Alexander Weinhold

Molecular Interactions between the Specialist Herbivore Manduca sexta (Lepidoptera, Sphigidae) and Its Natural Host Nicotiana attenuata. VIII. An Unbiased GCxGC-ToFMS Analysis of the Plant's Elicited Volatile Emissions

Treating wounds in Nicotiana attenuata leaves with Manduca sexta oral secretions (W+OS) mimics most changes elicited by M. sexta herbivory, but an unbiased analysis of the effect of the different OS constituents on volatile emissions is lacking. We used two-dimensional gas chromatography/time-of-flight (GCxGC-ToF) mass spectrometry combined with multivariate statistics to parse volatiles into regulatory patterns. Volatiles released by wounding alone and by the alkalinity of OS were assessed by applying a buffer known to mimic the pH-mediated changes of OS elicitation (pectin methyl esterase activation and methanol release). The activities of fatty acid amino acid conjugates, well-known elicitors of antiherbivore defenses, and of 2-hydroxyoctadecatrienoic acid, a newly discovered signal in OS, were determined. Approximately 400 analytes were detected after deconvolution and alignment of GCxGC data; 35 volatiles were significantly regulated upon W+OS. Two-thirds of these were specifically regulated by OS, being either amplified (most terpenoids and certain hexenylesters) or strongly repressed (many short-chain alcohols and some aromatic and hexenylester derivatives). Fatty acid amino acid conjugates played a central role in this pattern of regulation, since they induced the emission of half of OS-elicited volatiles and inhibited the production of almost all OS-repressed volatiles; 2-hydroxyoctadecatrienoic acid influenced emission of trans-α-bergamotene, while other unknown OS constituents amplified hexenylester production. We conclude that the complex bouquet of herbivory-elicited volatiles results from the complex modulations of the wound response by diverse cues found in OS. This work also underscores the value of ultra-high-resolution GCxGC-ToF analysis combined with the nontargeted mining of the resulting data.

Trichome-derived O-acyl sugars are a first meal for caterpillars that tags them for predation

Plant glandular trichomes exude secondary metabolites with defensive functions, but these epidermal protuberances are surprisingly the first meal of Lepidopteran herbivores on Nicotiana attenuata. O-acyl sugars, the most abundant metabolite of glandular trichomes, impart a distinct volatile profile to the body and frass of larvae that feed on them. The headspace composition of Manduca sexta larvae is dominated by the branched chain aliphatic acids hydrolyzed from ingested O-acyl sugars, which waxes and wanes rapidly with trichome ingestion. In native habitats a ground-hunting predator, the omnivorous ant Pogonomyrmex rugosus, but not the big-eyed bug Geocoris spp., use these volatile aliphatic acids to locate their prey.

O-Acyl Sugars Protect a Wild Tobacco from Both Native Fungal Pathogens and a Specialist Herbivore

O-Acyl sugars, trichome-specific metabolites, function as direct defenses against native pathogens and herbivores of a wild tobacco. O-Acyl sugars (O-AS) are abundant trichome-specific metabolites that function as indirect defenses against herbivores of the wild tobacco Nicotiana attenuata; whether they also function as generalized direct defenses against herbivores and pathogens remains unknown. We characterized natural variation in O-AS among 26 accessions and examined their influence on two native fungal pathogens, Fusarium brachygibbosum U4 and Alternaria sp. U10, and the specialist herbivore Manduca sexta. At least 15 different O-AS structures belonging to three classes were found in N. attenuata leaves. A 3-fold quantitative variation in total leaf O-AS was found among the natural accessions. Experiments with natural accessions and crosses between high- and low-O-AS accessions revealed that total O-AS levels were associated with resistance against herbivores and pathogens. Removing O-AS from the leaf surface increased M. sexta growth rate and plant fungal susceptibility. O-AS supplementation in artificial diets and germination medium reduced M. sexta growth and fungal spore germination, respectively. Finally, silencing the expression of a putative branched-chain α-ketoacid dehydrogenase E1 β-subunit-encoding gene (NaBCKDE1B) in the trichomes reduced total leaf O-AS by 20% to 30% and increased susceptibility to Fusarium pathogens. We conclude that O-AS function as direct defenses to protect plants from attack by both native pathogenic fungi and a specialist herbivore and infer that their diversification is likely shaped by the functional interactions among these biotic stresses.

High herbivore pressure favors constitutive over induced defense

Abstract Theoretical and empirical studies show that, when past or current herbivory is a reliable cue of future attack and defenses are costly, defenses can be induced only when needed and thereby permit investment in other functions such as growth or reproduction. Theory also states that, in environments where herbivory is constantly high, constitutive defenses should be favored. Here, we present data to support the second aspect of the induced resistance hypothesis. We examined herbivore‐induced responses for four species of Inga (Fabaceae), a common canopy tree in Neotropical forests. We quantified chemical defenses of expanding leaves, including phenolic, saponin and toxic amino acids, in experimental field treatments with and without caterpillars. Because young leaves lack fiber and are higher in protein than mature leaves, they typically lose >25% of their leaf area during the few weeks of expansion. We predicted that the high rates of attack would select for investment in constitutive defenses over induction. Our data show that chemical defenses were quite unresponsive to herbivory. We demonstrated that expanding leaves showed no or only small increases in investment in secondary metabolites, and no qualitative changes in the phenolic compound profile in response to herbivory. The proteinogenic amino acid tyrosine, which can be toxic at high concentrations, showed the greatest levels of induction. Synthesis: These results provide some of the first support for theoretical predictions that the evolution of induced vs. constitutive defenses depends on the risk of herbivory. In habitats with constant and high potential losses to herbivores, such as tropical rainforests, high investments in constitutive defenses are favored over induction.

Phaseoloidin, a Homogentisic Acid Glucoside from Nicotiana Attenuata Trichomes, Contributes to the Plant’s Resistance against Lepidopteran Herbivores

Plant trichomes are known for their capability to produce and store secondary metabolites that protect plants from biotic and abiotic stresses. 1H NMR studies on intact individual trichomes located on the leaf surface of Nicotiana attenuata revealed the presence of two major secondary metabolites: nicotine, the signature metabolite of the genus, and phaseoloidin, a homogentisic acid glucoside. This glucoside was reported originally from the seeds of Entada phaseoloides, and this is the first report of its occurrence in a Solanaceous plant. Artificial diet feeding bioassays with Manduca sexta and Spodoptera littoralis larvae, two important herbivores of N. attenuata, revealed that the ingestion of phaseoloidin negatively influenced caterpillar performance. This effect was more pronounced for the generalist, S. littoralis, than for the specialists, M. sexta.

Trichomes as dangerous lollipops: do lizards also use caterpillar body and frass odor to optimize their foraging?

When attacked by herbivores, plants produce toxic secondary metabolites that function as direct defenses, as well as indirect defenses that attract and reward predators of the offending herbivores. These indirect defenses include both nutritive rewards such as extra floral nectar, as well as informational rewards, such as the production and release of volatile compounds that betray the location of feeding herbivores to predators. Herbivory of Nicotiana attenuata by the tobacco hornworm (Manduca larvae) alters the volatile profiles of both the plant and larval headspace. Herbivory-elicited specific changes in the volatile profiles are detected by arthropod predators of Manduca larvae. The known predators that perceive volatile cues induced by Manduca herbivory of N. attenuata are insects that target Manduca at early developmental stages, when the larvae are still small; large, late-instar larvae may have outgrown these predation risks. However, here we offer evidence that branched chain aliphatic acids derived from the digestion of plant O-acyl sugars from trichomes may betray Manduca larvae to lizard predators during late developmental stages as well.

Glycoalkaloid composition explains variation in slug resistance in Solanum dulcamara

In natural environments, plants have to deal with a wide range of different herbivores whose communities vary in time and space. It is believed that the chemical diversity within plant species has mainly arisen from selection pressures exerted by herbivores. So far, the effects of chemical diversity on plant resistance have mostly been assessed for arthropod herbivores. However, also gastropods, such as slugs, can cause extensive damage to plants. Here we investigate to what extent individual Solanum dulcamara plants differ in their resistance to slug herbivory and whether this variation can be explained by differences in secondary metabolites. We performed a series of preference assays using the grey field slug (Deroceras reticulatum) and S. dulcamara accessions from eight geographically distinct populations from the Netherlands. Significant and consistent variation in slug preference was found for individual accessions within and among populations. Metabolomic analyses showed that variation in steroidal glycoalkaloids (GAs) correlated with slug preference; accessions with high GA levels were consistently less damaged by slugs. One, strongly preferred, accession with particularly low GA levels contained high levels of structurally related steroidal compounds. These were conjugated with uronic acid instead of the glycoside moieties common for Solanum GAs. Our results illustrate how intraspecific variation in steroidal glycoside profiles affects resistance to slug feeding. This suggests that also slugs should be considered as important drivers in the co-evolution between plants and herbivores.

Current Challenges in Plant Eco-Metabolomics

The relatively new research discipline of Eco-Metabolomics is the application of metabolomics techniques to ecology with the aim to characterise biochemical interactions of organisms across different spatial and temporal scales. Metabolomics is an untargeted biochemical approach to measure many thousands of metabolites in different species, including plants and animals. Changes in metabolite concentrations can provide mechanistic evidence for biochemical processes that are relevant at ecological scales. These include physiological, phenotypic and morphological responses of plants and communities to environmental changes and also interactions with other organisms. Traditionally, research in biochemistry and ecology comes from two different directions and is performed at distinct spatiotemporal scales. Biochemical studies most often focus on intrinsic processes in individuals at physiological and cellular scales. Generally, they take a bottom-up approach scaling up cellular processes from spatiotemporally fine to coarser scales. Ecological studies usually focus on extrinsic processes acting upon organisms at population and community scales and typically study top-down and bottom-up processes in combination. Eco-Metabolomics is a transdisciplinary research discipline that links biochemistry and ecology and connects the distinct spatiotemporal scales. In this review, we focus on approaches to study chemical and biochemical interactions of plants at various ecological levels, mainly plant–organismal interactions, and discuss related examples from other domains. We present recent developments and highlight advancements in Eco-Metabolomics over the last decade from various angles. We further address the five key challenges: (1) complex experimental designs and large variation of metabolite profiles; (2) feature extraction; (3) metabolite identification; (4) statistical analyses; and (5) bioinformatics software tools and workflows. The presented solutions to these challenges will advance connecting the distinct spatiotemporal scales and bridging biochemistry and ecology.

Gastropods and Insects Prefer Different Solanum dulcamara Chemotypes

Solanum dulcamara (Bittersweet nightshade) shows significant intraspecific variation in glycoalkaloid (GA) composition and concentration. We previously showed that constitutive differences in overall GA levels are correlated with feeding preference of the grey field slug (GFS; Deroceras reticulatum). One particularly preferred accession, ZD11, contained low GA levels, but high levels of previously unknown structurally related uronic acid conjugated compounds (UACs). Here we test whether different slug species as well as insect herbivores show similar feeding preferences among six S. dulcamara accessions with different GA chemotypes. In addition, we investigate whether slug feeding can lead to induced changes in the chemical composition and affect later arriving herbivores. A leaf disc assay using greenhouse-grown plants showed that three slug species similarly preferred accessions with low GA levels. Untargeted metabolomic analyses showed that previous slug feeding consistently increased the levels of N-caffeoyl-putrescine and a structurally related metabolite, but not the levels of GAs and UACs. Slug-induced responses only affected slug preference in one accession. A common garden experiment using the same six accessions revealed that ZD11 received the highest natural gastropod feeding damage, but suffered the lowest damage by specialist flea beetles. The latter preferred to feed on accessions with high GA levels. Our study indicates that different selection pressures imposed by generalist gastropods and specialist insects may explain part of the observed chemical diversity in S. dulcamara.

New perspectives on CO2, temperature and light effects on BVOC emissions using online measurements by PTR-MS and cavity ring-down spectroscopy

Volatile organic compounds (VOC) play important roles in atmospheric chemistry, plant ecology, and physiology, and biogenic VOC (BVOC) emitted by plants is the largest VOC source. Our knowledge about how environmental drivers (e.g., carbon, light, and temperature) may regulate BVOC emissions is limited because they are often not controlled. We combined a greenhouse facility to manipulate atmospheric CO2 ([CO2]) with proton-transfer-reaction mass spectrometry (PTR-MS) and cavity ring-down spectroscopy to investigate the regulation of BVOC in Norway spruce. Our results indicate a direct relationship between [CO2] and methanol and acetone emissions, and their temperature and light dependencies, possibly related to substrate availability. The composition of monoterpenes stored in needles remained constant, but emissions of mono-(linalool) and sesquiterpenes (β-farnesene) increased at lower [CO2], with the effects being most pronounced at the highest air temperature. Pulse-labeling suggested an immediate incorporation of recently assimilated carbon into acetone, mono- and sesquiterpene emissions even under 50 ppm [CO2]. Our results provide new perspectives on CO2, temperature and light effects on BVOC emissions, in particular how they depend on stored pools and recent photosynthetic products. Future studies using smaller but more seedlings may allow sufficient replication to examine the physiological mechanisms behind the BVOC responses.