Monitoring spatial and temporal metabolic dynamics of woody poplar root under mechanical stress conditions by NMR-based metabolomics

Introduction

Molecular factors are differentially observed in various bent sectors of poplar (Populus nigra) woody taproots. Responses to stress are modulated by a complex interplay among different hormones and signal transduction pathways. In recent years, metabolomics has been recognized as a powerful tool to characterize metabolic network regulation, and it has been widely applied to investigate plant responses to biotic and abiotic stresses.

Objectives

In this paper we used metabolomics to understand if long term-bending stress induces a “spatial” and a “temporal” metabolic reprogramming in woody poplar roots.

Methods

By NMR spectroscopy and statistical analysis we investigated the unstressed and three portions of stressed root (above-bent, bent, and below-bent) sectors collected at 12 (T₀), 13 (T₁) and 14 (T₂) months after stress induction.

Results

The data indicate a clear between-class separation of control and stressed regions, based on the metabolites regulation, during both spatial and temporal changes. We found that taproots, as a consequence of the stress, try to restore homeostasis and normal metabolic fluxes thorough the synthesis and/or accumulation of specific compounds related to mechanical forces distribution along the bent taproot.

Conclusion

The data demonstrate that the impact of mechanical stress on plant biology can efficiently be studied by NMR-based metabolomics.

     

Optimized GC–MS metabolomics for the analysis of kidney tissue metabolites

Introduction

Metabolomics is a promising approach for discovery of relevant biomarkers in cells, tissues, organs, and biofluids for disease identification and prediction. The field has mostly relied on blood-based biofluids (serum, plasma, urine) as non-invasive sources of samples as surrogates of tissue or organ-specific conditions. However, the tissue specificity of metabolites pose challenges in translating blood metabolic profiles to organ-specific pathophysiological changes, and require further downstream analysis of the metabolites.

Objectives

As part of this project, we aim to develop and optimize an efficient extraction protocol for the analysis of kidney tissue metabolites representative of key primate metabolic pathways.

Methods

Kidney cortex and medulla tissues of a baboon were homogenized and extracted using eight different extraction protocols including methanol/water, dichloromethane/methanol, pure methanol, pure water, water/methanol/chloroform, methanol/chloroform, methanol/acetonitrile/water, and acetonitrile/isopropanol/water. The extracts were analyzed by a two-dimensional gas chromatography time-of-flight mass-spectrometer (2D GC–ToF-MS) platform after methoximation and silylation.

Results

Our analysis quantified 110 shared metabolites in kidney cortex and medulla tissues from hundreds of metabolites found among the eight different solvent extractions spanning low to high polarities. The results revealed that medulla is metabolically richer compared to the cortex. Dichloromethane and methanol mixture (3:1) yielded highest number of metabolites across both the tissue types. Depending on the metabolites of interest, tissue type, and the biological question, different solvents can be used to extract specific groups of metabolites.

Conclusion

This investigation provides insights into selection of extraction solvents for detection of classes of metabolites in renal cortex and medulla, which is fundamentally important for identification of prognostic and diagnostic metabolic kidney biomarkers for future therapeutic applications.

     

A genome-scale metabolic model of potato late blight suggests a photosynthesis suppression mechanism

Background

Phytophthora infestans is a plant pathogen that causes an important plant disease known as late blight in potato plants (Solanum tuberosum) and several other solanaceous hosts. This disease is the main factor affecting potato crop production worldwide. In spite of the importance of the disease, the molecular mechanisms underlying the compatibility between the pathogen and its hosts are still unknown.

Results

To explain the metabolic response of late blight, specifically photosynthesis inhibition in infected plants, we reconstructed a genome-scale metabolic network of the S. tuberosum leaf, PstM1. This metabolic network simulates the effect of this disease in the leaf metabolism. PstM1 accounts for 2751 genes, 1113 metabolic functions, 1773 gene-protein-reaction associations and 1938 metabolites involved in 2072 reactions. The optimization of the model for biomass synthesis maximization in three infection time points suggested a suppression of the photosynthetic capacity related to the decrease of metabolic flux in light reactions and carbon fixation reactions. In addition, a variation pattern in the flux of carboxylation to oxygenation reactions catalyzed by RuBisCO was also identified, likely to be associated to a defense response in the compatible interaction between P. infestans and S. tuberosum.

Conclusions

In this work, we introduced simultaneously the first metabolic network of S. tuberosum and the first genome-scale metabolic model of the compatible interaction of a plant with P. infestans.

     

Proteases and Peptidases of Castor Bean Endosperm: Enzyme Characterization and Changes during Germination

The endosperm of castor bean seeds (Ricinus communis L.) contains two —SH-dependent aminopeptidases, one hydrolyzing l-leucine-β-naphthylamide optimally at pH 7.0, and the other hydrolyzing l-proline-β-naphthylamide optimally at pH 7.5. After germination the endosperm contains in addition an —SH-dependent hemoglobin protease, a serine-dependent carboxypeptidase, and at least two —SH-dependent enzymes hydrolyzing the model substrate α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). The carboxypeptidase is active on a variety of N-carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-l-alanine and N-carbobenzoxy-l-tyrosine-l-leucine. The pH optima for the protease, carboxypeptidase, and BANAase acivities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively.     

Changing trends in biotechnology of secondary metabolism in medicinal and aromatic plants

Main conclusion

Medicinal and aromatic plants are known to produce secondary metabolites that find uses as flavoring agents, fragrances, insecticides, dyes and drugs. Biotechnology offers several choices through which secondary metabolism in medicinal plants can be altered in innovative ways, to overproduce phytochemicals of interest, to reduce the content of toxic compounds or even to produce novel chemicals. Detailed investigation of chromatin organization and microRNAs affecting biosynthesis of secondary metabolites as well as exploring cryptic biosynthetic clusters and synthetic biology options, may provide additional ways to harness this resource. Plant secondary metabolites are a fascinating class of phytochemicals exhibiting immense chemical diversity. Considerable enigma regarding their natural biological functions and the vast array of pharmacological activities, amongst other uses, make secondary metabolites interesting and important candidates for research. Here, we present an update on changing trends in the biotechnological approaches that are used to understand and exploit the secondary metabolism in medicinal and aromatic plants. Bioprocessing in the form of suspension culture, organ culture or transformed hairy roots has been successful in scaling up secondary metabolite production in many cases. Pathway elucidation and metabolic engineering have been useful to get enhanced yield of the metabolite of interest; or, for producing novel metabolites. Heterologous expression of putative plant secondary metabolite biosynthesis genes in a microbe is useful to validate their functions, and in some cases, also, to produce plant metabolites in microbes. Endophytes, the microbes that normally colonize plant tissues, may also produce the phytochemicals produced by the host plant. The review also provides perspectives on future research in the field.

     

The correlation between leaf-surface and leaf-tissue secondary metabolites: a case study with pyrrolizidine alkaloids in <Emphasis Type="Italic">Jacobaea</Emphasis> hybrid plants

Introduction

Usually whole plant or whole leaf extracts are analyzed to study the chemical ecology of insect-plant interactions. For herbivore species the contact with the leaf surface enables them to estimate the quality of the plant. The relationship between the leaf-surface and leaf-tissue secondary metabolites (SMs) could offer important new insights in insect-plant interactions mediated by SMs. Pyrrolizidine alkaloids (PAs), typical defense chemicals in Jacobaea species, are repellent for generalist herbivores but are attractive to specialists.

Objectives

Explore whether the PAs on the leaf surface are a reliable representation of the PAs in the leaf tissue in PA-containing plants.

Method

The concentration of individual PAs present on the leaf surface and in the corresponding leaf tissue from 37 genotypes (one plant from each genotype) of an F₂ generation of a cross between Jacobaea vulgaris and Jacobaea aquatica was measured by high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). PAs were removed from the leaf surface by extraction with a slightly acidic aqueous solution.

Results

The total amount of PAs present on the surface of the leaves was only 0.015% (range 0.001–0.163%) of the total amount present in the leaf tissue. Most PAs present in the leaf tissue were also found on the surface, except for jaconine, dehydrojaconine, dehydrojacoline and usaramine N-oxide. Positive correlations between leaf-surface and leaf-tissue concentrations were found for most of the jacobine-like and otosenine-like PAs, but correlations for total PA, senecionine- and erucifoline-like PAs were not significant.

Conclusion

These results indicate that PA variation on the leaf surface only partially reflects the PA variation in the leaf tissue. Because most herbivores are affected in a different manner by individual PAs, this result means that the leaf surface does not give a reliable estimate of plant quality to herbivores.

     

Prediction of C-glycosylated apigenin (vitexin) biosynthesis in <Emphasis Type="Italic">Ficus deltoidea</Emphasis> based on plant proteins identified by LC-MS/MS

Background

Plant secondary metabolites act as defence molecules to protect plants from biotic and abiotic stresses. In particular, C-glycosylated flavonoids are more stable and reactive than their O-glycosylated counterparts. Therefore, vitexin (apigenin 8-C glucoside) present in Ficus deltoidea is well-known for its antioxidant, anti-inflammatory, and antidiabetic properties.

Methods

Phenol based extraction was used to extract proteins (0.05% yield) with less plant pigments. This can be seen from clear protein bands in gel electrophoresis. In-gel trypsin digestion was subsequently carried out and analysed for the presence of peptides by LC-MS/MS.

Results

Thirteen intact proteins are identified on a 12% polyacrylamide gel. The mass spectra matching was found to have 229 proteins, and 11.4% of these were involved in secondary metabolism. Proteins closely related to vitexin biosynthesis are listed and their functions are explained mechanistically. Vitexin synthesis is predicted to involve plant polyketide chalcone synthase, isomerization by chalcone isomerase, oxidation by cytochrome P450 to convert flavanone to flavone, and transfer of sugar moiety by C-glycosyltransferase, followed by dehydration to produce flavone-8-C-glucosides.

Conclusions

Phenol based extraction, followed by gel electrophoresis and LC-MS/MS could identify proteome explaining vitexin biosynthesis in F. deltoidea. Many transferases including β-1,3-galactosyltransferase 2 and glycosyl hydrolase family 10 protein were detected in this study. This explains the importance of transferase family proteins in Cglycosylated apigenin biosynthesis in medicinal plant.

     

Metabolic robustness and network modularity: a model study

Background

Several studies have mentioned network modularity—that a network can easily be decomposed into subgraphs that are densely connected within and weakly connected between each other—as a factor affecting metabolic robustness. In this paper we measure the relation between network modularity and several aspects of robustness directly in a model system of metabolism.

Methodology/Principal Findings

By using a model for generating chemical reaction systems where one can tune the network modularity, we find that robustness increases with modularity for changes in the concentrations of metabolites, whereas it decreases with changes in the expression of enzymes. The same modularity scaling is true for the speed of relaxation after the perturbations.

Conclusions/Significance

Modularity is not a general principle for making metabolism either more or less robust; this question needs to be addressed specifically for different types of perturbations of the system.     

Comprehensive metabolite profiling of onion bulbs (<Emphasis Type="Italic">Allium cepa</Emphasis>) using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry

Introduction

Onion (Allium cepa) represents one of the most important horticultural crops and is used as food, spice and medicinal plant almost worldwide. Onion bulbs accumulate a broad range of primary and secondary metabolites which impact nutritional, sensory and technological properties.

Objectives

To complement existing analytical methods targeting individual compound classes this work aimed at the development and validation of an analytical workflow for comprehensive metabolite profiling of onion bulbs.

Method

Metabolite profiling was performed by liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC/ESI-QTOFMS). For annotation of metabolites accurate mass tandem mass spectrometry experiments were carried out.

Results

On the basis of LC/ESI-QTOFMS and two chromatographic methods an analytical workflow was developed which facilitates profiling of polar and semi-polar onion metabolites including fructooligosaccharides, proteinogenic amino acids, peptides, S-substituted cysteine conjugates, flavonoids and saponins. To minimize enzymatic conversion of S-alk(en)ylcysteine sulfoxides, a sample preparation and extraction protocol for fresh onions was developed comprising cryohomogenization and a low-temperature quenching step. A total of 123 metabolites were annotated and characterized by chromatographic and tandem mass spectral data. For validation, recovery rates and matrix effects were determined for 15 model compounds. Repeatability and linearity were assessed for more than 80 endogenous metabolites.

Conclusion

As exemplarily demonstrated by comparative metabolic analysis of six onion cultivars the established analytical workflow in combination with targeted and non-targeted data analysis strategies can be successfully applied for comprehensive metabolite profiling of onion bulbs.

     

Successive Mineralization and Detoxification of Benzo[a]pyrene by the White Rot Fungus Bjerkandera sp. Strain BOS55 and Indigenous Microflora

White rot fungi can oxidize high-molecular-weight polycyclic aromatic hydrocarbons (PAH) rapidly to polar metabolites, but only limited mineralization takes place. The objectives of this study were to determine if the polar metabolites can be readily mineralized by indigenous microflora from several inoculum sources, such as activated sludge, forest soils, and PAH-adapted sediment sludge, and to determine if such metabolites have decreased mutagenicity compared to the mutagenicity of the parent PAH. ¹⁴C-radiolabeled benzo[a]pyrene was subjected to oxidation by the white rot fungus Bjerkandera sp. strain BOS55. After 15 days, up to 8.5% of the [¹⁴C]benzo[a]pyrene was recovered as ¹⁴CO₂ in fungal cultures, up to 73% was recovered as water-soluble metabolites, and only 4% remained soluble in dibutyl ether. Thin-layer chromatography analysis revealed that many polar fluorescent metabolites accumulated. Addition of indigenous microflora to fungal cultures with oxidized benzo[a]pyrene on day 15 resulted in an initially rapid increase in the level of ¹⁴CO₂ recovery to a maximal value of 34% by the end of the experiments (>150 days), and the level of water-soluble label decreased to 16% of the initial level. In fungal cultures not inoculated with microflora, the level of ¹⁴CO₂ recovery increased to 13.5%, while the level of recovery of water-soluble metabolites remained as high as 61%. No large differences in ¹⁴CO₂ production were observed with several inocula, showing that some polar metabolites of fungal benzo[a]pyrene oxidation were readily degraded by indigenous microorganisms, while other metabolites were not. Of the inocula tested, only PAH-adapted sediment sludge was capable of directly mineralizing intact benzo[a]pyrene, albeit at a lower rate and to a lesser extent than the mineralization observed after combined treatment with white rot fungi and indigenous microflora. Fungal oxidation of benzo[a]pyrene resulted in rapid and almost complete elimination of its high mutagenic potential, as observed in the Salmonella typhimurium revertant test performed with strains TA100 and TA98. Moreover, no direct mutagenic metabolite could be detected during fungal oxidation. The remaining weak mutagenic activity of fungal cultures containing benzo[a]pyrene metabolites towards strain TA98 was further decreased by subsequent incubations with indigenous microflora.Bioremediation of polycyclic aromatic hydrocarbon (PAH)-polluted soil is severely hampered by the low rate of degradation of the higher PAH, particularly the four- and five-ring PAH (6, 32). These higher PAH have very low water solubility and are often tightly bound to soil particles. This results in very low bioavailability for bacterial degradation. The observation that white rot fungi can oxidize PAH rapidly with their extracellular ligninolytic enzyme systems has therefore raised interest in the use of these organisms for bioremediation of PAH-polluted soils (3, 9). Although PAHs are extensively oxidized by white rot fungi, the degree of mineralization to CO₂ is always limited. In various studies evaluating the degradation of the potent carcinogen benzo[a]pyrene by several white rot fungal species, from 0.17 to 19% of the radiolabeled PAH was recovered as ¹⁴CO₂ (4, 5, 26). The major products of the oxidation were both nonpolar and polar metabolites. The accumulation of such metabolites could be a reason for concern, since mammalian and fungal monooxygenases can oxidize benzo[a]pyrene to epoxides and dihydrodiols, which are very potent carcinogens (28, 29). However, peroxidase-mediated extracellular oxidation of benzo[a]pyrene in cultures of white rot fungi results initially in benzo[a]pyrenediones, which show weak mutagenic activity (29). These primary metabolites are rapidly oxidized further to unidentified metabolites by Phanerochaete laevis and Phanerochaete chrysosporium (5, 26). Furthermore, the oxidized benzo[a]pyrene metabolites have a higher aqueous solubility. Since the low bioavailability of PAH is a major rate-limiting factor in the degradation of these compounds by bacteria (27, 31), the increased bioavailability of oxidized PAH metabolites suggests that these compounds can be more easily mineralized by bacteria.The aim of this study was to investigate the degradation and mineralization of the five-ring PAH benzo[a]pyrene by the white rot fungus Bjerkandera sp. strain BOS55 and the subsequent mineralization of the metabolites by natural mixed cultures of microorganisms. During the oxidation and mineralization of benzo[a]pyrene, the decrease in the mutagenicity of the metabolites was monitored. The white rot fungal strain Bjerkandera sp. strain BOS55 was used because of its outstanding ability to rapidly oxidize PAH (8, 19) and because extensive information concerning its physiology is available (7, 18, 20, 22, 23).     

Regulation of a Chemical Defense against Herbivory Produced by Symbiotic Fungi in Grass Plants

Neotyphodium uncinatum and Neotyphodium siegelii are fungal symbionts (endophytes) of meadow fescue (MF; Lolium pratense), which they protect from insects by producing loline alkaloids. High levels of lolines are produced following insect damage or mock herbivory (clipping). Although loline alkaloid levels were greatly elevated in regrowth after clipping, loline-alkaloid biosynthesis (LOL) gene expression in regrowth and basal tissues was similar to unclipped controls. The dramatic increase of lolines in regrowth reflected the much higher concentrations in young (center) versus older (outer) leaf blades, so LOL gene expression was compared in these tissues. In MF-N. siegelii, LOL gene expression was similar in younger and older leaf blades, whereas expression of N. uncinatum LOL genes and some associated biosynthesis genes was higher in younger than older leaf blades. Because lolines are derived from amino acids that are mobilized to new growth, we tested the amino acid levels in center and outer leaf blades. Younger leaf blades of aposymbiotic plants (no endophyte present) had significantly higher levels of asparagine and sometimes glutamine compared to older leaf blades. The amino acid levels were much lower in MF-N. siegelii and MF-N. uncinatum compared to aposymbiotic plants and MF with Epichloë festucae (a closely related symbiont), which lacked lolines. We conclude that loline alkaloid production in young tissue depleted these amino acid pools and was apparently regulated by availability of the amino acid substrates. As a result, lolines maximally protect young host tissues in a fashion similar to endogenous plant metabolites that conform to optimal defense theory.Loline alkaloids (LAs; Hofmeister, 1892; Siegel et al., 1990; TePaske et al., 1993; Blankenship et al., 2001) are protective secondary metabolites produced by some Epichloë and Neotyphodium spp. (epichloae), fungi that live as systemic symbionts in many cool season grasses (Poaceae subfamily Pooideae). The lolines are active against a broad spectrum of insects (Schardl et al., 2007) and are derived from l-Pro (Pro) and l-homoserine (Hse; Blankenship et al., 2005). Mock herbivory (clipping plants) is reported to induce higher levels of lolines in several grass-epichloë symbiota (Craven et al., 2001; Bultman et al., 2004; Gonthier et al., 2008), suggesting that the epichloae have evolved to regulate their metabolism in a manner appropriate for defense of their hosts. However, little is known of the regulation of LA synthesis in symbio and whether these symbionts follow prevailing models for how plants deploy chemical defenses against herbivores (McKey, 1979; Rhoades, 1979; Barto and Cipollini, 2005).The loline-alkaloid biosynthesis (LOL) gene cluster contains nine genes likely to direct LA production (Spiering et al., 2005). Neotyphodium uncinatum contains two highly similar LOL clusters (LOL1 and LOL2), and a single LOL cluster has been found in each of the LA-producing species, Neotyphodium coenophialum, Neotyphodium siegelii, and some strains of Epichloë festucae, among others (Spiering et al., 2005; Kutil et al., 2007). Fermentation cultures of N. uncinatum produce lolines, and studies involving application of labeled precursors and intermediates have almost completely elucidated the LA biosynthetic pathway (Blankenship et al., 2005; Spiering et al., 2005; Faulkner et al., 2006; Schardl et al., 2007). Putative roles of the LOL gene products—based on sequence relationships to known enzyme classes—fit well with the pathway. Furthermore, an RNA interference knockdown of lolC reduces LA levels, and a lolP knockout prevents conversion of N-methylloline to N-formylloline (Spiering et al., 2005, 2008). Expression kinetics of the LOL genes are tightly correlated with each other and with the LA production phase in N. uncinatum cultures (Zhang et al., 2009). This finding raises the question whether and how LOL gene expression in symbio relates to changes in LA levels in response to development and stresses in host plants.LA production in symbio may be influenced by physiological differences among plant tissues and developmental stages, as well as differences in nutritional status and environmental stresses (Kennedy and Bush, 1983; Belesky et al., 1987; Justus et al., 1997; Tong et al., 2006). Given the anti-insect activity of lolines, effects of plant damage on LA levels are of particular interest. Mock herbivory (clipping of leaves) leads to apparent increases in LA concentrations in regrowth tissues of tall fescue (TF; Lolium arundinaceum) symbiotic with N. coenophialum (Bultman et al., 2004; Sullivan et al., 2007) and of meadow fescue (MF; Lolium pratense) symbiotic with N. uncinatum or N. siegelii (Craven et al., 2001). Despite the higher LA levels, however, clipping or damage of TF-N. coenophialum by the herbivore Spodoptera frugiperda (fall armyworm) was reported to elicit only minor, marginally significant (P = 0.052) effects on expression of lolC (Sullivan et al., 2007). A study of the Glyceria striata-Epichloë glyceriae symbiotum demonstrated significantly higher expression of lolC and higher LA production when the grass was artificially damaged, whereas the effect of damage by S. frugiperda on LA concentrations and lolC expression was not significant (Gonthier et al., 2008).Prevailing concepts about how plants deploy chemical defenses include the optimal defense theory (ODT; McKey, 1979; Rhoades, 1979) and the growth differentiation balance hypothesis (GDBH; Barto and Cipollini, 2005). The ODT addresses the distribution of chemical defenses in the plant, predicting that such defenses will be concentrated in tissues that have relatively little means to physically inhibit herbivory (e.g. in young tissues) and are important in the fitness of the plant. The GBDH addresses the location of biosynthesis and predicts that mature tissues are more likely to produce secondary metabolites than are actively growing tissues, which instead need to use resources for biomass production. It is intriguing to consider whether the epichloae obey the predictions of ODT and GDBH, considering that many epichloae protect their hosts by synthesizing insecticidal alkaloids, but they are also evolutionarily derived from plant-pathogenic fungi (Moon et al., 2004) and do not always enhance host fitness (Faeth et al., 2004). In order to address these questions, it is necessary to understand how secondary metabolism of the epichloae is regulated in symbio. The production of lolines in MF-N. uncinatum and MF-N. siegelii is an ideal test case because the lolines accumulate to very high levels—up to 1.9% dry weight—in regrowth of clipped plants (Craven et al., 2001). Here, we test the hypotheses that LOL gene expression and substrate availability correlate with LA levels in younger versus older leaf tissues and in response to clipping in MF-N. uncinatum and MF-N. siegelii symbiota.     

Identifying diseases-related metabolites using random walk

Background

Metabolites disrupted by abnormal state of human body are deemed as the effect of diseases. In comparison with the cause of diseases like genes, these markers are easier to be captured for the prevention and diagnosis of metabolic diseases. Currently, a large number of metabolic markers of diseases need to be explored, which drive us to do this work.

Methods

The existing metabolite-disease associations were extracted from Human Metabolome Database (HMDB) using a text mining tool NCBO annotator as priori knowledge. Next we calculated the similarity of a pair-wise metabolites based on the similarity of disease sets of them. Then, all the similarities of metabolite pairs were utilized for constructing a weighted metabolite association network (WMAN). Subsequently, the network was utilized for predicting novel metabolic markers of diseases using random walk.

Results

Totally, 604 metabolites and 228 diseases were extracted from HMDB. From 604 metabolites, 453 metabolites are selected to construct the WMAN, where each metabolite is deemed as a node, and the similarity of two metabolites as the weight of the edge linking them. The performance of the network is validated using the leave one out method. As a result, the high area under the receiver operating characteristic curve (AUC) (0.7048) is achieved. The further case studies for identifying novel metabolites of diabetes mellitus were validated in the recent studies.

Conclusion

In this paper, we presented a novel method for prioritizing metabolite-disease pairs. The superior performance validates its reliability for exploring novel metabolic markers of diseases.

     

Duration of Priming of Two Indirect Plant Defenses

When plants are infested by herbivores, they emit herbivore-induced plant volatiles (HIPVs) that attract carnivorous natural enemies of herbivores. Furthermore, there are increasing evidences that defenses of intact plants against herbivores are primed when exposed to HIPVs. We previously reported that lima bean leaf volatiles induced by the herbivorous mites Tetranychus urticae primed two T. urtiae-induced indirect defenses in neighboring conspecific plants: HIPV emission and extrafloral nectar (EFN) secretion. An intriguing unanswered question is whether the durations of these two defenses are the same. Here, we show that the durations of the two defenses were the same for up to two days after the initiation of T. urticae damage. The two induced primed defense would act as a battery of defense in exposed plants.Key Words: herbivore-induced plant volatiles, indirect, defense, induced response, plant-plant interaction, primingWhen infested by herbivores, plants defend themselves indirectly by emitting herbivore-induced plant volatiles (HIPVs). One of the ecological functions of HIPVs is to attract carnivorous natural enemies of the herbivores.^1,2 Recently, it was reported that the emission of HIPVs primed defenses against herbivores in neighboring intact plants.^3–7 Thus, HIPVs also mediate interactions between infested and intact plants.⁸ The enhanced defense in response to HIPVs in intact plants is called ‘priming’, which has been studied intensively in plant-pathogen interactions,⁹ but not so in plant-insect interactions.We previously reported that exposure to HIPVs emitted from lima bean leaves infested by Tetranychus urticae primed HIPV production in detached intact conspecific leaves.³ We also reported that exposure to HIPVs, produced in response to T. urticae damage,⁴ primed the induced production of extrafloral nectar (EFN; an alternative food source for predators^10,11 in lima bean plants. An intriguing question is whether the two primed defenses work as a battery against T. urticae. To answer this, we examined the duration of primed HIPV production by lima bean plants using the same experimental set-up as our previous study of EFN priming by conspecific plants.⁴For exposure of plants to HIPVs, we used a 60 × 60 × 60 cm cage with two 30 × 30 cm windows on opposite sides of the cage.¹² As odor sources, we used eight plants that had been infested with 60 adult T. urticae females per plant for 1 day. Eight uninfested plants were used as control odor sources. Two uninfested plants were placed in a cage with the odor source plants and exposed to either HIPVs or uninfested plant volatiles (UPVs) for 10 days in a climate-controlled room (25 ± 2°C, 60–70% RH, 16:8; L:D).A Y-tube olfactometer¹³ was used to examine the response of the predators to HIPVs. Adult female P. persimilis were randomly selected from a colony and individually positioned at the beginning of the iron wire. When test mites reached the end of one arm of the olfactometer, their choice was recorded. We tested the olfactory responses of the predator toward (1) plants infested by T. urticae for two days after exposure to UPVs vs. plants infested by T. urticae for two days after exposure to HIPVs, and (2) plants infested by T. urticae for four days after exposure to UPVs vs. plants infested by T. urticae for four days after exposure to HIPVs.HIPV-exposed plants attracted more predators than UPV-exposed plants in a Y-tube olfactometer when infested by T. urticae for two days (Fig. 1A). By contrast, the predators did not distinguish between HIPV- and UPV-exposed plants when infested by T. urticae for four days (Fig. 1B). Our previous study showed that HIPV-exposed plants secreted significantly larger amounts of EFN secretion than UPV-exposed plants infested by T. urticae for two days under the same experimental condition as in this study.⁴ However, the difference was not significant when they were infested for four days.⁴Open in a separate windowFigure 1The olfactory response of P. persimilis females to volatiles from the odor-exposed plants, as determined in a Y-tube olfactometer: (A) plants infested by T. urticae for two days after exposure to UPVs (UPV-exposed—T. urticae 2d) vs. plants infested by T. urticae for two days after exposure to HIPVs (HIPV-exposed—T. urticae 2d), and (B) plants infested by T. urticae for four days after exposure to UPVs (UPV-exposed—T. urticae 4d) vs. plants infested by T. urticae for four days after the exposure to HIPVs (HIPV-exposed—T. urticae 4d). Asterisks beside each bar indicate a significant difference between the first trifoliate leaves and the primary leaves. Asterisks beside a bar indicate a significant difference (binomial test: p < 0.001).Lima bean plants increase the amount of endogenous jasmonic acid after exposure to HIPVs.¹⁴ Jasmonic acid, an important plant hormone regulating a defense signaling pathway against herbivores and pathogens,^15,16 is reported to be involved in the induction of both volatile emission^17,18 and EFN secretion¹⁹ in response to T. urticae damage in lima bean plants. The increase of endogenous jasmonic acid in HIPV-exposed plants may partly explain the simultaneous priming of the two defenses.In this study, we showed that the durations of priming of two indirect defenses were roughly the same for up to two days. Priming of these two indirect defenses would thus be a battery of defense at the outset of T. urticae damage. Further study is necessarily to test whether the primed battery of induced defense increases the fitness of the exposed plants.     

Inhibition of chloroplast reactions with phenylmercuric acetate

Phenylmercuric acetate is a selective inhibitor of the photosynthetic activities of isolated spinach (Spinacia oleracea) chloroplasts. At 5 μm concentration of phenylmercuric acetate, photophosphorylation is inhibited. At 33 μm phenylmercuric acetate, ferredoxin is inactivated. Ferredoxin-NADP oxidoreductase is 50% inhibited at 100 μm phenylmercuric acetate. Photosystem II reactions are 50% inhibited at 150 μm phenylmercuric acetate and very much higher cooncentrations—500 μm—are needed to approach complete inhibition. Phenylmercuric acetate inhibition of photosystem II appears to be selective, blocking a site between the 3-(3,4-dichlorophenyl)-1,1-dimethyl urea sensitive site and the site inactivated by high concentrations of tris buffer.     

Feed-forward neural network model for hunger and satiety related VAS score prediction

Background

An artificial neural network approach was chosen to model the outcome of the complex signaling pathways in the gastro-intestinal tract and other peripheral organs that eventually produce the satiety feeling in the brain upon feeding.

Methods

A multilayer feed-forward neural network was trained with sets of experimental data relating concentration-time courses of plasma satiety hormones to Visual Analog Scales (VAS) scores. The network successfully predicted VAS responses from sets of satiety hormone data obtained in experiments using different food compositions.

Results

The correlation coefficients for the predicted VAS responses for test sets having i) a full set of three satiety hormones, ii) a set of only two satiety hormones, and iii) a set of only one satiety hormone were 0.96, 0.96, and 0.89, respectively. The predicted VAS responses discriminated the satiety effects of high satiating food types from less satiating food types both in orally fed and ileal infused forms.

Conclusions

From this application of artificial neural networks, one may conclude that neural network models are very suitable to describe situations where behavior is complex and incompletely understood. However, training data sets that fit the experimental conditions need to be available.

     

Effect of Lake Trophic Status and Rooted Macrophytes on Community Composition and Abundance of Ammonia-Oxidizing Prokaryotes in Freshwater Sediments

Communities of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in freshwater sediments and those in association with the root system of the macrophyte species Littorella uniflora, Juncus bulbosus, and Myriophyllum alterniflorum were compared for seven oligotrophic to mesotrophic softwater lakes and acidic heathland pools. Archaeal and bacterial ammonia monooxygenase alpha-subunit (amoA) gene diversity increased from oligotrophic to mesotrophic sites; the number of detected operational taxonomic units was positively correlated to ammonia availability and pH and negatively correlated to sediment C/N ratios. AOA communities could be grouped according to lake trophic status and pH; plant species-specific communities were not detected, and no grouping was apparent for AOB communities. Relative abundance, determined by quantitative PCR targeting amoA, was always low for AOB (<0.05% of all prokaryotes) and slightly higher for AOA in unvegetated sediment and AOA in association with M. alterniflorum (0.01 to 2%), while AOA accounted for up to 5% in the rhizospheres of L. uniflora and J. bulbosus. These results indicate that (i) AOA are at least as numerous as AOB in freshwater sediments, (ii) aquatic macrophytes with substantial release of oxygen and organic carbon into their rhizospheres, like L. uniflora and J. bulbosus, increase AOA abundance; and (iii) AOA community composition is generally determined by lake trophy, not by plant species-specific interactions.Oxygen release from the roots of macrophyte species such as Littorella uniflora (L.) Asch. (shore weed), Lobelia dortmanna L. (water lobelia), and Glyceria maxima (Hartm.) Holmb. (reed sweet grass) stimulates nitrification and coupled nitrification-denitrification in the rhizosphere compared to that in unvegetated sediment (2, 36, 40). These interactions are of high ecological relevance especially in oligotrophic systems, since enhanced nitrogen loss due to rhizosphere-associated denitrification can retard natural eutrophication and succession of plant communities (1). While the microbial communities involved in coupled nitrification-denitrification have been well studied in rice paddy soils (7, 11), less information is available for natural freshwater sediments, especially those from oligotrophic lakes (2, 26).The first key step of coupled nitrification-denitrification, the oxidation of ammonia to nitrite, is catalyzed by two groups of prokaryotes—the ammonia-oxidizing bacteria (AOB) (24) and the only recently recognized ammonia-oxidizing archaea (AOA) (22). For both groups, the gene encoding the alpha-subunit of ammonia monooxygenase (amoA) has been widely used as a functional marker to analyze their community compositions (15, 25); recent studies demonstrated the ubiquity of AOA and their predominance over AOB in a broad range of environments (32, 38). AOA, but not AOB, were also strongly enriched in the rhizosphere of the freshwater macrophyte Littorella uniflora in a mesotrophic Danish lake, suggesting that AOA were primarily responsible for increased rates of nitrification in the rhizosphere of this plant species (19). Moreover, ammonia oxidizer communities differed between rhizosphere and unvegetated sediment, indicating a plant-specific effect on AOA and AOB community composition. The objectives of this study were therefore to test whether (i) AOA generally predominate over AOB in freshwater sediments and especially in macrophyte rhizospheres and (ii) macrophytes have species-specific effects on abundance and community composition of AOA and AOB in rhizosphere sediments and on root surfaces.To address these questions, two shallow heathland pools and five lakes in Denmark and Germany, ranging from low-pH and dystrophic sites to neutral-pH and oligotrophic and mesotrophic sites, were chosen, and three macrophyte species—Littorella uniflora, Juncus bulbosus L. (bulbous rush), and Myriophyllum alterniflorum DC. (alternate water milfoil)—were selected as model systems. These plant species differ in nitrogen nutrition, extent of radial oxygen loss, and lifestyle, presumably resulting in differential, plant species-specific effects on rhizosphere- and root-associated AOA and AOB communities. L. uniflora prefers nitrate as the nitrogen source, while J. bulbosus prefers ammonium (41, 45); oxygen release is high to moderate from the roots of L. uniflora and J. bulbosus (9, 12) but is minor from the roots of M. alterniflorum (M. Herrmann, P. Stief, and A. Schramm, unpublished results); L. uniflora and J. bulbosus remain photosynthetically active throughout the year, while only the below-ground parts of M. alterniflorum are retained during winter.Rhizosphere sediments and roots from each plant species were sampled from three different sites per species, and unvegetated sediment was obtained from all seven sites. The comparison of samples from these different sites and compartments (rhizosphere, root surface, unvegetated sediment) allowed an evaluation of the importance of plant species relative to that of environmental conditions related to lake trophic status and pH on ammonia oxidizer communities.     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Polymorphic Genes of Major Effect: Consequences for Variation,Selection and Evolution in Arabidopsis thaliana

The importance of genes of major effect for evolutionary trajectories within and among natural populations has long been the subject of intense debate. For example, if allelic variation at a major-effect locus fundamentally alters the structure of quantitative trait variation, then fixation of a single locus can have rapid and profound effects on the rate or direction of subsequent evolutionary change. Using an Arabidopsis thaliana RIL mapping population, we compare G-matrix structure between lines possessing different alleles at ERECTA, a locus known to affect ecologically relevant variation in plant architecture. We find that the allele present at ERECTA significantly alters G-matrix structure—in particular the genetic correlations between branch number and flowering time traits—and may also modulate the strength of natural selection on these traits. Despite these differences, however, when we extend our analysis to determine how evolution might differ depending on the ERECTA allele, we find that predicted responses to selection are similar. To compare responses to selection between allele classes, we developed a resampling strategy that incorporates uncertainty in estimates of selection that can also be used for statistical comparisons of G matrices.THE structure of the genetic variation that underlies phenotypic traits has important consequences for understanding the evolution of quantitative traits (Fisher 1930; Lande 1979; Bulmer 1980; Kimura 1983; Orr 1998; Agrawal et al. 2001). Despite the infinitesimal model''s allure and theoretical tractability (see Orr and Coyne 1992; Orr 1998, 2005a,b for reviews of its influence), evidence has accumulated from several sources (artificial selection experiments, experimental evolution, and QTL mapping) to suggest that genes of major effect often contribute to quantitative traits. Thus, the frequency and role of genes of major effect in evolutionary quantitative genetics have been a subject of intense debate and investigation for close to 80 years (Fisher 1930; Kimura 1983; Orr 1998, 2005a,b). Beyond the conceptual implications, the prevalence of major-effect loci also affects our ability to determine the genetic basis of adaptations and species differences (e.g., Bradshaw et al. 1995, 1998).Although the existence of genes of major effect is no longer in doubt, we still lack basic empirical data on how segregating variation at such genes affects key components of evolutionary process (but see Carrière and Roff 1995). In other words, How does polymorphism at genes of major effect alter patterns of genetic variation and covariation, natural selection, and the likely response to selection? The lack of data stems, in part, from the methods used to detect genes of major effect: experimental evolution (e.g., Bull et al. 1997; Zeyl 2005) and QTL analysis (see Erickson et al. 2004 for a review) often detect such genes retrospectively after they have become fixed in experimental populations or the species pairs used to generate the mapping population. The consequences of polymorphism at these genes on patterns of variation, covariation, selection, and the response to selection—which can be transient (Agrawal et al. 2001)—are thus often unobserved.A partial exception to the absence of data on the effects of major genes comes from artificial selection experiments, in which a substantial evolutionary response to selection in the phenotype after a plateau is often interpreted as evidence for the fixation of a major-effect locus (Frankham et al. 1968; Yoo 1980a,b; Frankham 1980; Shrimpton and Robertson 1988a,b; Caballero et al. 1991; Keightley 1998; see Mackay 1990 and Hill and Caballero 1992 for reviews). However, many of these experiments report only data on the selected phenotype (e.g., bristle number) or, alternatively, the selected phenotype and some measure of fitness (e.g., Frankham et al. 1968, Yoo 1980b; Caballero et al. 1991; Mackay et al. 1994; Fry et al. 1995; Nuzhdin et al. 1995; Zur Lage et al. 1997), making it difficult to infer how a mutation will affect variation, covariation, selection, and evolutionary responses for a suite of traits that might affect fitness themselves. One approach is to document how variation at individual genes of major effect affects the genetic variance–covariance matrix (“G matrix”; Lande 1979), which represents the additive genetic variance and covariance between traits.Although direct evidence for variation at major-effect genes altering patterns of genetic variation, covariation, and selection is rare, there is abundant evidence for the genetic mechanisms that could produce these dynamics. A gene of major effect could have these consequences due to any of at least three genetic mechanisms: (1) pleiotropy, where a gene of major effect influences several traits, including potentially fitness, simultaneously, (2) physical linkage or linkage disequilibrium (LD), in which a gene of major effect is either physically linked or in LD with other genes that influence other traits under selection, and (3) epistasis, in which the allele present at a major-effect gene alters the phenotypic effect of other loci and potentially phenotypes under selection. Evidence for these three evolutionary genetic mechanisms leading to changes in suites of traits comes from a variety of sources, including mutation accumulation experiments (Clark et al. 1995; Fernandez and Lopez-Fanjul 1996), mutation induction experiments (Keightley and Ohnishi 1998), artificial selection experiments (Long et al. 1995), and transposable element insertions (Rollmann et al. 2006). For pleiotropy in particular, major-effect genes that have consequences on several phenotypic traits are well known from the domestication and livestock breeding literature [e.g., myostatin mutations in Belgian blue cattle and whippets (Arthur 1995; Grobet et al. 1997; Mosher et al. 2007), halothane genes in pigs (Christian and Rothschild 1991; Fujii et al. 1991), and Booroola and Inverdale genes in sheep (Amer et al. 1999; Visscher et al. 2000)]. While these data suggest that variation at major-effect genes could—and probably does—influence variation, covariation, and selection on quantitative traits, data on the magnitude of these consequences remain lacking.Recombinant inbred line (RIL) populations are a promising tool for investigating the influence of major-effect loci. During advancement of the lines from F₂''s to RILs, alternate alleles at major-effect genes (and most of the rest of the genome) will be made homozygous, simplifying comparisons among genotypic classes. Because of the high homozygosity, individuals within RILs are nearly genetically identical, facilitating phenotyping of many genotypes under a range of environments. In addition, because of recombination, alternative alleles are randomized across genetic backgrounds—facilitating robust comparisons between sets of lines differing at a major-effect locus.Here we investigate how polymorphism at an artificially induced mutation, the erecta locus in Arabidopsis thaliana, affects the magnitude of these important evolutionary genetic parameters under ecologically realistic field conditions. We use the Landsberg erecta (Ler) × Columbia (Col) RIL population of A. thaliana to examine how variation at a gene of major effect influences genetic variation, covariation, and selection on quantitative traits in a field setting. The Ler × Col RIL population is particularly suitable, because it segregates for an artificially induced mutation at the erecta locus, which has been shown to influence a wide variety of plant traits. The Ler × Col population thus allows a powerful test of the effects of segregating variation at a gene—chosen a priori—with numerous pleiotropic effects. The ERECTA gene is a leucine-rich receptor-like kinase (LRR-RLK) (Torii et al. 1996) and has been shown to affect plant growth rates (El-Lithy et al. 2004), stomatal patterning and transpiration efficiency (Masle et al. 2005; Shpak et al. 2005), bacterial pathogen resistance (Godiard et al. 2003), inflorescence and floral organ size and shape (Douglas et al. 2002; Shpak et al. 2003, 2004), and leaf polarity (Xu et al. 2003; Qi et al. 2004).Specifically, we sought to answer the following questions: (1) Is variation at erecta significantly associated with changes to the G matrix? (2) Is variation at erecta associated with changes in natural selection on genetically variable traits? And (3) is variation at erecta associated with significantly different projected evolutionary responses to selection?