Metabolomic analysis of methyl jasmonate treated Brassica rapa leaves by 2-dimensional NMR spectroscopy

The metabolomic analysis of Brassica rapa leaves treated with methyl jasmonate was performed using 2-dimensional J-resolved NMR spectroscopy combined with multivariate data analysis. The principal component analysis of the J-resolved NMR spectra showed discrimination between control and methyl jasmonate treated plants by principal components 1 and 2. While the level of glucose, sucrose and amino acids showed a decrease after methyl jasmonate treatment, hydroxycinnamates and glucosinolate were highly increased. Methyl jasmonate treatment resulted in a long-term accumulation of indole glucosinolate and indole-3-acetic acid, lasting up to 14 days after treatment. Malate conjugated hydroxycinnamates also exhibited an increase until 14 days after methyl jasmonate treatment, these compounds might play an important role in plant defence responses mediated by methyl jasmonate.     

Metabolic characterization of Palatinate German white wines according to sensory attributes, varieties, and vintages using NMR spectroscopy and multivariate data analyses

(1)H NMR (nuclear magnetic resonance spectroscopy) has been used for metabolomic analysis of 'Riesling' and 'Mueller-Thurgau' white wines from the German Palatinate region. Diverse two-dimensional NMR techniques have been applied for the identification of metabolites, including phenolics. It is shown that sensory analysis correlates with NMR-based metabolic profiles of wine. (1)H NMR data in combination with multivariate data analysis methods, like principal component analysis (PCA), partial least squares projections to latent structures (PLS), and bidirectional orthogonal projections to latent structures (O2PLS) analysis, were employed in an attempt to identify the metabolites responsible for the taste of wine, using a non-targeted approach. The high quality wines were characterized by elevated levels of compounds like proline, 2,3-butanediol, malate, quercetin, and catechin. Characterization of wine based on type and vintage was also done using orthogonal projections to latent structures (OPLS) analysis. 'Riesling' wines were characterized by higher levels of catechin, caftarate, valine, proline, malate, and citrate whereas compounds like quercetin, resveratrol, gallate, leucine, threonine, succinate, and lactate, were found discriminating for 'Mueller-Thurgau'. The wines from 2006 vintage were dominated by leucine, phenylalanine, citrate, malate, and phenolics, while valine, proline, alanine, and succinate were predominantly present in the 2007 vintage. Based on these results, it can be postulated the NMR-based metabolomics offers an easy and comprehensive analysis of wine and in combination with multivariate data analyses can be used to investigate the source of the wines and to predict certain sensory aspects of wine.     

WRKY33-mediated indolic glucosinolate metabolic pathway confers resistance against Alternaria brassicicola in Arabidopsis and Brassica crops

The tryptophan (Trp)-derived plant secondary metabolites, including camalexin, 4-hydroxy-indole-3-carbonylnitrile, and indolic glucosinolate (IGS), show broad-spectrum antifungal activity. However, the distinct regulations of these metabolic pathways among different plant species in response to fungus infection are rarely studied. In this study, our results revealed that WRKY33 directly regulates IGS biosynthesis, notably the production of 4-methoxyindole-3-ylmethyl glucosinolate (4MI3G), conferring resistance to Alternaria brassicicola, an important pathogen which causes black spot in Brassica crops. WRKY33 directly activates the expression of CYP81F2, IGMT1, and IGMT2 to drive side-chain modification of indole-3-ylmethyl glucosinolate (I3G) to 4MI3G, in both Arabidopsis and Chinese kale (Brassica oleracea var. alboglabra Bailey). However, Chinese kale showed a more severe symptom than Arabidopsis when infected by Alternaria brassicicola. Comparative analyses of the origin and evolution of Trp metabolism indicate that the loss of camalexin biosynthesis in Brassica crops during evolution might attenuate the resistance of crops to Alternaria brassicicola. As a result, the IGS metabolic pathway mediated by WRKY33 becomes essential for Chinese kale to deter Alternaria brassicicola. Our results highlight the differential regulation of Trp-derived camalexin and IGS biosynthetic pathways in plant immunity between Arabidopsis and Brassica crops.     

Metabonomics evaluation of urine from rats given acute and chronic doses of acetaminophen using NMR and UPLC/MS

Urinary metabolic perturbations associated with acute and chronic acetaminophen-induced hepatotoxicity were investigated using nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography/mass spectrometry (UPLC/MS) metabonomics approaches to determine biomarkers of hepatotoxicity. Acute and chronic doses of acetaminophen (APAP) were administered to male Sprague-Dawley rats. NMR and UPLC/MS were able to detect both drug metabolites and endogenous metabolites simultaneously. The principal component analysis (PCA) of NMR or UPLC/MS spectra showed that metabolic changes observed in both acute and chronic dosing of acetaminophen were similar. Histopathology and clinical chemistry studies were performed and correlated well with the PCA analysis and magnitude of metabolite changes. Depletion of antioxidants (e.g. ferulic acid), trigonelline, S-adenosyl-l-methionine, and energy-related metabolites indicated that oxidative stress was caused by acute and chronic acetaminophen administration. Similar patterns of metabolic changes in response to acute or chronic dosing suggest similar detoxification and recovery mechanisms following APAP administration.     

基于GC-MS代谢组学技术委陵菜与星毛委陵菜的初生代谢比较研究

为明确药用植物委陵菜与其同属植物星毛委陵菜在不同基原之间的代谢差异，更有效的利用药用植物资源。基于气相色谱与质谱联用技术对两药用植物间的小分子初生代谢物进行非靶向代谢组学分析，利用主成分分析和偏最小二乘法分析，寻找可区分两物种的差异代谢物以及在初生代谢途径上的差异。结果：利用GC-MS在两植物中共检测到134个色谱峰，通过与数据库比对，共鉴定出43种初生代谢产物。PCA分析结果显示代谢物在委陵菜和星毛委陵菜间的分布有较大的差异；采用OPLS-DA筛选获得导致2物种区分的差异代谢物共33种，两物种在半乳糖代谢、淀粉和蔗糖代谢、氨酰生物合成、氨基糖和核苷酸糖代谢和果糖和甘露糖代谢途径有显著的差异。委陵菜和星毛委陵菜的初生代谢方面存在较大差异，虽然两植物来源于同科同属植物，但是星毛委陵菜不适合代替委陵菜作为药材使用。     

Infraspecific chemical variability of the leaf essential oil of Juniperus phoenicea var. turbinata from Portugal

The essential oil composition of 68 individual plants of Juniperus phoenicea from Portugal was investigated by GC, GC-MS and ¹³C NMR. -Pinene, β-phellandrene, -terpinyl acetate and myrcene were found to be the main constituents. Botanical and chemical data as well as phytogeographical distribution indicate J. phoenicea var. turbinata as the unique subspecies occurring in Portugal. Nevertheless, this taxon exhibits chemical polymorphism. The results of the oil compositions were processed by hierarchical clustering and principal component analysis allowing to establish three groups of essential oils differentiated by the content of -pinene, β-phellandrene and -terpinyl acetate.     

A metabolic system-wide characterisation of the pig: a model for human physiology

The pig is a single-stomached omnivorous mammal and is an important model of human disease and nutrition. As such, it is necessary to establish a metabolic framework from which pathology-based variation can be compared. Here, a combination of one and two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy (NMR) and high-resolution magic angle spinning (HR-MAS) NMR was used to provide a systems overview of porcine metabolism via characterisation of the urine, serum, liver and kidney metabolomes. The metabolites observed in each of these biological compartments were found to be qualitatively comparable to the metabolic signature of the same biological matrices in humans and rodents. The data were modelled using a combination of principal components analysis and Venn diagram mapping. Urine represented the most metabolically distinct biological compartment studied, with a relatively greater number of NMR detectable metabolites present, many of which are implicated in gut-microbial co-metabolic processes. The major inter-species differences observed were in the phase II conjugation of extra-genomic metabolites; the pig was observed to conjugate p-cresol, a gut microbial metabolite of tyrosine, with glucuronide rather than sulfate as seen in man. These observations are important to note when considering the translatability of experimental data derived from porcine models.     

Fruit metabolite networks in engineered and non-engineered tomato genotypes reveal fluidity in a hormone and agroecosystem specific manner

Introduction

Metabolomics provides a view of endogenous metabolic patterns not only during plant growth, development and senescence but also in response to genetic events, environment and disease. The effects of the field environment on plant hormone-specific metabolite profiles are largely unknown. Few studies have analyzed useful phenotypes generated by introducing single or multiple gene events alongside the non-engineered wild type control at field scale to determine the robustness of the genetic trait and its modulation in the metabolome as a function of specific agroecosystem environments.

Objectives

We evaluated the influence of genetic background (high polyamine lines; low methyl jasmonate line; low ethylene line; and isogenic genotypes carrying double transgenic events) and environments (hairy vetch, rye, plastic black mulch and bare soil mulching systems) on the metabolomic profile of isogenic reverse genetic mutations and selected mulch based cropping systems in tomato fruit. Net photosynthesis and fruit yield were also determined.

Methods

NMR spectroscopy was used for quantifying metabolites that are central to primary metabolism. We analyzed both the first moment (means) of metabolic response to genotypes and agroecosystems by traditional univariate/multivariate methods, and the second moment (covariances) of responses by creating networks that depicted changes in correlations of paired metabolites. This particular approach is novel and was necessary because our experimental material yielded highly variable metabolic responses that could not be easily understood using the traditional analytical approaches for first moment statistics.

Results

High endogenous spermidine and spermine content exhibited strong effects on amino acids, Krebs cycle intermediates and energy molecules (ADP + ATP) in ripening fruits of plants grown under different agroecosystem environments. The metabolic response to high polyamine genotypes was similar to the response to hairy vetch cover crop mulch; supported by the pattern of changes in correlation between metabolites. Changes in primary metabolites of genotypes mutated for the deficiency of ethylene or methyl jasmonate were unique under all growth conditions and opposite of high polyamine genotype results. The high polyamine trait was found to dominate the low ethylene and low jasmonate mutations under field conditions. For several metabolites low ethylene and low methyl jasmonate genotypes had an inverse relationship. Collectively, these results affirm that interactions between metabolite pathways and growth environments are affected by genotype, and influence the metabolite quality of a crop.

Conclusion

This study portrays how metabolite relationships change, both in mean and in correlation, under different genotypic and environmental conditions. Although these networks are surprisingly dynamic, we also find examples of selectively conserved associations.

     

NMR study of age dependent metabolic adjustments in wild type and pp2a-b’γ mutant Arabidopsis thaliana

The model plant Arabidopsis thaliana of the cruciferous Brassicaceae family has proven as an excellent tool for genetic modification and understanding of plant metabolic pathways. In this study we set up the methodology of ¹H NMR and principal component analysis (PCA) to address metabolic adjustments in Arabidopsis leaves. Wild type, together with a prematurely yellowing mutant deficient in a specific protein phosphatase 2A regulatory subunit PP2A-B’γ and a pp2a-b’γ 35S:PP2A-B’γ complementation line were analyzed at 4 and 6 weeks of age to reveal metabolic differences between vegetative and maturation phases of rosette growth. A PCA model revealed similar age-dependent metabolic adjustments in all genotypes. The contents of choline, sinapoyl malate, alanine and glutamine were high in four-week-old Arabidopsis plants and less abundant in six-week-old plants. Moreover, γ-aminobutyric acid (GABA) was observed only in four-week-old plants, whereas six-week-old plants were devoid of this metabolic compound. In contrast, the contents of fumaric acid, glucose and fructose became elevated at six weeks of age. β-Sitosterol was observed to be more abundant in 4 weeks old Arabidopsis rosettes, while α-linolenic and linoleic acids indicated higher content in 6 weeks old Arabidopsis rosettes. Total chlorophyll (Chl) content was higher in 4 weeks old Arabidopsis, whereas high content of Chl b was observed in 6 weeks old plants. We conclude that NMR based metabolomics analysis is a potential method for identification of age-dependent metabolites in Arabidopsis leaves.     

NMR metabolomics of berry quality in sea buckthorn (Hippophae L.)

Sea buckthorn (Hippophae L.) is a woody perennial shrub or small tree whose berries are rich in bioactive compounds with powerful nutritional and medicinal properties. Untargeted ¹H nuclear magnetic resonance (NMR) spectroscopy and principal component analysis (PCA) were used to characterize the metabolic profile of berry quality. There was strong separation in the NMR signal intensity of bioactive compounds between pulp and seeds, such as amino acids [gamma-aminobutyric acid (GABA), aspartate, glutamate, theanine, and proline], organic acids (citrate, succinate, malate, acetate, quinate, and heriguard), and carbohydrates (sucrose, fructose, glucose, and melibiose). Sea buckthorn cultivars could be clearly separated into two groups using principal component analysis (PCA) based on NMR spectroscopy of bioactive compounds in the pulp and seeds. Several metabolic compounds such as sugar, organic acids, and amino acids could serve as biomarkers for prediction of berry quality and for classification of germplasm collections. This dataset provides potential information concerning the mechanisms of berry quality formation and contributes to increasing the breeding efficiency of sea buckthorn quality improvement.     

Metal ion-inducing metabolite accumulation in Brassica rapa

Plants face a number of biotic and abiotic environmental stress factors during growth. Among the abiotic factors, in particular, a great deal of attention has been paid to metals not only because of their increasing amounts in the environment due to rapid industrial development but also because of the variation of metal composition in soil. Cultivation of crops close to industrial areas or irrigation with contaminated water may result in both growth inhibition and tissue accumulation of metals. Brassica species are well known as metal accumulators and are being used for phytoremediation of contaminated soils. However, the metal tolerance mechanism in the plant still remains unclear. In order to investigate the metabolomic changes induced by metal ions in Brassica, plants were subjected to concentrations 50, 100, 250 and 500mmol of copper (Cu), iron (Fe) and manganese (Mn) in separate treatments. (1)H NMR and two-dimensional NMR spectra coupled with principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) were applied to investigate the metabolic change in Brassica rapa (var. Raapstelen). The (1)H-NMR analysis followed by the application of chemometric methods revealed a number of metabolic consequences. Among the metabolites that showed variation, glucosinolates and hydroxycinnamic acids conjugated with malates were found to be the discriminating metabolites as were primary metabolites like carbohydrates and amino acids. This study shows that the effects of Cu and Fe on plant metabolism were larger than those of Mn and that the metabolomic changes varied not only according to the type of metal but also according to its concentration.     

Metabolic Discrimination of Catharanthus roseus Calli According to Their Relative Locations Using 1H-NMR and Principal Component Analysis

Creating a plant-cell suspension culture involves first transferring the callus into liquid media, but there are no objective criteria for selecting the location of the callus to be transferred. In this study, inner and outer cells of Catharanthus roseus with various elicitors in solid-state cultures were differentiated by ¹H NMR (nuclear magnetic resonance) spectrometry and principal component analysis (PCA). It was found that the samples of various elicitors and relative locations could be separated in PCA-derived score plots. Especially, there was a clear separation between nontreated samples and those cotreated with silver nitrate and methyl jasmonate. Loading-plot analysis was therefore applied to data obtained from nontreated samples and those cotreated with silver nitrate and methyl jasmonate to determine the separation of major metabolites on score plots. The levels of valine, lactic acid, threonine, alanine, arginine, acetic acid, malic acid, succinic acid, citric acid, asparagine, choline, lactose, fumaric acid, phenylalanine, tryptophan, and formic acid were higher in the inner callus than in the outer callus, whereas 2-oxoglutaric acid, oxalacetic acid, sucrose, and glucose dominated in the outer callus. The results obtained in this study suggest that inner and outer calli can be differentiated by ¹H-NMR-based metabolomic analysis.     

NMR-based metabonomic study of transgenic maize

The aim of this research was to verify the possibility of identifying and classifying maize seeds obtained from transgenic plants, in different classes according to the modification, on the basis of the concerted variation in metabolite levels detected by NMR spectra. It was possible to recognise the discriminant metabolites of transgenic samples as well as to classify non-a priori defined samples of maize. It is important to underline that the obtained results are useful to point out the metabolic consequences of a specific genic modification on a plant, without using a targeted analysis of the different metabolites, in fact it was possible to classify the seeds also without the complete assignment of the spectra. The analysis was performed by applying multivariate techniques (principal component analysis and partial least squares-discriminant analysis) to NMR data.     

双管基因枪介导的基因瞬时表达技术在拟南芥中的应用

基因瞬时表达是植物中研究目标基因功能的常用手段。在模式植物拟南芥(Arabidopsis thaliana)中, 相比原生质体和农杆菌介导的基因异源表达技术, 利用粒子轰击进行基因瞬时表达一直鲜有报道。其主要原因是拟南芥叶型相对较小、基因枪操作相对烦琐以及基因表达效率差异较大。该研究通过优化双管基因枪系统, 在营养生长旺盛的拟南芥莲座叶中实现GFP和GUS基因高效表达。同时, 通过GUS报告基因明确了坏死诱导因子BAX、Avh238和ATR13/Rpp13激发拟南芥细胞坏死的表型。但在本氏烟(Nicotiana benthamiana)中明显诱导细胞坏死的Avrblb1/RB基因对, 在拟南芥中却丧失了诱导细胞坏死的活性。由于双管基因枪系统每次轰击时设置平行对照, 可有效降低转化实验中的样本变异度, 为拟南芥及其突变体研究中准确评价基因功能和高通量筛选目标基因提供新的技术参考。     

茉莉酸甲酯对菊花抗蚜性的影响

本研究以杭白菊为试验材料,分析茉莉酸甲酯对菊花抗蚜性的影响。供试幼苗叶面喷施不同浓度(0.01、0.05、0.1、0.5、1 mmol·L^-1)茉莉酸甲酯后接种菊姬长管蚜,测定外源茉莉酸甲酯对蚜虫胁迫下菊花叶片的保护酶、防御酶活性、渗透性物质、次生代谢物和茉莉酸途径关键酶基因表达的影响,探究菊花抗蚜性与茉莉酸信号途径的关系。结果表明: 0.01、0.05、0.1、0.5、1 mmol·L^-1浓度的茉莉酸甲酯均显著提高了杭白菊叶片的保护酶、防御酶活性及次生代谢物含量,降低了丙二醛和可溶性糖含量,外源茉莉酸甲酯处理诱导杭白菊CmAOS和CmCOI1的表达,并使内源茉莉酸含量显著增加,杭白菊的抗蚜性增强。     

A metabonomic approach to the investigation of drug-induced phospholipidosis: an NMR spectroscopy and pattern recognition study

¹H NMR spectroscopy of urine and pattern recognition analysis have been used to study the metabolic perturbations caused following dosing of five novel drug candidates, two of which (GWA, GWB) caused mild lung and liver phospholipidosis, whilst the rest (GWC-GWE) did not cause any detectable toxicity. Urine samples were collected predose, 0-8 h, 8-16 h, 16-24 h and 24-32 h after single, oral dosing with each compound to Han Wistar rats (n = 3 per group), and liver and lung samples for were taken at 48 h for histology. ¹H NMR spectra of whole urine were acquired, processed and subsequently analysed using principal component analysis. All animals administered the drug candidates showed a significant reduction in serum triglycerides and those animals administered either GWA or GWB were observed to have foamy alveolar macrophages and the presence of multilamellar bodies in hepatocytes by electron microscopy. In the plot of the first two principal components, urinary spectra of those animals dosed with GWA or GWB mapped separately to controls, all pre-dose samples and animals dosed with GWC-GWE. Inspection of the principal components loadings indicated an increase in urinary phenylacetylglycine with a concomitant decrease in urinary citrate and 2-oxoglutarate, possibly constituting a novel urinary biomarker set for phospholipidosis. This work exemplifies the use of NMR spectroscopy and pattern recognition methods for the detection of novel biomarker combinations for poorly understood toxicity types and the potential in screening novel drugs for toxicity.     

Structurally related guaianolides from Inula thapsoides

The whole plant extract of Inula thapsoides afforded seven new structurally related guaianolides. The structures and stereochemistry were determined by spectroscopic methods, particularly one- and two-dimensional NMR spectroscopy, and extensive NOE experiments.     

Class targeted metabolomics: ESI ion trap screening methods for glucosinolates based on MSn fragmentation

Glucosinolates are naturally occurring anionic secondary plant metabolites incorporating a thioglucosidic link to the carbon of a sulphonated oxime. There are a large number of naturally occurring glucosinolates and they are found in relatively large quantities in many plant species within the family Crucifereae. These metabolites are of interest for both their anticancer and flavour properties and in the study of nitrogen and sulphur metabolism in model plants such as Arabidopsis. Parent ion mapping is an analytical mass spectrometry approach that allows rapid assessment of glucosinolate content. Ion mapping proved to be highly sensitive and the glucosinolate sinigrin could be detected at three parts per trillion. This method takes advantage of the glucosinolate anion fragmentation which consistently produces a sulphonate ring-opened glucose moiety in the ion trap mass spectrometer, m/z 259. An intramolecular transfer mechanism for this fragmentation is presented here for the first time. This fragmentation can be exploited as a general identifier of the glucosinolate class of metabolites in plant extracts and in LCMSⁿ can be employed provide positive identification and quantification of individual glucosinolates. Such approaches offer sensitive tools for focused metabolomics analysis and screening of plant breeding lines.