A "Magic Mushroom" Multi-Product Sesquiterpene Synthase.

Psilocybe "magic mushrooms" are chemically well understood for their psychotropic tryptamines. However, the diversity of their other specialized metabolites, in particular terpenoids, has largely remained an open question. Yet, knowledge on the natural product background is critical to understand if other compounds modulate the psychotropic pharmacological effects. CubA, the single clade II sesquiterpene synthase of P. cubensis, was heterologously produced in Escherichia coli and characterized in vitro, complemented by in vivo product formation assays in Aspergillus niger as a heterologous host. Extensive GC-MS analyses proved a function as multi-product synthase and, depending on the reaction conditions, cubebol, ß-copaene, δ-cadinene, and germacrene D were detected as the major products of CubA. In addition, mature P. cubensis carpophores were analysed chromatographically which led to the detection of ß-copaene and δ-cadinene. Enzymes closely related to CubA are encoded in the genomes of various Psilocybe species. Therefore, our results provide insight into the metabolic capacity of the entire genus.

Tetranorsesquiterpenoids as Attractants of Yucca Moths to Yucca Flowers.

The obligate pollination mutualism between Yucca and yucca moths is a classical example of coevolution. Oviposition and active pollination by female yucca moths occur at night when Yucca flowers are open and strongly scented. Thus, floral volatiles have been suggested as key sensory signals attracting yucca moths to their host plants, but no bioactive compounds have yet been identified. In this study, we showed that both sexes of the pollinator moth Tegeticula yuccasella are attracted to the floral scent of the host Yucca filamentosa. Chemical analysis of the floral headspace from six Yucca species in sections Chaenocarpa and Sarcocarpa revealed a set of novel tetranorsesquiterpenoids putatively derived from (E)-4,8-dimethyl-1,3,7-nonatriene. Their structure elucidation was accomplished by NMR analysis of the crude floral scent sample of Yucca treculeana along with GC/MS analysis and confirmed by total synthesis. Since all these volatiles are included in the floral scent of Y. filamentosa, which has been an important model species for understanding the pollination mutualism, we name these compounds filamentolide, filamentol, filamental, and filamentone. Several of these compounds elicited antennal responses in pollinating (Tegeticula) and non-pollinating (Prodoxus) moth species upon stimulation in electrophysiological recordings. In addition, synthetic (Z)-filamentolide attracted significant numbers of both sexes of two associated Prodoxus species in a field trapping experiment. Highly specialized insect-plant interactions, such as obligate pollination mutualisms, are predicted to be maintained through "private channels" dictated by specific compounds. The identification of novel bioactive tetranorsesquiterpenoids is a first step in testing such a hypothesis in the Yucca-yucca moth interaction.

Chromane Derivatives from Underground Parts of Iris tenuifolia and Their In Vitro Antimicrobial, Cytotoxicity and Antiproliferative Evaluation.

Phytochemical investigation of the ethanol extract of underground parts of Iris tenuifolia Pall. afforded five new compounds; an unusual macrolide termed moniristenulide (1), 5-methoxy-6,7-methylenedioxy-4-O-2'-cycloflavan (2), 5,7,2',3'-tetrahydroxyflavanone (3), 5-hydroxy-6,7-dimethoxyisoflavone-2'-O-ß-d-glucopyranoside (9), 5,2',3'-dihydroxy-6,7-dimethoxyisoflavone (10), along with seven known compounds (4-8, 11-12). The structures of all purified compounds were established by analysis of 1D and 2D NMR spectroscopy and HR-ESI-MS. The antimicrobial activity of the compounds 1-3, 5, 9, and 10 was investigated using the agar diffusion method against fungi, Gram-positive and Gram-negative bacteria. In consequence, new compound 3 was found to possess the highest antibacterial activity against Enterococcus faecalis VRE and Mycobacterium vaccae. Cell proliferation and cytotoxicity tests were also applied on all isolated compounds and plant crude extract in vitro with the result of potent inhibitory effect against leukemia cells. In particular, the newly discovered isoflavone 10 was active against both of the leukemia cells K-562 and THP-1 while 4-6 of the flavanone type compounds were active against only THP-1.

Mechanistic studies of sesquiterpene cyclases based on their carbon isotope ratios at natural abundance.

During the process of terpene biosynthesis, C-C bond breaking and forming steps are subjected to kinetic carbon isotope effects, leading to distinct carbon isotopic signatures of the products. Accordingly, carbon isotopic signatures could be used to reveal the 'biosynthetic history' of the produced terpenoids. Five known sesquiterpene cyclases, regulating three different pathways, representing simple to complex biosynthetic sequences, were heterologously expressed and used for in vitro assays with farnesyl diphosphate as substrate. Compound specific isotope ratio mass spectrometry measurements of the enzyme substrate farnesyl diphosphate (FDP) and the products of all the five cyclases were performed. The calculated δ13 C value for FDP, based on δ13 C values and relative amounts of the products, was identical with its measured δ13 C value, confirming the reliability of the approach and the precision of measurements. The different carbon isotope ratios of the products reflect the complexity of their structure and are correlated with the frequency of carbon-carbon bond forming and breaking steps on their individual biosynthetic pathways. Thus, the analysis of carbon isotopic signatures of terpenes at natural abundance can be used as a powerful tool in elucidation of associated biosynthetic mechanisms of terpene synthases and in future in vivo studies even without 'touching' the plant.

Elevated Carbon Dioxide Concentration Reduces Alarm Signaling in Aphids.

Insects often rely on olfaction to communicate with conspecifics. While the chemical language of insects has been deciphered in recent decades, few studies have assessed how changes in atmospheric greenhouse gas concentrations might impact pheromonal communication in insects. Here, we hypothesize that changes in the concentration of atmospheric carbon dioxide affect the whole dynamics of alarm signaling in aphids, including: (1) the production of the active compound (E)-ß-farnesene (Eßf), (2) emission behavior when under attack, (3) perception by the olfactory apparatus, and (4) the escape response. We reared two strains of the pea aphid, Acyrthosiphon pisum, under ambient and elevated CO2 concentrations over several generations. We found that an increase in CO2 concentration reduced the production (i.e., individual content) and emission (released under predation events) of Eßf. While no difference in Eßf neuronal perception was observed, we found that an increase in CO2 strongly reduced the escape behavior expressed by an aphid colony following exposure to natural doses of alarm pheromone. In conclusion, our results confirm that changes to greenhouse gases impact chemical communication in the pea aphid, and could potentially have a cascade effect on interactions with higher trophic levels.

Biosynthesis of 8-hydroxyquinoline-2-carboxylic acid, an iron chelator from the gut of the lepidopteran Spodoptera littoralis.

In the regurgitate (foregut content) of Spodoptera larvae we found high concentrations (0.5-5 mM) of 8-hydroxyquinoline-2-carboxylic acid (8-HQA). In a survey of different lepidopteran species, this compound was only detected in species belonging to the family of Noctuidae. 8-HQA was shown to derive from tryptophan metabolism. The amount of 8-HQA in the regurgitate was strongly dependent on the tryptophan content of the diet. In the insect 8-HQA is generated from tryptophan via kynurenine and 3-hydroxykynurenine. 8-HQA is produced by the larvae and not by their commensal gut bacteria. Analysis of different life stages of Spodoptera larvae revealed that 8-HQA is formed during the larval stage, probably acting as an iron chelator to control the gut microbiome.

Quantification of growth-defense trade-offs in a common currency: nitrogen required for phenolamide biosynthesis is not derived from ribulose-1,5-bisphosphate carboxylase/oxygenase turnover.

Induced defenses are thought to be economical: growth and fitness-limiting resources are only invested into defenses when needed. To date, this putative growth-defense trade-off has not been quantified in a common currency at the level of individual compounds. Here, a quantification method for ¹5N-labeled proteins enabled a direct comparison of nitrogen (N) allocation to proteins, specifically, ribulose-1,5-bisposphate carboxylase/oxygenase (RuBisCO), as proxy for growth, with that to small N-containing defense metabolites (nicotine and phenolamides), as proxies for defense after herbivory. After repeated simulated herbivory, total N decreased in the shoots of wild-type (WT) Nicotiana attenuata plants, but not in two transgenic lines impaired in jasmonate defense signaling (irLOX3) and phenolamide biosynthesis (irMYB8). N was reallocated among different compounds within elicited rosette leaves: in the WT, a strong decrease in total soluble protein (TSP) and RuBisCO was accompanied by an increase in defense metabolites, irLOX3 showed a similar, albeit attenuated, pattern, whereas irMYB8 rosette leaves were the least responsive to elicitation, with overall higher levels of RuBisCO. Induced defenses were higher in the older compared with the younger rosette leaves, supporting the hypothesis that tissue developmental stage influences defense investments. We propose that MYB8, probably by regulating the production of phenolamides, indirectly mediates protein pool sizes after herbivory. Although the decrease in absolute N invested in TSP and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis, ¹5N flux studies revealed that N for phenolamide synthesis originates from recently assimilated N, rather than from RuBisCO turnover.

8-HQA adjusts the number and diversity of bacteria in the gut microbiome of Spodoptera littoralis.

Quinolinic carboxylic acids are known for their metal ion chelating properties in insects, plants and bacteria. The larval stages of the lepidopteran pest, Spodoptera littoralis, produce 8-hydroxyquinoline-2-carboxylic acid (8-HQA) in high concentrations from tryptophan in the diet. At the same time, the larval midgut is known to harbor a bacterial population. The motivation behind the work was to investigate whether 8-HQA is controlling the bacterial community in the gut by regulating the concentration of metal ions. Knocking out the gene for kynurenine 3-monooxygenase (KMO) in the insect using CRISPR/Cas9 eliminated production of 8-HQA and significantly increased bacterial numbers and diversity in the larval midgut. Adding 8-HQA to the diet of knockout larvae caused a dose-dependent reduction of bacterial numbers with minimal effects on diversity. Enterococcus mundtii dominates the community in all treatments, probably due to its highly efficient iron uptake system and production of the colicin, mundticin. Thus host factors and bacterial properties interact to determine patterns of diversity and abundance in the insect midgut.

Determination of ¹5N-incorporation into plant proteins and their absolute quantitation: a new tool to study nitrogen flux dynamics and protein pool sizes elicited by plant-herbivore interactions.

Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.

MAPK-dependent JA and SA signalling in Nicotiana attenuata affects plant growth and fitness during competition with conspecifics.

BACKGROUND: Induced defense responses to herbivores are generally believed to have evolved as cost-saving strategies that defer the fitness costs of defense metabolism until these defenses are needed. The fitness costs of jasmonate (JA)-mediated defenses have been well documented. Those of the early signaling units mediating induced resistance to herbivores have yet to be examined. Early signaling components that mediate herbivore-induced defense responses in Nicotiana attenuata, have been well characterized and here we examine their growth and fitness costs during competition with conspecifics. Two mitogen-activated protein kinases (MAPKs), salicylic acid (SA)-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) are rapidly activated after perception of herbivory and both kinases regulate herbivory-induced JA levels and JA-mediated defense metabolite accumulations. Since JA-induced defenses result in resource-based trade-offs that compromise plant productivity, we evaluated if silencing SIPK (irSIPK) and WIPK (irWIPK) benefits the growth and fitness of plants competiting with wild type (WT) plants, as has been shown for plants silenced in JA-signaling by the reduction of Lipoxygenase 3 (LOX3) levels. RESULTS: As expected, irWIPK and LOX3-silenced plants out-performed their competing WT plants. Surprisingly, irSIPK plants, which have the largest reductions in JA signaling, did not. Phytohormone profiling of leaves revealed that irSIPK plants accumulated higher levels of SA compared to WT. To test the hypothesis that these high levels of SA, and their presumed associated fitness costs of pathogen associated defenses in irSIPK plants had nullified the JA-deficiency-mediated growth benefits in these plants, we genetically reduced SA levels in irSIPK plants. Reducing SA levels partially recovered the biomass and fitness deficits of irSIPK plants. We also evaluated whether the increased fitness of plants with reduced SA or JA levels resulted from increased nitrogen or CO2 assimilation rates, and found no evidence that greater intake of these fitness-limiting resources were responsible. CONCLUSIONS: Signaling mediated by WIPK, but not SIPK, is associated with large fitness costs in competing N. attenuata plants, demonstrating the contrasting roles that these two MAPKs play in regulating the plants' growth-defense balance. We discuss the role of SIPK as an important regulator of plant fitness, possibly by modulating SA-JA crosstalk as mediated through ethylene signaling.

Reductive dehalogenation of brominated ethenes by Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE-S.

Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE-S are anaerobes that can utilize tetrachloroethene (PCE) as an electron acceptor in their energy metabolism. The end-product of PCE reduction for both organisms is cis-1,2-dichloroethene, which is formed via trichloroethene as the intermediate. The bacteria were able to dehalogenate cis- and trans-1,2-dibromoethene (cDBE and tDBE) in growing cultures and cell extracts. Dibromoethene supported growth of both organisms. The organisms debrominated cDBE and tDBE to vinyl bromide (VB); D. hafniense PCE-S also produced ethene in addition to VB. The PCE reductive dehalogenases (PCE dehalogenases) of S. multivorans and D. hafniense PCE-S mediated the debromination of tribromoethene (TBE) and both isomers of 1,2-DBE, indicating that this enzyme was responsible for the reductive dehalogenation of brominated ethenes. cDBE, tDBE, 1,1-DBE and VB were formed upon TBE debromination; VB was the major end-product. The PCE dehalogenase of D. hafniense PCE-S also formed ethene. With the purified enzymes from both organisms the kinetic properties of dehalogenation of brominated alkenes were studied and compared with those of their chlorinated analogues.

Conformational studies on the Delta8(E,Z)-sphingolipid desaturase from Helianthus annuus with chiral fluoropalmitic acids as mechanistic probes.

The Delta(8)-sphingolipid desaturase from sunflower (Helianthus annuus) converts phytosphinganine into a mixture of Delta(8)-(E)- and -(Z)-phytosphingenines by removal of two syn-hydrogen atoms from anti-, and gauche-conformations of the substrate. With chiral (R)-6-, (S)-6-, (R)-7-, and (S)-7-fluoropalmitic acids the importance of conformations for the formation of (E)- and (Z)-isomers was investigated by using growing yeast cells expressing the desaturase from H. annuus. The fluoropalmitic acids were readily incorporated into a series of fluorinated phytosphinganines. The desaturation products of the major C(18)-fluorophytosphinganine demonstrate that different conformations of the relevant aliphatic segment of the sphingolipids can be exposed to the active center of the enzyme resulting in (E)- or (Z)-fluoroalkenes. The presence of a fluorine atom at the position of the initial hydrogen removal C8-H(R) led to a complete suppression of the desaturation reaction, while replacement of C8-H(S) with fluorine generated a mixture of mainly (Z)- and trace amounts of (E)-fluoroolefine. Fluorine at C9 of the phytosphinganine precursors did not interfere with the initial C-H activation step and produced (E)- and (Z)-fluoroalkenes in the same ratio as observed for the nonfluorinated precursors. Hydroxylated byproducts of the desaturation process were not observed. These results strongly support the importance of conformations of the transition states during desaturation as the relevant criterion for the relative ratio of (E)- and (Z)-alkenes.

SpitWorm, a Herbivorous Robot: Mechanical Leaf Wounding with Simultaneous Application of Salivary Components.

Induction of jasmonate-mediated plant defense against insect herbivory is initiated by a combination of both mechanical wounding and chemical factors. In order to study both effects independently on plant defense induction, SpitWorm, a computer-controlled device which mimics the damage pattern of feeding insect larvae on leaves and, in addition, can apply oral secretions (OS) or other solutions to the 'biting site' during 'feeding,' was developed and evaluated. The amount of OS left by a Spodoptera littoralis larva during feeding on Phaseolus lunatus (lima bean) leaves was estimated by combining larval foregut volume, biting rate, and quantification of a fluorescent dye injected into the larvae's foregut prior to feeding. For providing OS amounts by SpitWorm equivalent to larval feeding, dilution and delivery rate were optimized. The effectiveness of SpitWorm was tested by comparing volatile organic compounds (VOC) emissions of P. lunatus leaves treated with either SpitWorm, MecWorm, or S. littoralis larvae. Identification and quantification of emitted VOCs revealed that SpitWorm induced a volatile bouquet that is qualitatively and quantitatively similar to herbivory. Additionally, RT-qPCR of four jasmonic acid responsive genes showed that SpitWorm, in contrast to MecWorm, induces the same regulation pattern as insect feeding. Thus, SpitWorm mimics insect herbivory almost identically to real larvae feeding.

Volatile DMNT systemically induces jasmonate-independent direct anti-herbivore defense in leaves of sweet potato (Ipomoea batatas) plants.

Plants perceive and respond to volatile signals in their environment. Herbivore-infested plants release volatile organic compounds (VOCs) which can initiate systemic defense reactions within the plant and contribute to plant-plant communication. Here, for Ipomoea batatas (sweet potato) leaves we show that among various herbivory-induced plant volatiles, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) had the highest abundance of all emitted compounds. This homoterpene was found being sufficient for a volatile-mediated systemic induction of defensive Sporamin protease inhibitor activity in neighboring sweet potato plants. The systemic induction is jasmonate independent and does not need any priming-related challenge. Induced emission and responsiveness to DMNT is restricted to a herbivory-resistant cultivar (Tainong 57), while a susceptible cultivar, Tainong 66, neither emitted amounts comparable to Tainong 57, nor showed reaction to DMNT. This is consistent with the finding that Spodoptera larvae feeding on DMNT-exposed cultivars gain significantly less weight on Tainong 57 compared to Tainong 66. Our results indicate a highly specific, single volatile-mediated plant-plant communication in sweet potato.

De novo biosynthesis versus sequestration: a network of transport systems supports in iridoid producing leaf beetle larvae both modes of defense.

In the larval chrysomelines the de novo synthesis of monoterpenoids (iridoids) is believed to represent the ancestral state in the evolution of chemical defenses. Here we demonstrate that the iridoid producing larvae of Plagiodera versicolora and Phratora laticollis have the potential to sequester precursors from food. In nature, iridoids may even have a dual origin, namely plant-derived and de novo produced. The ability to sequester plant-derived precursors was proved by (i) (13)C-labelling of the terpenoids in the food plant, (ii) by larval feeding on leaves impregnated with analogs and labelled putative precursors for iridoid biosynthesis; and (iii) by injection of the precursors into the hemolymph followed by mass spectroscopic analysis of their distribution in the hemolymph, defensive secretion, and faeces. The experimental findings support a network of transport systems which allows a broader range of glucosides to enter and to leave the hemocoel, while only the appropriate precursor, 8-hydroxygeraniol-8-O-beta-d-glucoside, is channelled to the reservoir and processed to iridoids. The dual system of de novo biosynthesis and sequestration of phytogenic precursors may have favoured the larvae to shift from one host plant to another without losing their defense.

Corrigendum: In Vivo Isotopic Labeling of Symbiotic Bacteria Involved in Cellulose Degradation and Nitrogen Recycling within the Gut of the Forest Cockchafer (Melolontha hippocastani).

[This corrects the article on p. 1970 in vol. 8, PMID: 29075241.].

One Pathway Is Not Enough: The Cabbage Stem Flea Beetle Psylliodes chrysocephala Uses Multiple Strategies to Overcome the Glucosinolate-Myrosinase Defense in Its Host Plants.

The cabbage stem flea beetle (Psylliodes chrysocephala) is a key pest of oilseed rape in Europe, and is specialized to feed on Brassicaceae plants armed with the glucosinolate-myrosinase defense system. Upon tissue damage, the ß-thioglucosidase enzyme myrosinase hydrolyzes glucosinolates (GLS) to form toxic isothiocyanates (ITCs) which deter non-adapted herbivores. Here, we show that P. chrysocephala selectively sequester GLS from their host plants and store these throughout their life cycle. In addition, P. chrysocephala metabolize GLS to desulfo-GLS, which implies the evolution of GLS sulfatase activity in this specialist. To assess whether P. chrysocephala can largely prevent GLS hydrolysis in ingested plant tissue by sequestration and desulfation, we analyzed the metabolic fate of 4-methylsulfinylbutyl (4MSOB) GLS in adults. Surprisingly, intact and desulfo-GLS together accounted for the metabolic fate of only 26% of the total ingested GLS in P. chrysocephala, indicating that most ingested GLS are nevertheless activated by the plant myrosinase. The presence of 4MSOB-ITC and the corresponding nitrile in feces extracts confirmed the activation of ingested GLS, but the detected amounts of unmetabolized ITCs were low. P. chrysocephala partially detoxifies ITCs by conjugation with glutathione via the conserved mercapturic acid pathway. In addition to known products of the mercapturic acid pathway, we identified two previously unknown cyclic metabolites derived from the cysteine-conjugate of 4MSOB-ITC. In summary, the cabbage stem flea beetle avoids ITC formation by specialized strategies, but also relies on and extends the conserved mercapturic acid pathway to prevent toxicity of formed ITCs.

In Vivo Isotopic Labeling of Symbiotic Bacteria Involved in Cellulose Degradation and Nitrogen Recycling within the Gut of the Forest Cockchafer (Melolontha hippocastani).

The guts of insects harbor symbiotic bacterial communities. However, due to their complexity, it is challenging to relate a specific symbiotic phylotype to its corresponding function. In the present study, we focused on the forest cockchafer (Melolontha hippocastani), a phytophagous insect with a dual life cycle, consisting of a root-feeding larval stage and a leaf-feeding adult stage. By combining in vivo stable isotope probing (SIP) with 13C cellulose and 15N urea as trophic links, with Illumina MiSeq (Illumina-SIP), we unraveled bacterial networks processing recalcitrant dietary components and recycling nitrogenous waste. The bacterial communities behind these processes change between larval and adult stages. In 13C cellulose-fed insects, the bacterial families Lachnospiraceae and Enterobacteriaceae were isotopically labeled in larvae and adults, respectively. In 15N urea-fed insects, the genera Burkholderia and Parabacteroides were isotopically labeled in larvae and adults, respectively. Additionally, the PICRUSt-predicted metagenome suggested a possible ability to degrade hemicellulose and to produce amino acids of, respectively, 13C cellulose- and 15N urea labeled bacteria. The incorporation of 15N from ingested urea back into the insect body was confirmed, in larvae and adults, by isotope ratio mass spectrometry (IRMS). Besides highlighting key bacterial symbionts of the gut of M. hippocastani, this study provides example on how Illumina-SIP with multiple trophic links can be used to target microorganisms embracing different roles within an environment.

Coprophagous features in carnivorous Nepenthes plants: a task for ureases.

Most terrestrial carnivorous plants are specialized on insect prey digestion to obtain additional nutrients. Few species of the genus Nepenthes developed mutualistic relationships with mammals for nitrogen supplementation. Whether dietary changes require certain enzymatic composition to utilize new sources of nutrients has rarely been tested. Here, we investigated the role of urease for Nepenthes hemsleyana that gains nitrogen from the bat Kerivoula hardwickii while it roosts inside the pitchers. We hypothesized that N. hemsleyana is able to use urea from the bats' excrements. In fact, we demonstrate that 15N-enriched urea provided to Nepenthes pitchers is metabolized and its nitrogen is distributed within the plant. As ureases are necessary to degrade urea, these hydrolytic enzymes should be involved. We proved the presence and enzymatic activity of a urease for Nepenthes plant tissues. The corresponding urease cDNA from N. hemsleyana was isolated and functionally expressed. A comprehensive phylogenetic analysis for eukaryotic ureases, including Nepenthes and five other carnivorous plants' taxa, identified them as canonical ureases and reflects the plant phylogeny. Hence, this study reveals ureases as an emblematic example for an efficient, low-cost but high adaptive plasticity in plants while developing a further specialized lifestyle from carnivory to coprophagy.

Dynamic pathway allocation in early terpenoid biosynthesis of stress-induced lima bean leaves.

Two independent pathways contribute in higher plants to the formation of isopenteny1 diphosphate (IDP), the central building block of isoprenoids. In general, the cytosolic mevalonate pathway (MVA) provides the precursors for sesquiterpenes and sterols, whereas the plastidial methylerythritol pathway (MEP) furnishes the monoterpene-, diterpene- and carotenoids. Administration of deuterium labeled 1-deoxy-d-xylulose and mevalolactone to lima beans (Phaseolus lunatus), followed by gas chromatographic separation and mass spectrometric analysis of de novo produced volatiles revealed that the strict separation of both pathways does not exist. This could be confirmed by blocking the pathways individually with cerivastatin((R)) (MVA) and fosmidomycin (MEP), respectively. Isotopic ratio mass spectrometry (IRMS) at natural abundance levels demonstrated independently and without the need for labeled precursors a dynamic allocation of the MVA- or the MEP-pathway in the biosynthesis of the nerolidol-derived homoterpene 4,8-dimethy1-nona-1,3,7-triene (DMNT). Insect-feeding upregulated predominantly the MVA-pathway, while the fungal elicitor alamethicin stimulated the biosynthesis of DMNT via the MEP-pathway.