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Glandular trichomes (GTs) are epidermal structures that provide the first line of chemical defense against arthropod herbivores and other biotic threats. The most conspicuous structure on leaves of cultivated tomato (Solanum lycopersicum) is the type-VI GT (tVI-GT), which accumulates both flavonoids and volatile terpenoids. Although these classes of specialized metabolites are derived from distinct metabolic pathways, previous studies with a chalcone isomerase 1 (CHI1)-deficient mutant called anthocyanin free (af) showed that flavonoids are required for terpenoid accumulation in tVI-GTs. Here, we combined global transcriptomic and proteomic analyses of isolated trichomes as a starting point to show that the lack of CHI1 is associated with reduced levels of terpenoid biosynthetic transcripts and enzymes. The flavonoid deficiency in af trichomes also resulted in the upregulation of abiotic stress-responsive genes associated with DNA damage and repair. Several lines of biochemical and genetic evidence indicate that the terpenoid defect in af mutants is specific for the tVI-GT and is associated with the absence of bulk flavonoids rather than loss of CHI1 per se. A newly developed genome-scale model of metabolism in tomato tVI-GTs helped identify metabolic imbalances caused by the loss of flavonoid production. We provide evidence that flavonoid deficiency in this cell type leads to increased production of reactive oxygen species (ROS), which may impair terpenoid biosynthesis. Collectively, our findings support a role for flavonoids as ROS-scavenging antioxidants in GTs.

Following decades of tight restrictions, recent legislative adjustments have decriminalized the use of products derived from cannabis (Cannabis sativa L.) in many countries and jurisdictions. This has led to a renewed interest in better understanding the chemical basis of physiological effects attributed to cannabis use. The present review article summarizes our current knowledge regarding the 130 structures of cannabinoids that have been characterized from cannabis extracts to date. We are also providing information on the methods employed for structure determination to help the reader assess the quality of the original structural assignments. Cannabinoid chemical diversity is discussed in the context of current knowledge regarding the enzymes involved in cannabinoid biosynthesis. We briefly assess to what extent cannabinoid levels are determined by the genotype of a given chemovar and discuss the limits of enzymatic control over the cannabinoid profile.

Monoterpene synthases catalyze the first committed step in the biosynthesis of monoterpenes and are in part responsible for the enormous structural diversity among this class of metabolites. Here, we explore the structure-function relationships underlying the formation of limonene enantiomers in limonene synthases that bind geranyl diphosphate as a common substrate. On the basis of analyses that consider both crystal structure data and amino acid sequence divergence, we identified candidate active site residues with potential roles in catalyzing reactions that involve accommodating reaction intermediates of opposite enantiomeric series. We demonstrate that spearmint (-)-limonene synthase [which generates99% (-)-limonene over (+)-limonene] can be converted into a mutant enzyme, by exchanging four residues (C321S, N345I, I453V, and M458V), which produces (+)-limonene with reversed enantiospecificity [80% (+)-limonene and 3% (-)-limonene; the remainder are mostly bicyclic monoterpenes]. This study provides the foundation for a more in-depth understanding of the formation of enantiomeric series of monoterpenes, which can have vastly different olfactory properties.

Monoterpenoids are the main components of plant essential oils and the active components of some traditional Chinese medicinal herbs like Mentha haplocalyx Briq., Nepeta tenuifolia Briq., Perilla frutescens (L.) Britt and Pogostemin cablin (Blanco) Benth. Pulegone reductase is the key enzyme in the biosynthesis of menthol and is required for the stereoselective reduction of the Δ2,8 double bond of pulegone to produce the major intermediate menthone, thus determining the stereochemistry of menthol. However, the structural basis and mechanism underlying the stereoselectivity of pulegone reductase remain poorly understood. In this study, we characterized a novel (−)-pulegone reductase from Nepeta tenuifolia (NtPR), which can catalyze (−)-pulegone to (+)-menthone and (−)-isomenthone through our RNA-seq, bioinformatic analysis in combination with in vitro enzyme activity assay, and determined the structure of (+)-pulegone reductase from M. piperita (MpPR) by using X-ray crystallography, molecular modeling and docking, site-directed mutagenesis, molecular dynamics simulations, and biochemical analysis. We identified and validated the critical residues in the crystal structure of MpPR involved in the binding of the substrate pulegone. We also further identified that residues Leu56, Val282, and Val284 determine the stereoselectivity of the substrate pulegone, and mainly contributes to the product stereoselectivity. This work not only provides a starting point for the understanding of stereoselectivity of pulegone reductases, but also offers a basis for the engineering of menthone/menthol biosynthetic enzymes to achieve high-titer, industrial-scale production of enantiomerically pure products.

Peppermint (Mentha x piperita L.) and Japanese catnip (Schizonepeta tenuifolia (Benth.) Briq.) accumulate p-menthane monoterpenoids with identical functionalization patterns but opposite stereochemistry. In the present study, we investigate the enantioselectivity of multiple enzymes involved in monoterpenoid biosynthesis in these species. Based on kinetic assays, mint limonene synthase, limonene 3-hydroxylase, isopiperitenol dehydrogenase, isopiperitenone reductase, and menthone reductase exhibited significant enantioselectivity toward intermediates of the pathway that proceeds through (-)-4S-limonene. Limonene synthase, isopiperitenol dehydrogenase and isopiperitenone reductase of Japanese catnip preferred intermediates of the pathway that involves (+)-4R-limonene, whereas limonene 3-hydroxylase was not enantioselective, and the activities of pulegone reductase and menthone reductase were too low to acquire meaningful kinetic data. Molecular modeling studies with docked ligands generally supported the experimental data obtained with peppermint enzymes, indicating that the preferred enantiomer was aligned well with the requisite cofactor and amino acid residues implicated in catalysis. A striking example for enantioselectivity was peppermint (-)-menthone reductase, which binds (-)-menthone with exquisite affinity but was predicted to bind (+)-menthone in a non-productive orientation that positions its carbonyl functional group at considerable distance to the NADPH cofactor. The work presented here lays the groundwork for structure-function studies aimed at unraveling how enantioselectivity evolved in closely related species of the Lamiaceae and beyond.

Glandular trichomes are specialized anatomical structures that accumulate secretions with important biological roles in plant-environment interactions. These secretions also have commercial uses in the flavor, fragrance, and pharmaceutical industries. The capitate-stalked glandular trichomes of Cannabis sativa (cannabis), situated on the surfaces of the bracts of the female flowers, are the primary site for the biosynthesis and storage of resins rich in cannabinoids and terpenoids. In this study, we profiled nine commercial cannabis strains with purportedly different attributes, such as taste, color, smell, and genetic origin. Glandular trichomes were isolated from each of these strains, and cell type-specific transcriptome data sets were acquired. Cannabinoids and terpenoids were quantified in flower buds. Statistical analyses indicated that these data sets enable the high-resolution differentiation of strains by providing complementary information. Integrative analyses revealed a coexpression network of genes involved in the biosynthesis of both cannabinoids and terpenoids from imported precursors. Terpene synthase genes involved in the biosynthesis of the major monoterpenes and sesquiterpenes routinely assayed by cannabis testing laboratories were identified and functionally evaluated. In addition to cloning variants of previously characterized genes, specifically CsTPS14CT [(−)-limonene synthase] and CsTPS15CT (β-myrcene synthase), we functionally evaluated genes that encode enzymes with activities not previously described in cannabis, namely CsTPS18VF and CsTPS19BL (nerolidol/linalool synthases), CsTPS16CC (germacrene B synthase), and CsTPS20CT (hedycaryol synthase). This study lays the groundwork for developing a better understanding of the complex chemistry and biochemistry underlying resin accumulation across commercial cannabis strains.

The Mentha germplasm collection housed at the USDA National Clonal Germplasm Repository is a valuable source of diversity for genetic studies and mint breeding. We surveyed phenotypes and genotypes of accessions belonging to two species ancestral to commercial peppermint: M. aquatica and M. suaveolens. Morphology, ploidy, essential oil composition, and relative Verticillium wilt resistance were assessed. Genotyping with simple sequence repeat (SSR) markers was performed in order to establish a set of informative markers for distinguishing accessions from each other. M. suaveolens accessions were triploid or tetraploid, while M. aquatica accessions were octoploid or nonaploid. Holoploid genome sizes differed significantly among accessions within both species. Half of the M. aquatica accessions had (+)-menthofuran as the primary oil constituent, while other accessions showed atypical oil profiles. Most M. suaveolens accessions had high levels of either piperitenone oxide, (−)-carvone, or trans-piperitenone oxide. M. aquatica accessions showed a range of Verticillium wilt resistance to susceptibility, while most M. suaveolens accessions were highly wilt-resistant. Results from genotyping the accessions with nine SSR markers distinguished three groups: one mainly M. suaveolens, one mostly M. aquatica, and one with a mixture of the species. This study enables updates of accession descriptions in the Germplasm Resources Information Network database, and increases the utility of the Mentha collection to the research community.

Land-use intensification is a major driver of biodiversity loss. However, understanding how different components of land use drive biodiversity loss requires the investigation of multiple trophic levels across spatial scales. Using data from 150 agricultural grasslands in central Europe, we assess the influence of multiple components of local- and landscape-level land use on more than 4,000 above- and belowground taxa, spanning 20 trophic groups. Plot-level land-use intensity is strongly and negatively associated with aboveground trophic groups, but positively or not associated with belowground trophic groups. Meanwhile, both above- and belowground trophic groups respond to landscape-level land use, but to different drivers: aboveground diversity of grasslands is promoted by diverse surrounding land-cover, while belowground diversity is positively related to a high permanent forest cover in the surrounding landscape. These results highlight a role of landscape-level land use in shaping belowground communities, and suggest that revised agroecosystem management strategies are needed to conserve whole-ecosystem biodiversity., Nature Communications, 12 (1), ISSN:2041-1723

Mentha is a strongly scented herb of the Lamiaceae (formerly Labiatae) and includes about 30 species and hybrid species that are distributed or introduced throughout the globe. These fragrant plants have been selected throughout millennia for use by humans as herbs, spices, and pharmaceutical needs. The distilling of essential oils from mint began in Japan and England but has become a significant industrial product for the US, China, India, and other countries. The US Department of Agriculture (USDA), Agricultural Research Service, National Clonal Germplasm Repository (NCGR) maintains a mint genebank in Corvallis, Oregon. This facility preserves and distributes about 450 clones representing 34 taxa, hybrid species, advanced breeder selections, and F1 hybrids. Mint crop wild relatives are included in this unique resource. The majority of mint accessions and hybrids in this collection were initially donated in the 1970s by the A.M. Todd Company, located in Kalamazoo, Michigan. Other representatives of diverse mint taxa and crop wild relatives have since been obtained from collaborators in Australia, New Zealand, Europe, and Vietnam. These mints have been evaluated for cytology, oil components, verticillium wilt resistance, and key morphological characters. Pressed voucher specimens have been prepared for morphological identity verification. An initial set of microsatellite markers has been developed to determine clonal identity and assess genetic diversity. Plant breeders at private and public institutions are using molecular analysis to determine identity and diversity of the USDA mint collection. Evaluation and characterization includes essential oil content, disease resistance, male sterility, and other traits for potential breeding use. These accessions can be a source for parental genes for enhancement efforts to produce hybrids, or for breeding new cultivars for agricultural production. Propagules of Mentha are available for distribution to international researchers as stem cuttings, rhizome cuttings, or seed, which can be requested through the GRIN-Global database of the US National Plant Germplasm System, subject to international treaty and quarantine regulations.

The shoot system of all loblolly pine (Pinus taeda L.) contains abundant resin ducts, and the oleoresins contained within them have demonstrated roles in constitutive defenses. This study is providing a quantitative assessment of oleoresin biosynthesis and accumulation in resin ducts. Morphometric analyses of representative tissue sections indicate that the fractional volume of resin ducts is particularly high in the cortex of young stems and their needles, representing a major portion of total pine resins from primary growth of the canopy. We demonstrate that it is possible to extrapolate oleoresin formation from the microscopic scale (tissues sections) to the macroscale (entire trees), which has implications for assessing resins as renewable feedstocks for bioproducts.

Sterols serve essential functions as membrane constituents and hormones (brassinosteroids) in plants, while non-sterol triterpenoids have been implicated in defense responses. Surprisingly little is known about the sterol and triterpene profiles in different plant organs. To enhance our understanding of organ-specific sterol and triterpene accumulation, we quantified these metabolite classes in four different organs (root, stem, leaf, seed) ofArabidopsis thaliana(L.). Based on these data sets we developed kinetic mathematical models of sterol biosynthesis to capture flux distribution and pathway regulation in different organs. Simulations indicated that an increased flux through the sterol pathway would not only result in an increase of sterol end products but also a concomitant build-up of certain intermediates. These computational predictions turner out to be consistent with experimental data obtained with transgenic plants ectopically overexpressing 3-hydroxy-3-methylglutary-coenzyme A reductase (HMG1gene). The opportunities and limitations of incorporating mathematical modeling into the design of approaches to engineer sterol biosynthesis are discussed.

Taxol (paclitaxel) is a very widely used anticancer drug, but its commercial sources mainly consist of stripped bark or suspension cultures of members of the plant genus Taxus. Taxol accumulates as part of a complex mixture of chemical analogs, termed taxoids, which complicates its production in pure form, highlighting the need for metabolic engineering approaches for high-level Taxol production in cell cultures or microbial hosts. Here, we report on the characterization of acyl-activating enzymes (AAEs) that catalyze the formation of CoA esters of different organic acids relevant for the N-substitution of the 3-phenylisoserine side chain of taxoids. On the basis of similarities to AAE genes of known function from other organisms, we identified candidate genes in publicly available transcriptome data sets obtained with Taxus × media. We cloned 17 AAE genes, expressed them heterologously in Escherichia coli, purified the corresponding recombinant enzymes, and performed in vitro assays with 27 organic acids as potential substrates. We identified TmAAE1 and TmAAE5 as the most efficient enzymes for the activation of butyric acid (Taxol D side chain), TmAAE13 as the best candidate for generating a CoA ester of tiglic acid (Taxol B side chain), TmAAE3 and TmAAE13 as suitable for the activation of 4-methylbutyric acid (N-debenzoyl-N-(2-methylbutyryl)taxol side chain), TmAAE15 as a highly efficient candidate for hexanoic acid activation (Taxol C side chain), and TmAAE4 as suitable candidate for esterification of benzoic acid with CoA (Taxol side chain). This study lays important groundwork for metabolic engineering efforts aimed at improving Taxol production in cell cultures.

Many aromatic plants accumulate mixtures of secondary (or specialized) metabolites in anatomical structures called glandular trichomes (GTs). Different GT types may also synthesize different mixtures of secreted metabolites, and this contributes to the enormous chemical diversity reported to occur across species. Over the past two decades, significant progress has been made in characterizing the genes and enzymes that are responsible for the unique metabolic capabilities of GTs in different lineages of flowering plants. Less is known about the processes that regulate flux distribution through precursor pathways toward metabolic end-products. We discuss here the results from a meta-analysis of genome-scale models that were developed to capture the unique metabolic capabilities of different GT types.

Biodiversity-ecosystem function experiments test how species diversity influences fundamental ecosystem processes. Historically, arthropod driven functions, such as herbivory and pest-control, have been thought to be influenced by direct and indirect associations among species. Although a number of studies have evaluated how plant diversity affects arthropod communities and arthropod-mediated ecosystem processes, it remains unclear whether diversity effects on arthropods are sufficiently consistent over time such that observed responses can be adequately predicted by classical hypotheses based on associational effects. By combining existing results from a long-term grassland biodiversity experiment (Jena Experiment) with new analyses, we evaluate the consistency of consumer responses within and across taxonomic, trophic, and trait-based (i.e. vertical stratification) groupings, and we consider which changes in arthropod community composition are associated with changes in consumer-mediated ecosystem functions. Overall, higher plant species richness supported more diverse and complex arthropod communities and this pattern was consistent across multiple years. Vegetation-associated arthropods responded more strongly to changes in plant species richness than ground-dwelling arthropods. Additionally, increases in plant species richness were associated with shifts in the species-abundance distributions for many, but not all taxa. For example, highly specialized consumers showed a decrease in dominance and an increase in the number of rare species with increasing plant species richness. Most ecosystem processes investigated responded to increases in plant species richness in the same way as the trophic group mediating the process, e.g. both herbivory and herbivore diversity increase with increasing plant species richness. In the Jena Experiment and other studies, inconsistencies between predictions based on classic hypotheses of associational effects and observed relationships between plant species richness and arthropod diversity likely reflect the influence of multi-trophic community dynamics and species functional trait distributions. Future research should focus on testing a broader array of mechanisms to unravel the biological processes underlying the biodiversity-ecosystem functioning relationships.

The pseudoalkaloid diterpene Taxol® (paclitaxel) emerged as a best-selling anti-cancer drug in the mid-1990s. The compound attracted considerable interest because of its unique mechanism to stabilize microtubules, thus reducing dynamicity and ultimately promoting mitotic arrest. Taxol was originally isolated from members of the genus Taxus. Over the last 50 years, close to 600 metabolites with taxane scaffolds were isolated from various Taxus species and their structures reported. The present review article provides an overview of the known chemical diversity of taxanes, with an emphasis on the functionalization of diterpene scaffolds. The implications of the occurrence of chemically diverse taxane metabolites for unraveling Taxol biosynthesis and enabling pathway engineering are discussed as well.

Mountain grasslands have recently been exposed to substantial changes in land use and climate and in the near future will likely face an increased frequency of extreme droughts. To date, how the drought responses of carbon (C) allocation, a key process in the C cycle, are affected by land‐use changes in mountain grassland is not known.We performed an experimental summer drought on an abandoned grassland and a traditionally managed hay meadow and traced the fate of recent assimilates through the plant–soil continuum. We applied two 13 CO 2 pulses, at peak drought and in the recovery phase shortly after rewetting.Drought decreased total C uptake in both grassland types and led to a loss of above‐ground carbohydrate storage pools. The below‐ground C allocation to root sucrose was enhanced by drought, especially in the meadow, which also held larger root carbohydrate storage pools.The microbial community of the abandoned grassland comprised more saprotrophic fungal and Gram(+) bacterial markers compared to the meadow. Drought increased the newly introduced AM and saprotrophic (A+S) fungi:bacteria ratio in both grassland types. At peak drought, the 13C transfer into AM and saprotrophic fungi, and Gram(−) bacteria was more strongly reduced in the meadow than in the abandoned grassland, which contrasted the patterns of the root carbohydrate pools.In both grassland types, the C allocation largely recovered after rewetting. Slowest recovery was found for AM fungi and their 13C uptake. In contrast, all bacterial markers quickly recovered C uptake. In the meadow, where plant nitrate uptake was enhanced after drought, C uptake was even higher than in control plots. Synthesis. Our results suggest that resistance and resilience (i.e. recovery) of plant C dynamics and plant‐microbial interactions are negatively related, that is, high resistance is followed by slow recovery and vice versa. The abandoned grassland was more resistant to drought than the meadow and possibly had a stronger link to AM fungi that could have provided better access to water through the hyphal network. In contrast, meadow communities strongly reduced C allocation to storage and C transfer to the microbial community in the drought phase, but in the recovery phase invested C resources in the bacterial communities to gain more nutrients for regrowth. We conclude that the management of mountain grasslands increases their resilience to drought.

Changes to primary producer diversity can cascade up to consumers and affect ecosystem processes. Although the effect of producer diversity on higher trophic groups have been studied, these studies often quantify taxonomy-based measures of biodiversity, like species richness, which do not necessarily reflect the functioning of these communities. In this study, we assess how plant species richness affects the functional composition and diversity of higher trophic levels and discuss how this might affect ecosystem processes, such as herbivory, predation and decomposition. Based on six different consumer traits, we examined the functional composition of arthropod communities sampled in experimental plots that differed in plant species richness. The two components we focused on were functional variation in the consumer community structure (functional structure) and functional diversity, expressed as functional richness, evenness and divergence. We found a consistent positive effect of plant species richness on the functional richness of herbivores, carnivores, and omnivores, but not decomposers, and contrasting patterns for functional evenness and divergence. Increasing plant species richness shifted the omnivore community to more predatory and less mobile species, and the herbivore community to more specialized and smaller species. This was accompanied by a shift towards more species occurring in the vegetation than in the ground layer. Our study shows that plant species richness strongly affects the functional structure and diversity of aboveground arthropod communities. The observed shifts in body size (herbivores), specialization (herbivores), and feeding mode (omnivores) together with changes in the functional diversity may underlie previously observed increases in herbivory and predation in plant communities of higher diversity.

Adventitious root cultures were developed from Tripterygium regelii, and growth conditions were optimized for the abundant production of diterpenoids, which can be collected directly from the medium. An analysis of publicly available transcriptome data sets collected with T. regelii roots and root cultures indicated the presence of a large gene family (with 20 members) for terpene synthases (TPSs). Nine candidate diterpene synthase genes were selected for follow-up functional evaluation, of which two belonged to the TPS-c, three to the TPS-e/f, and four to the TPS-b subfamilies. These genes were characterized by heterologous expression in a modular metabolic engineering system in Escherichia coli Members of the TPS-c subfamily were characterized as copalyl diphosphate (diterpene) synthases, and those belonging to the TPS-e/f subfamily catalyzed the formation of precursors of kaurane diterpenoids. The TPS-b subfamily encompassed genes coding for enzymes involved in abietane diterpenoid biosynthesis and others with activities as monoterpene synthases. The structural characterization of diterpenoids accumulating in the medium of T. regelii adventitious root cultures, facilitated by searching the Spektraris online spectral database, enabled us to formulate a biosynthetic pathway for the biosynthesis of triptolide, a diterpenoid with pharmaceutical potential. Considering the significant enrichment of diterpenoids in the culture medium, fast-growing adventitious root cultures may hold promise as a sustainable resource for the large-scale production of triptolide.

1. For managed temperate forests, conservationists and policymakers favour fine-grained uneven-aged management over more traditional coarse-grained even-aged management, based on the assumption that within-stand habitat heterogeneity enhances biodiversity. There is, however, little empirical evidence to support this assumption. We investigated for the first time how differently grained forest management systems affect the biodiversity of multiple above- and below-ground taxa across spatial scales. 2. We sampled 15 taxa of animals, plants, fungi and bacteria within the largest contiguous beech forest landscape of Germany and classified them into functional groups. Selected forest stands have been managed for more than a century at different spatial grains. The even-aged (coarse-grained management) and uneven-aged (fine-grained) forests are comparable in spatial arrangement, climate and soil conditions. These were compared to forests of a nearby national park that have been unmanaged for at least 20 years. We used diversity accumulation curves to compare γ-diversity for Hill-numbers 0D (species richness), 1D (Shannon diversity) and 2D (Simpson diversity) between the management systems. Beta diversity was quantified as multiple-site dissimilarity. 3. Gamma diversity was higher in even-aged than in uneven-aged forests for at least one of the three Hillnumbers for six taxa (up to 77%), while eight showed no difference. Only bacteria showed the opposite pattern. Higher γ-diversity in even-aged forests was also found for forest specialists and saproxylic beetles. 4. Between-stand β-diversity was higher in even-aged than in uneven-aged forests for one third (all species) and half (forest specialists) of all taxa, driven by environmental heterogeneity between age-classes, while α-diversity showed no directional response across taxa or for forest specialists. 5. Synthesis and applications. Comparing even-aged and uneven-aged forest management in Central European beech forests, our results show that a mosaic of different age-classes is more important for regional biodiversity than high within-stand heterogeneity. We suggest reconsidering the current trend of replacing even-aged management in temperate forests. Instead, the variability of stages and stand structures should be increased to promote landscape scale biodiversity.

The genus Mentha encompasses mint species cultivated for their essential oils, which are formulated into a vast array of consumer products. Desirable oil characteristics and resistance to the fungal disease Verticillium wilt are top priorities for the mint industry. However, cultivated mints have complex polyploid genomes and are sterile. Breeding efforts, therefore, require the development of genomic resources for fertile mint species. Here, we present draft de novo genome and plastome assemblies for a wilt-resistant South African accession of Mentha longifolia (L.) Huds., a diploid species ancestral to cultivated peppermint and spearmint. The 353 Mb genome contains 35 597 predicted protein-coding genes, including 292 disease resistance gene homologs, and nine genes determining essential oil characteristics. A genetic linkage map ordered 1397 genome scaffolds on 12 pseudochromosomes. More than two million simple sequence repeats were identified, which will facilitate molecular marker development. The M. longifolia genome is a valuable resource for both metabolic engineering and molecular breeding. This is exemplified by employing the genome sequence to clone and functionally characterize the promoters in a peppermint cultivar, and demonstrating the utility of a glandular trichome-specific promoter to increase expression of a biosynthetic gene, thereby modulating essential oil composition.

Latent nucleophiles are compounds that are themselves not nucleophilic but can produce a strong nucleophile when activated. Such nucleophiles can expand the scope of Lewis base catalyzed reactions. As a proof of concept, we report that N-silyl pyrroles, indoles, and carbazoles serve as latent N-centered nucleophiles in substitution reactions of allylic fluorides catalyzed by Lewis bases. The reactions feature broad scopes for both reaction partners, excellent regioselectivities, and produce enantioenriched N-allyl pyrroles, indoles, and carbazoles when chiral cinchona alkaloid catalysts are used.

Many experiments have shown that loss of biodiversity reduces the capacity of ecosystems to provide the multiple services on which humans depend. However, experiments necessarily simplify the complexity of natural ecosystems and will normally control for other important drivers of ecosystem functioning, such as the environment or land use. In addition, existing studies typically focus on the diversity of single trophic groups, neglecting the fact that biodiversity loss occurs across many taxa and that the functional effects of any trophic group may depend on the abundance and diversity of others. Here we report analysis of the relationships between the species richness and abundance of nine trophic groups, including 4,600 above- and below-ground taxa, and 14 ecosystem services and functions and with their simultaneous provision (or multifunctionality) in 150 grasslands. We show that high species richness in multiple trophic groups (multitrophic richness) had stronger positive effects on ecosystem services than richness in any individual trophic group; this includes plant species richness, the most widely used measure of biodiversity. On average, three trophic groups influenced each ecosystem service, with each trophic group influencing at least one service. Multitrophic richness was particularly beneficial for 'regulating' and 'cultural' services, and for multifunctionality, whereas a change in the total abundance of species or biomass in multiple trophic groups (the multitrophic abundance) positively affected supporting services. Multitrophic richness and abundance drove ecosystem functioning as strongly as abiotic conditions and land-use intensity, extending previous experimental results to real-world ecosystems. Primary producers, herbivorous insects and microbial decomposers seem to be particularly important drivers of ecosystem functioning, as shown by the strong and frequent positive associations of their richness or abundance with multiple ecosystem services. Our results show that multitrophic richness and abundance support ecosystem functioning, and demonstrate that a focus on single groups has led to researchers to greatly underestimate the functional importance of biodiversity.

Plant diversity positively impacts ecosystem services such as biomass production and soil organic matter (SOM) storage. Both processes counteract increasing atmospheric CO 2 concentration and global warming and consequently need better understanding. In general it is assumed that complementary resource use is driving the positive biomass effect and that the rhizospheric microbial community provides the necessary nutrients mineralizing SOM. So far however, it remains unclear how this link between the above and the belowground system is functioning; in detail it remains unclear if a more efficient CO 2 uptake at higher diversity levels leads to higher root exudation that stimulate the microbial mineralization. Contrastingly we show here for the first time that more diverse grassland communities provide a better access to root exudates for the rhizospheric community. We applied a continuous 13 CO 2 label in a controlled environment (The Montpellier European Ecotron) to ecosystem monoliths from the long-term The Jena Experiment and showed analyzing the δ 13 C content of phospholipid fatty acids and neutral lipid fatty acid that plant diversity increased the plant-derived C uptake of Gram negative bacteria and arbuscular mycorrhizal fungi (AMF). Root biomass but not the amount and δ 13 C content of root sugars positively influenced the plant diversity effect observed on Gram negative bacteria whereas the specific interaction between plant and AMF was independent from any plant trait. Our results demonstrate that plant diversity facilitated the accessibility of plant derived C but not the above-belowground transfer rates. This facilitating effect enabled more diverse plant communities to use complementary C and most likely nutrient resources both from soil organic matter mineralization for better growth. We anticipate from our results that plant diversity effects are less driven by the performance of individuals in mixtures (trait plasticity) but by the combination of individuals that interact independently (trait complementarity).

Membrane lipids and their related acyl and/or alkyl moieties are important biomarkers of the microbial community in environmental samples. Intact polar lipids, which are described by their lipid head groups and fatty acid tails, are major membrane components of living cells only and may therefore provide information about the living soil microbial community. However, the intact polar lipids in soil organic matter may originate from non-microbial organisms (e.g., roots, soil animals) as well as microorganisms. We report on the profiles of more than 300 membrane-derived intact polar lipids in grassland soil organic matter from sites with diverse plant communities and soil properties. The origin of the soil lipid profiles was deduced by comparisons with the intact polar lipids of plant roots, amoebae, collembolans, fungi and bacteria. In addition, we investigated the impact of plant and soil-related drivers on the composition of the intact polar lipids in soil organic matter. Soil organic matter lipid profiles were considerably more diverse and evenly distributed than the profiles of roots and soil organisms (amoebae, bacteria, collembolans and fungi). Moreover, the lipid profiles in soil organic matter had more odd-carbon numbered acyl and alkyl chains, ornithine lipids, and phospholipids with low levels of unsaturation, possibly because they are of bacterial origin. We suggest that most of the intact polar lipids in soil organic matter (45–80%) were derived from bacteria, rather than plants, fungi, or soil animals. The PE (31:0) and PE (32:1) lipids were considered as biomarkers for all bacteria, whereas PE (29:0, 29:2 and 30:0) are specific to Gram (+) bacteria and PG (33; 0, 34:1 and 36:2) and OL (36:0, 37:0, 37:1, 39:1 and 39:2) were used to identify Gram (−) bacteria. Bacterial lipids were differentiated from intact lipids of plant roots that typically contain MGDG (36:4) and MGDG (36:6), while fungi have indicative markers of PC (36:4) and PE (36:4). Thus, intact polar lipid profiles complement and advance other approaches, such as fatty acid-based analysis of soil microbial community composition, and may allow for analysis of soil food web structure.

Members of the Psilotales (whisk ferns) have a unique anatomy, with conducting tissues but lacking true leaves and roots. Based on recent phyogenies, these features appear to represent a reduction from a more typical modern fern plant rather than the persistence of ancestral features. In this study, extracts of several Psilotum organs and tissues were analyzed by Gas Chromatography - Mass Spectrometry (GC-MS) and High Performance Liquid Chromatography - Quadrupole Time of Flight - Mass Spectrometry (HPLC-QTOF-MS). Some arylpyrones and biflavonoids had previously been reported to occur in Psilotum and these metabolite classes were found to be prominent constituents in the present study. Some of these were enriched and further characterized by Nuclear Magnetic Resonance (NMR) spectroscopy. HPLC-QTOF-MS and NMR data were searched against an updated Spektraris database (expanded by incorporating over 300 new arylpyrone and biflavonoid spectral records) to aid significantly with peak annotation. Principal Component Analysis (PCA) with combined GC-MS and HPLC-QTOF-MS data sets obtained with several Psilotum organs and tissues indicated a clear separation of the sample types. The principal component scores for below-ground rhizome samples corresponded to the vectors for carbohydrate monomers and dimers and small organic acids. Above-ground rhizome samples had principal component scores closer to the direction of vectors for arylpyrone glycosides and sucrose (which had high concentrations in above-and below-ground rhizomes). The unique position of brown synangia in a PCA plot correlated with the vector for biflavonoid glycosides. Principal component scores for green and yellow synangia correlated with the direction of vectors for arylpyrone glycosides and biflavonoid aglycones. Localization studies with cross sections of above-ground rhizomes, using Matrix-Assisted Laser Desorption/Ionization - Mass Spectrometry (MALDI-MS), provided evidence for a preferential accumulation of arylpyrone glycosides and biflavonoid aglycones in cells of the chlorenchyma. Our results indicate a differential localization of metabolites with potentially tissue-specific functions in defenses against biotic and abiotic stresses. The data are also a foundation for follow-up work to better understand chemical diversity in the Psilotales and other members of the fern lineage.

International audience; Many scientific disciplines are currently experiencing a 'reproducibility crisis' because numerous scientific findings cannot be repeated consistently. A novel but controversial hypothesis postulates that stringent levels of environmental and biotic standardization in experimental studies reduce reproducibility by amplifying the impacts of laboratory-specific environmental factors not accounted for in study designs. A corollary to this hypothesis is that a deliberate introduction of controlled systematic variability (CSV) in experimental designs may lead to increased reproducibility. To test this hypothesis, we had 14 European laboratories run a simple microcosm experiment using grass (Brachypodium distachyon L.) monocultures and grass and legume (Medicago truncatula Gaertn.) mixtures. Each laboratory introduced environmental and genotypic CSV within and among replicated microcosms established in either growth chambers (with stringent control of environmental conditions) or glasshouses (with more variable environmental conditions). The introduction of genotypic CSV led to 18% lower among-laboratory variability in growth chambers, indicating increased reproducibility, but had no significant effect in glasshouses where reproducibility was generally lower. Environmental CSV had little effect on reproducibility. Although there are multiple causes for the 'reproducibility crisis', deliberately including genetic variability may be a simple solution for increasing the reproducibility of ecological studies performed under stringently controlled environmental conditions.

The elucidation of pathways leading to the signature metabolites accumulated in aromatic and medicinal plants has traditionally been fairly slow in comparison to progress made with much simpler microbes, where gene clustering for natural product biosynthesis is very common. However, recent advances in next-generation sequencing and innovative new approaches to gene discovery have narrowed this gap, and the elucidation of even complex plant pathways has now become significantly faster. A wealth of sequence data has been generated with aromatic and medicinal plants, and it has become increasingly difficult for researchers to stay abreast of new developments. In this review article, I will summarize the functionality of currently available genomics databases and will comment on future needs to maximize their utility.

Premise of research. The genus Citrus includes species that are among the most important tree fruit crops. Although fruit juice is the primary product, monoterpenoid essential oil obtained from the peel is an important value-added commodity. Peel monoterpenes are synthesized by subepidermal secretory cavities that consist of glandular cells surrounding an extracellular oil storage space. Several previous studies have focused on early secretory cavity development, ending with the initiation of secretion. In order to better understand the process of monoterpene formation, transport, and storage, it is important to obtain detailed information concerning plant oil glands during all phases of development.Methodology. TEM was performed on secretory cavities isolated from the exocarp of grapefruit (Citrus × paradisi Macfad. ‘Duncan’) preserved by microwave-assisted fixation or high-pressure freezing and freeze substitution. Immunocytochemistry was employed to localize (+)-limonene synthase. Tomography was used t...

Land-use intensification is a key driver of biodiversity change. However, little is known about how it alters relationships between the diversities of different taxonomic groups, which are often correlated due to shared environmental drivers and trophic interactions. Using data from 150 grassland sites, we examined how land-use intensification (increased fertilization, higher livestock densities, and increased mowing frequency) altered correlations between the species richness of 15 plant, invertebrate, and vertebrate taxa. We found that 54% of pairwise correlations between taxonomic groups were significant and positive among all grasslands, while only one was negative. Higher land-use intensity substantially weakened these correlations (35% decrease in r and 43% fewer significant pairwise correlations at high intensity), a pattern which may emerge as a result of biodiversity declines and the breakdown of specialized relationships in these conditions. Nevertheless, some groups (Coleoptera, Heteroptera, Hymenoptera and Orthoptera) were consistently correlated with multidiversity, an aggregate measure of total biodiversity comprised of the standardized diversities of multiple taxa, at both high and low land-use intensity. The form of intensification was also important; increased fertilization and mowing frequency typically weakened plant–plant and plant–primary consumer correlations, whereas grazing intensification did not. This may reflect decreased habitat heterogeneity under mowing and fertilization and increased habitat heterogeneity under grazing. While these results urge caution in using certain taxonomic groups to monitor impacts of agricultural management on biodiversity, they also suggest that the diversities of some groups are reasonably robust indicators of total biodiversity across a range of conditions. Read More: http://www.esajournals.org/doi/10.1890/14-1307.1

Development and testing of Spektraris-NMR an online spectral resource, is reported for the NMR-based structural identification of plant natural products (PNPs). Spektraris-NMRallows users to search with multiple spectra at once and returns a table with alist of hits arranged according to the goodness of fit between query data and database entries. For each hit, a link to a tabulated alignment of 1H-NMR and 13C-NMR spectroscopic peaks (query versus database entry) is provided. Furthermore, full spectroscopic records and experimental meta information about each database entry can be accessed online. To test the utility of Spektraris-NMR for PNP identification, the database was populated with NMR data (total of 466 spectra) for ∼250 taxanes, which are structurally complex diterpenoids (including the anticancer drug taxol) commonly found in the genus Taxus. NMR data generated used was then generated with metabolites purified from Taxus cell suspension cultures to search Spektraris-NMR, and were able to identify eight taxanes with high confidence. A ninth isolated metabolite could be assigned, based on spectral searches, to a taxane skeletal class, but no high confidence hit was produced. Using various spectroscopic methods, this metabolite was characterized as the taxane 2-deacetylbaccatin IV, a novel taxane. These results indicate that Spektraris-NMR is a valuable resource for rapid and reliable identification of known metabolites and has the potential to contribute to de-replication efforts in the search for novel PNPs.

Secretory structures in terrestrial plants appear to have first emerged as intracellular oil bodies in liverworts. In vascular plants, internal secretory structures, such as resin ducts and laticifers, are usually found in conjunction with vascular bundles, whereas subepidermal secretory cavities and epidermal glandular trichomes generally have more complex tissue distribution patterns. The primary function of plant secretory structures is related to defense responses, both constitutive and induced, against herbivores and pathogens. The ability to sequester secondary (or specialized) metabolites and defense proteins in secretory structures was a critical adaptation that shaped plant-herbivore and plant-pathogen interactions. Although this review places particular emphasis on describing the evolution of pathways leading to terpenoids, it also assesses the emergence of other metabolite classes to outline the metabolic capabilities of different plant lineages.

Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (-)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.

Metabolomics enables quantitative evaluation of metabolic changes caused by genetic or environmental perturbations. However, little is known about how perturbing a single gene changes the metabolic system as a whole and which network and functional properties are involved in this response. To answer this question, we investigated the metabolite profiles from 136 mutants with single gene perturbations of functionally diverse Arabidopsis (Arabidopsis thaliana) genes. Fewer than 10 metabolites were changed significantly relative to the wild type in most of the mutants, indicating that the metabolic network was robust to perturbations of single metabolic genes. These changed metabolites were closer to each other in a genome-scale metabolic network than expected by chance, supporting the notion that the genetic perturbations changed the network more locally than globally. Surprisingly, the changed metabolites were close to the perturbed reactions in only 30% of the mutants of the well-characterized genes. To determine the factors that contributed to the distance between the observed metabolic changes and the perturbation site in the network, we examined nine network and functional properties of the perturbed genes. Only the isozyme number affected the distance between the perturbed reactions and changed metabolites. This study revealed patterns of metabolic changes from large-scale gene perturbations and relationships between characteristics of the perturbed genes and metabolic changes.

During the last decades, a number of biodiversity experiments have been established to study the effects of plant diversity on ecosystem functioning. This research has been accompanied by a controversial discussion how “diversity effects” in experimental communities are related to the role of biodiversity in “real-world” ecosystems. To explore potential relationships, we compared plant community characteristics of 12 semi-natural managed reference grasslands to selected 10-year old communities of a large grassland biodiversity experiment (Jena Experiment): two communities initially sown with 60 species (JE60), and two communities assembled naturally during succession from bare soil (JESuc). Compared to semi-natural grasslands (17.6 ± 5.6), JE60 had higher species richness (27.5 ± 0.8), while species richness in JESuc (15.2 ± 0.5) was not different on subplots of 0.64 m2 size. Evenness and spatial heterogeneity were similar among grassland types, but biomass proportions of legumes and forbs were higher in JE60, while JESuc and semi-natural grasslands were dominated by grasses. Structural equation modelling applied to identify the drivers of biomass production in mixtures of the Jena Experiment with similar species richness (sown with 8, 16, and 60 species) and reference grasslands, showed no direct relationships between observed species richness and biomass production. In contrast, functional characteristics related to nitrogen acquisition and use were the most important variables explaining community biomass production. These functional characteristics were either driven by management intensity (fertilisation) in the “real world” reference grasslands or established by sowing in the experimental grasslands. Our results imply that species functional characteristics are key for a better understanding of the mechanisms underlying community assembly and ecosystem functioning and that the drivers of ecosystem functioning are not fundamentally different between experimental and “real-world” grasslands. Thus, experimental studies with designed extinction scenarios may help to predict the consequences of species loss for ecosystem functioning in “real-world” ecosystems.

The genus Mentha has considerable economic importance as a source of essential oils for the flavor, fragrance, and aromatherapy industries (Lange and Ahkami, 2013). The history of commercial mint cultivars is complex and involves various polyploid hybrids. The most widely grown cultivars are essentially sterile, which has impeded the development of genetic resources. The work presented here aims to shed more light into the factors that determine oil composition, which is important for clonal selection strategies.

Plant species richness (PSR) increases nutrient uptake which depletes bioavailable nutrient pools in soil. No such relationship between plant uptake and availability in soil was found for phosphorus (P). We explored PSR effects on P mobilisation [phosphatase activity (PA)] in soil. PA increased with PSR. The positive PSR effect was not solely due to an increase in Corg concentrations because PSR remained significant if related to PA:Corg . An increase in PA per unit Corg increases the probability of the temporal and spatial match between substrate, enzyme and microorganism potentially serving as an adaption to competition. Carbon use efficiency of microorganisms (Cmic :Corg ) increased with increasing PSR while enzyme exudation efficiency (PA:Cmic ) remained constant. These findings suggest the need for efficient C rather than P cycling underlying the relationship between PSR and PA. Our results indicate that the coupling between C and P cycling in soil becomes tighter with increasing PSR.

For nearly 30 years, coenzyme M (CoM) was assumed to be present solely in methanogenic archaea. In the late 1990s, CoM was reported to play a role in bacterial propene metabolism, but no biosynthetic pathway for CoM has yet been identified in bacteria. Here, using bioinformatics and proteomic approaches in the metabolically versatile bacterium Xanthobacter autotrophicus Py2, we identified four putative CoM biosynthetic enzymes encoded by the xcbB1, C1, D1, and E1 genes. Only XcbB1 was homologous to a known CoM biosynthetic enzyme (ComA), indicating that CoM biosynthesis in bacteria involves enzymes different from those in archaea. We verified that the ComA homolog produces phosphosulfolactate from phosphoenolpyruvate (PEP), demonstrating that bacterial CoM biosynthesis is initiated similarly as the phosphoenolpyruvate-dependent methanogenic archaeal pathway. The bioinformatics analysis revealed that XcbC1 and D1 are members of the aspartase/fumarase superfamily (AFS) and that XcbE1 is a pyridoxal 5'-phosphate-containing enzyme with homology to d-cysteine desulfhydrases. Known AFS members catalyze β-elimination reactions of succinyl-containing substrates, yielding fumarate as the common unsaturated elimination product. Unexpectedly, we found that XcbC1 catalyzes β-elimination on phosphosulfolactate, yielding inorganic phosphate and a novel metabolite, sulfoacrylic acid. Phosphate-releasing β-elimination reactions are unprecedented among the AFS, indicating that XcbC1 is an unusual phosphatase. Direct demonstration of phosphosulfolactate synthase activity for XcbB1 and phosphate β-elimination activity for XcbC1 strengthened their hypothetical assignment to a CoM biosynthetic pathway and suggested functions also for XcbD1 and E1. Our results represent a critical first step toward elucidating the CoM pathway in bacteria.

Schizonepeta tenuifolia Briquet, commonly known as Japanese catnip, is used for the treatment of colds, headaches, fevers, and skin rashes in traditional Asian medicine (China, Japan and Korea). The volatile oil and its constituents have various demonstrated biological activities, but there is currently limited information regarding the site of biosynthesis. Light microscopy and scanning electron microscopy indicated the presence of three distinct glandular trichome types which, based on their morphological features, are referred to as peltate, capitate and digitiform glandular trichomes. Laser scanning microscopy and 3D reconstruction demonstrated that terpenoid-producing peltate glandular trichomes contain a disk of twelve secretory cells. The oil of peltate glandular trichomes, collected by laser microdissection or using custom-made micropipettes, was demonstrated to contain (−)-pulegone, (+)-menthone and (+)-limonene as major constituents. Digitiform and capitate glandular trichomes did not contain appreciable levels of terpenoid volatiles. The yield of distilled oil from spikes was significantly (44%) higher than that from leaves, while the composition of oils was very similar. Oils collected directly from leaf peltate glandular trichomes over the course of a growing season contained primarily (−)-pulegone (>80% at 32 days after germination) in young plants, while (+)-menthone began to accumulate later (>75% at 80 days after germination), at the expense of (−)-pulegone (the levels of (+)-limonene remained fairly stable at 3–5%). The current study establishes the morphological and chemical characteristics of glandular trichome types of S. tenuifolia, and also provides the basis for unraveling the biosynthesis of essential oil in this popular medicinal plant.

Numerous important ecosystem functions and services depend on soil biodiversity. However, little is known about the mechanisms which maintain the vast belowground biodiversity and about the filters shaping soil community composition. Yet, biotic interactions like facilitation and dispersal by animals are assumed to play a crucial role, particularly as most soil animal taxa are strongly limited in their active dispersal abilities. Here, we report on a newfound interaction of potentially high ubiquity and importance in soil communities: the endozoochorous dispersal of soil fauna by gastropods. We focus on the dispersal-limited group of oribatid mites, one of the most diverse and abundant soil animal groups. In a field survey in a German riparian forest, 73% of 40 collected slugs (Arion vulgaris) egested a total of 135 oribatid mites, belonging to 35 species. Notably, 70% of the egested mites were alive and survived the gut passage through slugs. Similar results were found for Roman snails (Helix pomatia), indicating the generality of our findings across different gastropod taxa. Complementary laboratory experiments confirmed our field observations, revealing that oribatid mites are, indeed, ingested and egested alive by slugs, and that they are able to independently escape the faeces and colonise new habitats. Our results strongly indicate that gastropods may help soil organisms to disperse within habitats, to overcome dispersal barriers, and to reach short-lived resource patches. Gastropods might even disperse whole multi-trophic micro-ecosystems, a discovery that could have profound implications for our understanding of dispersal mechanisms and the distribution of soil biodiversity.

The commercially important essential oils of peppermint (Mentha × piperita) and its relatives in the mint family (Lamiaceae) are accumulated in specialized anatomical structures called glandular trichomes (GTs). A genome-scale stoichiometric model of secretory phase metabolism in peppermint GTs was constructed based on current biochemical and physiological knowledge. Fluxes through the network were predicted based on metabolomic and transcriptomic data. Using simulated reaction deletions, this model predicted that two processes, the regeneration of ATP and ferredoxin (in its reduced form), exert substantial control over flux toward monoterpenes. Follow-up biochemical assays with isolated GTs indicated that oxidative phosphorylation and ethanolic fermentation were active and that cooperation to provide ATP depended on the concentration of the carbon source. We also report that GTs with high flux toward monoterpenes express, at very high levels, genes coding for a unique pair of ferredoxin and ferredoxin-NADP+ reductase isoforms. This study provides, to our knowledge, the first evidence of how bioenergetic processes determine flux through monoterpene biosynthesis in GTs.

Summary 1. As a rule, communities consist of few abundant and many rare species, which is reflected in the characteristic shape of species abundance distributions (SADs). The processes that shape these SADs have been a longstanding problem for ecological research. Although many studies found strong negative effects of increasing land-use intensity on diversity, few reports consider land-use effects on SADs. 2. Arthropods (insects and spiders) were sampled on 142 grassland plots in three regions in Germany, which were managed with different modes (mowing, fertilization and/or grazing) and intensities of land use. We analysed the effect of land use on three parameters characterizing the shape of SADs: abundance decay rate (the steepness of the rank abundance curve, represented by the niche-preemption model parameter), dominance (Berger-Parker dominance) and rarity (Fisher’s alpha). Furthermore, we tested the core-satellite hypothesis by comparing the species’ rank within the SAD to their distribution over the land-use gradient. 3. When data on Araneae, Cicadina, Coleoptera, Heteroptera and Orthoptera were combined, abundance decay rate increased with combined land-use intensity (including all modes). Among the single land-use modes, increasing fertilization and grazing intensity increased the decay rate of all taxa, while increasing mowing frequency significantly affected the decay rate only in interaction with fertilization. Results of single taxa differed in their details, but all significant interaction effects included fertilization intensity. Dominance generally increased with increasing fertilization and rarity decreased with increasing grazing or mowing intensity, despite small differences among taxa and regions. The majority of species found on

Cyclooxygenase (COX)-2 mediates ischemic pre- and postconditioning as well as anesthetic-induced preconditioning. However, the role of COX-1 and -2 in anesthetic-induced postconditioning has not been investigated. We evaluated the role of COX-1 and -2 in sevoflurane-induced postconditioning in vivo. Pentobarbital-anaesthetized male C57BL/6 mice were subjected to 45 minutes of coronary artery occlusion and 3 hours of reperfusion. Animals received either no intervention, the vehicle dimethyl sulfoxide (DMSO, 10 µL/g intraperitoneally), acetylsalicylic acid (ASA, 5 µg/g intraperitoneally), the selective COX-1 inhibitor SC-560 (10 µg/g intraperitoneally), or the selective COX-2 inhibitor NS-398 (5 µg/g intraperitoneally). 1.0 MAC (minimum alveolar concentration) sevoflurane was administered for 18 minutes during early reperfusion either alone or in combination with ASA, SC-560, and NS-398. Infarct size was determined with triphenyltetrazolium chloride. Statistical analysis was performed using 1-way and 2-way analyses of variance with post hoc Duncan testing. The infarct size in the control group was 44% ± 9%. DMSO (42% ± 7%), ASA (36% ± 6%), and NS-398 (44% ± 18%) had no effect on infarct size. Sevoflurane (17% ± 4%; P < .05) and SC-560 (26% ± 10%; P < .05) significantly reduced the infarct size compared with control condition. Sevoflurane-induced postconditioning was not abolished by ASA (16% ± 5%) and SC-560 (22% ± 4%). NS-398 abolished sevoflurane-induced postconditioning (33% ± 14%). It was concluded that sevoflurane induces postconditioning in mice. Inhibition of COX-1 elicits a myocardial infarct size reduction and does not abolish sevoflurane-induced postconditioning. Blockade of COX-2 abolishes sevoflurane-induced postconditioning. These results indicate that sevoflurane-induced postconditioning is mediated by COX-2.

Because arthropods dominate terrestrial ecosystems in species number and biomass, they can potentially play a major role as environmental indicators in applied ecology and nature conservation. We tested the suitability of arthropods as indicators for particular forest types based on a comprehensive standardized sampling of various taxa by different trap types in 93 mature temperate forest sites in three regions of Germany. We tested whether indicator species (1) can be derived for different forest types across regions, (2) are more widespread and more abundant than non-indicator ones, (3) belong to a particular taxon or trophic guild, and (4) are consistent between regions and years. Among 2041 sampled arthropod species, only four were significant indicator species for the same forest type in all region, and no single taxon or guild performed better than other groups. Indicators were generally more abundant and more widespread than non-indicators, but both abundance and distribution varied widely between species. When the analysis was repeated using data from the next year, indicator values of species significantly correlated between years, but the identity of more than 50 % of significant indicators changed, suggesting high among-year variability. We conclude that overall, arthropods did not turn out to be reliable indicators, at least at the scale of Germany. If anything, arthropod indicator species should be defined at the regional scale. Furthermore, indicators should be selected across taxa and trophic levels. Future evaluation of indicator species among arthropods should be conducted over several years based on standardized sampling protocols to develop a reliable definition of indicator species despite the high fluctuations in abundance of species among arthropods, which might either mask or overestimate the indicator value of particular species.

The metabolic underpinnings of plant survival under severe drought-induced senescence conditions are poorly understood. In this study, we assessed the morphological, physiological and metabolic responses to sustained water deficit in Brachypodium distachyon, a model organism for research on temperate grasses. Relative to control plants, fresh biomass, leaf water potential, and chlorophyll levels decreased rapidly in plants grown under drought conditions, demonstrating an early onset of senescence. The leaf C/N ratio and protein content showed an increase in plants subjected to drought stress. The concentrations of several small molecule carbohydrates and amino acid-derived metabolites previously implicated in osmotic protection increased rapidly in plants experiencing water deficit. Malic acid, a low molecular weight organic acid with demonstrated roles in stomatal closure, also increased rapidly as a response to drought treatment. The concentrations of prenyl lipids, such as phytol and α-tocopherol, increased early during the drought treatment but then dropped dramatically. Surprisingly, continued changes in the quantities of metabolites were observed, even in samples harvested from visibly senesced plants. The data presented here provide insights into the processes underlying persistent metabolic activity during sustained water deficit and can aid in identifying mechanisms of drought tolerance in plants.

The importance of spatial scale for β-diversity has been shown in several studies, but it is unclear how spatial diversity patterns correlate among different organismic groups. We studied spatial diversity organization of plants and several trophic guilds of beetles in beech-dominated forests in two regions of Germany to test whether different trophic guilds are organized independently in space. We applied multiplicative diversity partitioning using a nested hierarchical design of four increasingly broader spatial levels (subplot, plot, forest class, region) and tested for correlations among trophic guilds by using Pearson product moment correlations and Mantel-tests. We observed similar general diversity patterns at different trophic guilds showing a high contribution of β-diversity to total γ-diversity and found β-diversity to be higher at different spatial scales and α-diversity to be lower than expected by random distributions of individuals. Results, however, partly depended on the weighting of rare and abundant species. Beta-diversity in our study was caused mainly by species spatial turnover rather than by nestedness. Correlations of α-diversity between trophic guilds were low whereas correlations of β-diversity above subplot level were high. Importantly, more strongly connected trophic guilds revealed not generally stronger relationships than less strongly connected guilds. Three important implications for conservation can be deduced from our results: (1) heterogeneity of beech forests at different spatial scales should be supported in conservation strategies to enhance biodiversity and related functions; (2) the observed high importance of spatial turnover in relation to nestedness implies a concentration of conservation efforts to a large number of not necessarily the richest sites, and (3) recommendation for particular conservation strategies (e.g. selection of priority sites for conservation at regional scale) based on single indicator taxa or functional guild is difficult because of the varied response of the species in our study.

Organic management is one of the most popular strategies to reduce negative environmental impacts of intensive agriculture. However, little is known about benefits for biodiversity and potential worsening of yield under organic grasslands management across different grassland types, i.e. meadow, pasture and mown pasture. Therefore, we studied the diversity of vascular plants and foliage-living arthropods (Coleoptera, Araneae, Heteroptera, Auchenorrhyncha), yield, fodder quality, soil phosphorus concentrations and land-use intensity of organic and conventional grasslands across three study regions in Germany. Furthermore, all variables were related to the time since conversion to organic management in order to assess temporal developments reaching up to 18 years. Arthropod diversity was significantly higher under organic than conventional management, although this was not the case for Araneae, Heteroptera and Auchenorrhyncha when analyzed separately. On the contrary, arthropod abundance, vascular plant diversity and also yield and fodder quality did not considerably differ between organic and conventional grasslands. Analyses did not reveal differences in the effect of organic management among grassland types. None of the recorded abiotic and biotic parameters showed a significant trend with time since transition to organic management, except soil organic phosphorus concentrations which decreased with time. This implies that permanent grasslands respond slower and probably weaker to organic management than crop fields do. However, as land-use intensity and inorganic soil phosphorus concentrations were significantly lower in organic grasslands, overcoming seed and dispersal limitation by re-introducing plant species might be needed to exploit the full ecological potential of organic grassland management. We conclude that although organic management did not automatically increase the diversity of all studied taxa, it is a reasonable and useful way to support agro-biodiversity.

In the past two decades, a large number of studies have investigated the relationship between biodiversity and ecosystem functioning, most of which focussed on a limited set of ecosystem variables. The Jena Experiment was set up in 2002 to investigate the effects of plant diversity on element cycling and trophic interactions, using a multi-disciplinary approach. Here, we review the results of 15 years of research in the Jena Experiment, focussing on the effects of manipulating plant species richness and plant functional richness. With more than 85,000 measures taken from the plant diversity plots, the Jena Experiment has allowed answering fundamental questions important for functional biodiversity research.[br/] First, the question was how general the effect of plant species richness is, regarding the many different processes that take place in an ecosystem. About 45% of different types of ecosystem processes measured in the 'main experiment', where plant species richness ranged from 1 to 60 species, were significantly affected by plant species richness, providing strong support for the view that biodiversity is a significant driver of ecosystem functioning. Many measures were not saturating at the 60-species level, but increased linearly with the logarithm of species richness. There was, however, great variability in the strength of response among different processes. One striking pattern was that many processes, in particular belowground processes, took several years to respond to the manipulation of plant species richness, showing that biodiversity experiments have to be long-term, to distinguish trends from transitory patterns. In addition, the results from the Jena Experiment provide further evidence that diversity begets stability, for example stability against invasion of plant species, but unexpectedly some results also suggested the opposite, e.g. when plant communities experience severe perturbations or elevated resource availability. This highlights the need to revisit diversity-stability theory.[br/] Second, we explored whether individual plant species or individual plant functional groups, or biodiversity itself is more important for ecosystem functioning, in particular biomass production. We found strong effects of individual species and plant functional groups on biomass production, yet these effects mostly occurred in addition to, but not instead of, effects of plant species richness.[br/] Third, the Jena Experiment assessed the effect of diversity on multitrophic interactions. The diversity of most organisms responded positively to increases in plant species richness, and the effect was stronger for above-than for belowground organisms, and stronger for herbivores than for carnivores or detritivores. Thus, diversity begets diversity. In addition, the effect on organismic diversity was stronger than the effect on species abundances.[br/] Fourth, the Jena Experiment aimed to assess the effect of diversity on N, P and C cycling and the water balance of the plots, separating between element input into the ecosystem, element turnover, element stocks, and output from the ecosystem. While inputs were generally less affected by plant species richness, measures of element stocks, turnover and output were often positively affected by plant diversity, e.g. carbon storage strongly increased with increasing plant species richness. Variables of the N cycle responded less strongly to plant species richness than variables of the C cycle.[br/] Fifth, plant traits are often used to unravel mechanisms underlying the biodiversity-ecosystem functioning relationship. In the Jena Experiment, most investigated plant traits, both above-and belowground, were plastic and trait expression depended on plant diversity in a complex way, suggesting limitation to using database traits for linking plant traits to particular functions.[br/] Sixth, plant diversity effects on ecosystem processes are often caused by plant diversity effects on species interactions. Analyses in the Jena Experiment including structural equation modelling suggest complex interactions that changed with diversity, e.g. soil carbon storage and greenhouse gas emission were affected by changes in the composition and activity of the belowground microbial community. Manipulation experiments, in which particular organisms, e.g. belowground invertebrates, were excluded from plots in split-plot experiments, supported the important role of the biotic component for element and water fluxes.[br/] Seventh, the Jena Experiment aimed to put the results into the context of agricultural practices in managed grasslands. The effect of increasing plant species richness from 1 to 16 species on plant biomass was, in absolute terms, as strong as the effect of a more intensive grassland management, using fertiliser and increasing mowing frequency. Potential bioenergy production from high-diversity plots was similar to that of conventionally used energy crops. These results suggest that diverse 'High Nature Value Grasslands' are multifunctional and can deliver a range of ecosystem services including production-related services.[br/] A final task was to assess the importance of potential artefacts in biodiversity-ecosystem functioning relationships, caused by the weeding of the plant community to maintain plant species composition. While the effort ( in hours) needed to weed a plot was often negatively related to plant species richness, species richness still affected the majority of ecosystem variables. Weeding also did not negatively affect monoculture performance; rather, monocultures deteriorated over time for a number of biological reasons, as shown in plant-soil feedback experiments.[br/] To summarize, the Jena Experiment has allowed for a comprehensive analysis of the functional role of biodiversity in an ecosystem. A main challenge for future biodiversity research is to increase our mechanistic understanding of why the magnitude of biodiversity effects differs among processes and contexts. It is likely that there will be no simple answer. For example, among the multitude of mechanisms suggested to underlie the positive plant species richness effect on biomass, some have received limited support in the Jena Experiment, such as vertical root niche partitioning. However, others could not be rejected in targeted analyses. Thus, from the current results in the Jena Experiment, it seems likely that the positive biodiversity effect results from several mechanisms acting simultaneously in more diverse communities, such as reduced pathogen attack, the presence of more plant growth promoting organisms, less seed limitation, and increased trait differences leading to complementarity in resource uptake. Distinguishing between different mechanisms requires careful testing of competing hypotheses. Biodiversity research has matured such that predictive approaches testing particular mechanisms are now possible.