our results indicate that lacking isoprene emission is of minor importance for C. populi herbivory under natural conditions, and that the lack of isoprene is not expected to change the economic losses in poplar plantations caused by C. populi infestation.

UV-B mediated metabolic rearrangements in poplar revealed by non-targeted metabolomics.

Plants have to cope with various abiotic stresses including UV-B radiation (280-315 nm). UV-B radiation is perceived by a photoreceptor, triggers morphological responses and primes plant defence mechanisms such as antioxidant levels, photoreapir or accumulation of UV-B screening pigments. As poplar is an important model system for trees, we elucidated the influence of UV-B on overall metabolite patterns in poplar leaves grown under high UV-B radiation. Combining non-targeted metabolomics with gas exchange analysis and confocal microscopy, we aimed understanding how UV-B radiation triggers metabolome-wide changes, affects isoprene emission, photosynthetic performance, epidermal light attenuation and finally how isoprene-free poplars adjust their metabolome under UV-B radiation. Exposure to UV-B radiation caused a comprehensive rearrangement of the leaf metabolome. Several hundreds of metabolites were up- and down-regulated over various pathways. Our analysis, revealed the up-regulation of flavonoids, anthocyanins and polyphenols and the down-regulation of phenolic precursors in the first 36 h of UV-B treatment. We also observed a down-regulation of steroids after 12 h. The accumulation of phenolic compounds leads to a reduced light transmission in UV-B-exposed plants. However, the accumulation of phenolic compounds was reduced in non-isoprene-emitting plants suggesting a metabolic- or signalling-based interaction between isoprenoid and phenolic pathways.