Opposite variations in fumarate and malate dominate metabolic phenotypes of Arabidopsis salicylate mutants with abnormal biomass under chilling

In chilling conditions (5 degrees C), salicylic acid (SA)-deficient mutants (sid2, eds5 and NahG) of Arabidopsis thaliana produced more biomass than wild type (Col-0), whereas the SA overproducer cpr1 was extremely stunted. The hypothesis that these phenotypes were reflected in metabolism was explored using 600MHz H-1 nuclear magnetic resonance (NMR) analysis of unfractionated polar shoot extracts. Biomass-related metabolic phenotypes were identified as multivariate data models of these NMR fingerprints'. These included principal components that correlated with biomass. Also, partial least squares-regression models were found to predict the relative size of plants in previously unseen experiments in different light intensities, or relative size of one genotype from the others. The dominant signal in these models was fumarate, which was high in SA-deficient mutants, intermediate in Col-0 and low in cpr1 at 5 degrees C. Among signals negatively correlated with biomass, malate was prominent. Abundance of transcripts of the FUM2 cytosolic fumarase (At5g50950) showed strong positive correlation with fumarate levels and with biomass, whereas no significant differences were found for the FUM1 mitochondrial fumarase (At2g47510). It was confirmed that the morphological effects of SA under chilling find expression in the metabolome, with a role of fumarate highlighted

Defining genetic and chemical diversity in wheat grain by 1H-NMR spectroscopy of polar metabolites

Scope The application of high‐throughput 1H nuclear magnetic resonance (1H‐NMR) of unpurified extracts to determine genetic diversity and the contents of polar components in grain of wheat. Methods and results Milled whole wheat grain was extracted with 80:20 D2O:CD3OD containing 0.05% d4–trimethylsilylpropionate. 1H‐NMR spectra were acquired under automation at 300°K using an Avance Spectrometer operating at 600.0528 MHz. Regions for individual metabolites were identified by comparison to a library of known standards run under identical conditions. The individual 1H‐NMR peaks or levels of known metabolites were then compared by Principal Component Analysis using SIMCA‐P software. Conclusions High‐throughput 1H‐NMR is an excellent tool to compare the extent of genetic diversity within and between wheat species, and to quantify specific components (including glycine betaine, choline, and asparagine) in individual genotypes. It can also be used to monitor changes in composition related to environmental factors and to support comparisons of the substantial equivalence of transgenic lines

Investigating the beneficial traits of Trichoderma hamatum GD12 for sustainable agriculture : insights from genomics

Trichoderma hamatum strain GD12 is unique in that it can promote plant growth, activate biocontrol against pre- and post-emergence soil pathogens and can induce systemic resistance to foliar pathogens. This study extends previous work in lettuce to demonstrate that GD12 can confer beneficial agronomic traits to other plants, providing examples of plant growth promotion in the model dicot, Arabidopsis thaliana and induced foliar resistance to Magnaporthe oryzae in the model monocot rice. We further characterize the lettuce-T. hamatum interaction to show that bran extracts from GD12 and an N-acetyl-β-D-glucosamindase-deficient mutant differentially promote growth in a concentration dependent manner, and these differences correlate with differences in the small molecule secretome. We show that GD12 mycoparasitises a range of isolates of the pre-emergence soil pathogen Sclerotinia sclerotiorum and that this interaction induces a further increase in plant growth promotion above that conferred by GD12. To understand the genetic potential encoded by T. hamatum GD12 and to facilitate its use as a model beneficial organism to study plant growth promotion, induced systemic resistance and mycoparasitism we present de novo genome sequence data. We compare GD12 with other published Trichoderma genomes and show that T. hamatum GD12 contains unique genomic regions with the potential to encode novel bioactive metabolites that may contribute to GD12's agrochemically important traits. Read Full Tex

Pretreatment with Naltrindole exhibits robust cardioprotection in an isolated rat heart model of ischemia-reperfusion injury

Previously, a novel three-amino acid peptide (tripeptide) that is structurally similar to other amino acid-based delta and kappa-opioid receptor agonists (the predominant opioid receptor subtypes in heart tissue) demonstrated a significant reduction in infarct size and improved cardiac function when administered during preconditioning in isolated rat hearts using the Langendorf model. The cardioprotective effects of tripeptide were blocked by naloxone (NX, broad-spectrum opioid antagonist) and nor-binaltorphine (BNI, kappa-opioid receptor antagonist), whereas naltrindole (NTI, delta-opioid receptor antagonist) seemed to augment the effects of tripeptide. To determine whether the cardioprotective effects of the combination were due to tripeptide or NTI, the effects of NTI and other opioid antagonists were evaluated individually in the same model. Therefore, the goal was to evaluate the effects of NTI, BNI, and NX independently. Hearts isolated from male Sprague-Dawley rats (~300g) were subjected to global ischemia (I, 30min)/reperfusion (R, 50min). NX (10 μM, n=6), BNI (5 μM, n=7), NTI (5 μM, n=8), or Krebs’ buffer control (control n=10) were given to the hearts 5 min prior to ischemia and during the first 5 min of reperfusion in a Langendorf model, perfused at a constant pressure of 80mmHg. Left ventricular (LV) functional indices were measured using an indwelling pressure transducer-tipped catheter. At the end of reperfusion (50min), hearts were frozen, sectioned (2 mm), and stained with 1% triphenyltetrazolium chloride (TTC). To determine infarct size, the weight of infarcted tissue was compared to total tissue at risk. All data were evaluated using ANOVA Student-Newman-Keuls post-hoc analysis. Control (untreated) hearts showed an elevated final LV end-diastolic pressure (LVEDP) of 60 ± 5 mmHg compared to an average initial baseline of 8 ± 1 mmHg (similar in all groups) and a mean infarct size of 36 ± 3% at 50 min post-reperfusion. NX and BNI demonstrated no significant cardioprotective effects compared to control with final LVEDP measures of 64 ± 8 mmHg and 61 ± 2 mmHg, respectively. Infarct size for NX (35 ± 5%) and BNI (36 ± 5%) treated hearts were similar to control hearts. By contrast, NTI significantly improved final LVEDP (17 ± 3mmHg) to near baseline values and reduced infarct size to 7 ± 2% compared to all groups (p\u3c 0.01). The results indicate that pretreatment with NTI can prevent I/R injury and restore post-reperfused heart function to near pre-ischemic levels. In future studies, we will evaluate the cardioprotective effects of NTI in an acute in vivo heart I/R model. In separate studies, we will attempt to determine the mechanism of action of NTI preconditioning, specifically whether cardioprotection is indeed mediated via delta receptor antagonism or via some other mechanism

Miyabeacin: A new cyclodimer presents a potential role for willow in cancer therapy

Willow (Salix spp.) is well known as a source of medicinal compounds, the most famous being salicin, the progenitor of aspirin. Here we describe the isolation, structure determination, and anti-cancer activity of a cyclodimeric salicinoid (miyabeacin) from S. miyabeana and S. dasyclados. We also show that the capability to produce such dimers is a heritable trait and how variation in structures of natural miyabeacin analogues is derived via cross-over Diels-Alder reactions from pools of ortho-quinol precursors. These transient ortho-quinols have a role in the, as yet uncharacterised, biosynthetic pathways around salicortin, the major salicinoid of many willow genotypes

An inter-laboratory comparison demonstrates that [1H]-NMR metabolite fingerprinting is a robust technique for collaborative plant metabolomic data collection

In any metabolomics experiment, robustness and reproducibility of data collection is of vital importance. These become more important in collaborative studies where data is to be collected on multiple instruments. With minimisation of variance in sample preparation and instrument performance it is possible to elucidate even subtle differences in metabolite fingerprints due to genotype or biological treatment. In this paper we report on an inter laboratory comparison of plant derived samples by [1H]-NMR spectroscopy across five different sites and within those sites utilising instruments with different probes and magnetic field strengths of 9.4 T (400 MHz), 11.7 T (500 MHz) and 14.1 T (600 MHz). Whilst the focus of the study is on consistent data collection across laboratories, aspects of sample stability and the requirement for sample rotation within the NMR magnet are also discussed. Comparability of the datasets from participating laboratories was exceptionally good and the data were amenable to comparative analysis by multivariate statistics. Field strength differences can be adjusted for in the data pre-processing and multivariate analysis demonstrating that [1H]-NMR fingerprinting is the ideal technique for large scale plant metabolomics data collection requiring the participation of multiple laboratories

Successful sample preparation for serial crystallography experiments

Serial crystallography, at both synchrotron and X-ray free-electron laser light sources, is becoming increasingly popular. However, the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography. This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization. These case studies aim to show (i) how vapour diffusion conditions can be converted into batch by optimizing the length of time crystals take to appear; (ii) how an understanding of the crystallization phase diagram can act as a guide when designing batch crystallization protocols; and (iii) an accessible methodology when attempting to scale batch conditions to larger volumes. These methods are needed to minimize the sample preparation gap between standard rotation crystallography and dedicated serial laboratories, ultimately making serial crystallography more accessible to all crystallographers

Co-ordinated expression of amino acid metabolism in response to N and S deficiency during wheat grain filling

Increasing demands for productivity together with environmental concerns about fertilizer use dictate that the future sustainability of agricultural systems will depend on improving fertilizer use efficiency. Characterization of the biological processes responsible for efficient fertilizer use will provide tools for crop improvement under reduced inputs. Transcriptomic and metabolomic approaches were used to study the impact of nitrogen (N) and sulphur (S) deficiency on N and S remobilization from senescing canopy tissues during grain filling in winter wheat (Triticum aestivum). Canopy tissue N was remobilized effectively to the grain after anthesis. S was less readily remobilized. Nuclear magnetic resonance (NMR) metabolite profiling revealed significant effects of suboptimal N or S supply in leaves but not in developing grain. Analysis of amino acid pools in the grain and leaves revealed a strategy whereby amino acid biosynthesis switches to the production of glutamine during grain filling. Glutamine accumulated in the first 7 d of grain development, prior to conversion to other amino acids and protein in the subsequent 21 d. Transcriptome analysis indicated that a down-regulation of the terminal steps in many amino acid biosynthetic pathways occurs to control pools of amino acids during leaf senescence. Grain N and S contents increased in parallel after anthesis and were not significantly affected by S deficiency, despite a suboptimal N:S ratio at final harvest. N deficiency resulted in much slower accumulation of grain N and S and lower final concentrations, indicating that vegetative tissue N has a greater control of the timing and extent of nutrient remobilization than S