Phytochemistry (v.62, #6)

Phytochemistry meets genome analysis, and beyond......... by Richard A. Dixon; Dieter Strack (815-816).

Plant metabolomics: large-scale phytochemistry in the functional genomics era by Lloyd W Sumner; Pedro Mendes; Richard A Dixon (817-836).
Metabolomics or the large-scale phytochemical analysis of plants is reviewed in relation to functional genomics and systems biology. A historical account of the introduction and evolution of metabolite profiling into today's modern comprehensive metabolomics approach is provided. Many of the technologies used in metabolomics, including optical spectroscopy, nuclear magnetic resonance, and mass spectrometry are surveyed. The critical role of bioinformatics and various methods of data visualization are summarized and the future role of metabolomics in plant science assessed.The critical role of metabolomics in functional genomics and systems biology of plants is reviewed. The development of metabolomics, current literature, technological approaches, bioinformatic tools and plant applications are discussed.
Keywords: Metabolomics; Metabolic profiling; Metabolite profiling; Analytical instrumentation; Mass spectrometry; MS; Bioinformatics; Metabolite databases; Medicago truncatula;

The multitude of metabolites found in living organisms and the calculated, unexpected small number of genes identified during genome sequencing projects discomfit biologists. Several processes on the transcription and translation level lead to the formation of isoenzymes and can therefore explain at least parts of this surprising result. However, poor enzyme specificity may also contribute to metabolome diversity. In former studies, when enzymes were isolated from natural sources, impure protein preparations were hold responsible for broad enzyme specificity. Nowadays, highly purified enzymes are available by molecular biological methods such as heterologous expression in host organisms and they can be thoroughly analyzed. During biochemical analysis of heterologously expressed enzymes poor specificity was observed for enzymes involved in fruit ripening, e.g. in flavour and color formation. Surprisingly broad specificity was shown for the reactants in the case of alcohol acyl-CoA transferase, O-methyltransferase, glucosyltransferase, P450 monooxygenases as well as polyketide synthases and for the product in the case of monoterpene synthases. Literature data confirm the assumption of limited specificity for enzymes involved in metabolism and bioformation of secondary metabolites. It is concluded that metabolome diversity is caused by low enzyme specificity but availability of suitable substrates due to compartmentation has also taken into account.The multitude of metabolites found in living organisms and the calculated small number of genes identified during genome sequencing projects discomfort biologists. Several processes on the transcription and translation level lead to the formation of isoenzymes and can therefore explain at least parts of this unexpected result. In this review it is assumed that metabolome diversity is governed by enzyme specificity and also by availability of suitable substrates as multifunctional enzymes are involved in various metabolic pathways.
Keywords: Plant secondary metabolites; Enzyme specificity; Substrate availability; Diversity;

Metabolomic analysis of the consequences of cadmium exposure in Silene cucubalus cell cultures via 1H NMR spectroscopy and chemometrics by Nigel J.C Bailey; Matjaz Oven; Elaine Holmes; Jeremy K Nicholson; Meinhart H Zenk (851-858).
Several essential and non-essential metals (typically those from periods 4, 5 and 6 in groups 11–15 in the periodic table) are commonly detoxified in higher plants by complexation with phytochelatin. The genetic and gross metabolic basis of metal tolerance in plants is, however, poorly understood. Here, we have analyzed plant cell extracts using 1H NMR spectroscopy combined with multivariate statistical analysis of the data to investigate the biochemical consequences of Cd2+ exposure in Silene cucubalus cell cultures. Principal components analysis of 1H NMR spectra showed clear discrimination between control and Cd2+ dosed groups, demonstrating the metabolic effects of Cd2+ and thus allowing the identification of increases in malic acid and acetate, and decreases in glutamine and branched chain amino acids as consequences of Cd2+ exposure. This work shows the value of NMR-based metabolomic approaches to the determination of biochemical effects of pollutants in naturally selected populations. Silene cucubalus plant cell cultures were exposed to cadmium chloride. By using a combination of NMR spectroscopy and principal components analysis, it was possible to discriminate between dosed (open triangles) and control (closed triangles) groups based on differences in their overall metabolic profile.
Keywords: Cadmium; Metabolomics; NMR spectroscopy; Silene cucubalus; Metabolite;

Chemometric discrimination of unfractionated plant extracts analyzed by electrospray mass spectrometry by Royston Goodacre; Emma V. York; James K. Heald; Ian M. Scott (859-863).
Metabolic fingerprints were obtained from unfractionated Pharbitis nil leaf sap samples by direct infusion into an electrospray ionization mass spectrometer. Analyses took less than 30 s per sample and yielded complex mass spectra. Various chemometric methods, including discriminant function analysis and the machine-learning methods of artificial neural networks and genetic programming, could discriminate the metabolic fingerprints of plants subjected to different photoperiod treatments. This rapid automated analytical procedure could find use in a variety of phytochemical applications requiring high sample throughput.Chemometric methods including discriminant function analysis, artificial neural networks, and genetic programming, could discriminate the metabolic fingerprints obtained from unfractionated Pharbitis nil leaf sap by direct infusion into an electrospray ionization MS.
Keywords: Pharbitis nil; Convolvulaceae; Japanese Morning Glory; Electrospray ionization mass spectrometry; Neural networks; Genetic programming; Metabolic fingerprinting;

Lactofen induces isoflavone accumulation and glyceollin elicitation competency in soybean by Serena Landini; Madge Y. Graham; Terrence L. Graham (865-874).
Lactofen, the active ingredient of the soybean disease resistance-inducing herbicide, Cobra, induces large accumulations of isoflavone conjugates and aglycones in soybean tissues. The predominant isoflavones induced in cotyledon tissues are daidzein (and its conjugates) and formononetin and glycitein aglycones. The latter two isoflavones are usually present only at very low levels in soybean seedling tissues. In leaves, the predominant lactofen-induced isoflavones are daidzein and formononetin aglycones and the malonyl-glucosyl conjugate of genistein. Isoflavone induction also occurs in cells distal to the point of treatment, but is only weakly systemic. Lactofen also induces elicitation competency, the capacity of soybean cells to accumulate the pterocarpan phytoalexin glyceollin in response to glucan elicitors from the cell wall of the pathogen Phytophthora sojae. Comparison of the activity of a series of diphenyl ether herbicides demonstrated that while all diphenyl ethers tested induced some degree of elicitation competency, only certain ones induced isoflavone accumulation in the absence of glucan elicitor. As a group the diphenyl ethers are thought to inhibit protoporhyrinogen oxidase, eventually leading to singlet oxygen generation. Another singlet oxygen generator, rose bengal, also induced elicitation competency, but little isoflavone accumulation. It is hypothesized that diphenyl ether-induced activated oxygen species mimic some aspects of hypersensitive cell death, which leads to elicitation competency in infected tissues.The disease protectant herbicide lactofen has several complementary effects on phenylpropanoid defense metabolism in soybean.
Keywords: Glycine max; Papilionoideae; Soybean; Phenylpropanoid; Isoflavone; Genistein; Daidzein; Diphenyl ether; Herbicide;

Metabolomic analysis aims at a comprehensive characterization of biological samples. Yet, biologically meaningful interpretations are often limited by the poor spatial and temporal resolution of the acquired data sets. One way to remedy this is to limit the complexity of the cell types being studied. Cucurbita maxima Duch. vascular exudates provide an excellent material for metabolomics in this regard. Using automated mass spectral deconvolution, over 400 components have been detected in these exudates, but only 90 of them were tentatively identified. Many amino compounds were found in vascular exudates from leaf petioles at concentrations several orders of magnitude higher than in tissue disks from the same leaves, whereas hexoses and sucrose were found in far lower amounts. In order to find the expected impact of assimilation rates on sugar levels, total phloem composition of eight leaves from four plants was followed over 4.5 days. Surprisingly, no diurnal rhythm was found for any of the phloem metabolites that was statistically valid for all eight leaves. Instead, each leaf had its own distinct vascular exudate profile similar to leaves from the same plant, but clearly different from leaves harvested from plants at the same developmental stage. Thirty to forty per cent of all metabolite levels of individual leaves were different from the average of all metabolite profiles. Using metabolic co-regulation analysis, similarities and differences between the exudate profiles were more accurately characterized through network computation, specifically with respect to nitrogen metabolism.Co-regulatory networks comprising of 400 metabolites are shown to be robust compared to the large biological variation found for Cucurbita maxima vascular exudates.
Keywords: Metabolite profiling; Sieve elements; Companion cells; GC/MS; Mass spectrometry; Extrafascicular phloem; LC/MS; Cucurbita maxima; Cucurbitaceae;

The non-supervised construction of a mass spectral and retention time index data base (MS/RI library) from a set of plant metabolic profiles covering major organs of potato (Solanum tuberosum), tobacco (Nicotiana tabaccum), and Arabidopsis thaliana, was demonstrated. Typically 300–500 mass spectral components with a signal to noise ratio ⩾75 were obtained from GC/EI-time-of-flight (TOF)-MS metabolite profiles of methoxyaminated and trimethylsilylated extracts. Profiles from non-sample controls contained approximately 100 mass spectral components. A MS/RI library of 6205 mass spectral components was accumulated and applied to automated identification of the model compounds galactonic acid, a primary metabolite, and 3-caffeoylquinic acid, a secondary metabolite. Neither MS nor RI alone were sufficient for unequivocal identification of unknown mass spectral components. However library searches with single bait mass spectra of the respective reference substance allowed clear identification by mass spectral match and RI window. Moreover, the hit lists of mass spectral searches were demonstrated to comprise candidate components of highly similar chemical nature. The search for the model compound galactonic acid allowed identification of gluconic and gulonic acid among the top scoring mass spectral components. Equally successful was the exemplary search for 3-caffeoylquinic acid, which led to the identification of quinic acid and of the positional isomers, 4-caffeoylquinic acid, 5-caffeoylquinic acid among other still non-identified conjugates of caffeic and quinic acid. All identifications were verified by co-analysis of reference substances. Finally we applied hierarchical clustering to a complete set of pair-wise mass spectral comparisons of unknown components and reference substances with known chemical structure. We demonstrated that the resulting clustering tree depicted the chemical nature of the reference substances and that most of the nearest neighbours represented either identical components, as judged by co-elution, or conformational isomers exhibiting differential retention behaviour. Unknown components could be classified automatically by grouping with the respective branches and sub-branches of the clustering tree.A mass spectral and retention time index library of 6205 mass spectral components was generated in a non-supervised manner from plant metabolic profiles covering major organs of potato, tobacco, and Arabidopsis thaliana. Using the two model compounds, 3-caffeoyl-quinic acid and galactonic acid, this database was employed to automated identification and classification of unknown components via mass spectral matching and hierarchical clustering.
Keywords: Arabidopsis thaliana; Brassicaceae; Solanum tuberosum; Nicotiana tabacum; Solanaceae; Caffeoylquinic acids; Chlorogenic acid; Caffeic acid; Quinic acid; Galactonic acid; Gluconic acid; Glucaric acid; Gulonic acid; Ascorbic acid; Metabolite profiling; Mass spectral library; Gas chromatography (GC); Time-of-flight mass spectrometry (TOF-MS); Retention time index (RI);

Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C-13 in Taxus x media cell cultures by Raymond E.B. Ketchum; Christopher D. Rithner; Deyou Qiu; You Sun Kim; Robert M. Williams; Rodney B. Croteau (901-909).
Cells from suspension cultures of Taxus cuspidata were extracted with pentane as a source of relatively non-polar taxoids. Of the 13 taxoids identified in this fraction, eight were oxygenated at C-14 and two had not been previously described. These taxoids, along with existing taxoid standards, were employed to profile the metabolites of Taxus x media cv. Hicksii cell suspension cultures induced with methyl jasmonate to produce paclitaxel (Taxol®). The majority of the taxoid metabolites produced in these induced cultures were oxygenated at C-13, and not C-14.Methyl jasmonate elicitation of Taxus cell suspension cultures preferentially increased production of taxoids oxygenated at C-13, including paclitaxel (Taxol®).
Keywords: Taxus x media cv. Hicksii; Taxus cuspidata; Taxaceae; Yew; Metabolomics; Metabolic profiling; Taxane diterpenoids; Taxoids; Paclitaxel; Taxol®;

The role of chemical fingerprinting: application to Ephedra by Brian T Schaneberg; Sara Crockett; Erdal Bedir; Ikhlas A Khan (911-918).
Ephedra sinica, known as Ma Huang, is one of the oldest medicinal herbs in Traditional Chinese Medicine (TCM). Preparations, namely teas, of E. sinica have been used for over 5000 years as a stimulant and as an antiasthmatic. In the West, extracts of E. sinica, E. intermedia or E. equisetina are most commonly used in dietary supplements as a stimulant and to promote weight loss. More than 50 species of Ephedra are native to both hemispheres, but the detection of ephedrine alkaloids has been limited to species in Eurasia. Currently, methods exist to quantitate the ephedrine alkaloids in extracts of plant material or dietary supplements, but the methods are not able to verify the extract is of an Ephedra species. Reverse phase high performance liquid chromatography with photodiode array detection was applied for the chemical fingerprinting of the Ephedra species. Two regions of comparison were determined in the chromatograms at 320 nm. The series of peaks between 52 and 64 min confirms an Ephedra species is being analyzed. The aforementioned peaks also could distinguish between Ephedra species from Eurasia, North America and South America. Peaks at ca. 57 and 59 min were isolated and determined to be two new compounds, 4-(2-eicosyloxycarbonyl-vinyl)-benzoic acid and 4-(2-docosyloxycarbonyl-vinyl)-benzoic acid respectively. Authentication of ground plant material as Ephedra can be achieved by this chemical fingerprinting method. Ephedra sinica, known as Ma Huang, is one of the oldest medicinal herbs in traditional Chinese medicine. Reverse phase high performance liquid chromatography with photodiode array detection was applied for the chemical fingerprinting of the Ephedra species. Two compounds, 4-(2-eicosyloxycarbonyl-vinyl)-benzoic acid and 4-(2-docosyloxycarbonyl-vinyl)-benzoic acid, were isolated.
Keywords: Ephedra; Ma Huang; Ephedrine alkaloids; High performance liquid chromatography; Chemical fingerprinting;

Metabolic fingerprinting of salt-stressed tomatoes by Helen E. Johnson; David Broadhurst; Royston Goodacre; Aileen R. Smith (919-928).
The aim of this study was to adopt the approach of metabolic fingerprinting through the use of Fourier transform infrared (FT-IR) spectroscopy and chemometrics to study the effect of salinity on tomato fruit. Two varieties of tomato were studied, Edkawy and Simge F1. Salinity treatment significantly reduced the relative growth rate of Simge F1 but had no significant effect on that of Edkawy. In both tomato varieties salt-treatment significantly reduced mean fruit fresh weight and size class but had no significant affect on total fruit number. Marketable yield was however reduced in both varieties due to the occurrence of blossom end rot in response to salinity. Whole fruit flesh extracts from control and salt-grown tomatoes were analysed using FT-IR spectroscopy. Each sample spectrum contained 882 variables, absorbance values at different wavenumbers, making visual analysis difficult and therefore machine learning methods were applied. The unsupervised clustering method, principal component analysis (PCA) showed no discrimination between the control and salt-treated fruit for either variety. The supervised method, discriminant function analysis (DFA) was able to classify control and salt-treated fruit in both varieties. Genetic algorithms (GA) were applied to identify discriminatory regions within the FT-IR spectra important for fruit classification. The GA models were able to classify control and salt-treated fruit with a typical error, when classifying the whole data set, of 9% in Edkawy and 5% in Simge F1. Key regions were identified within the spectra corresponding to nitrile containing compounds and amino radicals. The application of GA enabled the identification of functional groups of potential importance in relation to the response of tomato to salinity.The metabolic fingerprinting approach using Fourier transform infrared spectroscopy, chemometrics and evolutionary computing has been adopted to study the effect of salt stress on tomato fruit.
Keywords: Lycopersicon esculentum; Tomato; Fourier transform infrared spectroscopy; Salt stress; Metabolic fingerprinting;

An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry by Juan I Castrillo; Andrew Hayes; Shabaz Mohammed; Simon J Gaskell; Stephen G Oliver (929-937).
A method for the global analysis of yeast intracellular metabolites, based on electrospray mass spectrometry (ES-MS), has been developed. This has involved the optimization of methods for quenching metabolism in Saccharomyces cerevisiae and extracting the metabolites for analysis by positive-ion electrospray mass spectrometry. The influence of cultivation conditions, sampling, quenching and extraction conditions, concentration step, and storage have all been studied and adapted to allow direct infusion of samples into the mass spectrometer and the acquisition of metabolic profiles with simultaneous detection of more than 25 intracellular metabolites. The method, which can be applied to other micro-organisms and biological systems, may be used for comparative analysis and screening of metabolite profiles of yeast strains and mutants under controlled conditions in order to elucidate gene function via metabolomics. Examples of the application of this analytical strategy to specific yeast strains and single-ORF yeast deletion mutants generated through the EUROFAN programme are presented.A method of quenching and extraction of cellular metabolites is optimized for direct analysis of samples by electrospray mass spectrometry (ES-MS) and application in functional genomics studies.
Keywords: Yeast; Quenching and extraction method; Intracellular metabolite analysis; Electrospray mass spectrometry (ES-MS); Direct infusion; Metabolic profiles; Metabolic fingerprinting; Metabolome; Metabolomics; Functional genomics;

The carotenoid, tocopherol and chlorophyll metabolic profiles and content of a selection of fruits and vegetables found commonly in the diet, have been determined using a rapid RP-HPLC technique with on-line PDA detection. Information gathered from the screening of secondary plant metabolites is vital for the accurate determination of the dietary intake of these micro-nutrients, and in the development of comprehensive food tables. Determination of basal levels is also necessary for the rational engineering of health-promoting phytochemicals in food crops. In addition this approach can also be applied to the routine screening of products to determine metabolic differences between varieties and cultivars, as well as between genetically modified and the corresponding non-genetically modified tissue.The identity and levels of carotenoids, tocopherols and chlorophylls in fruits and vegetables have been achieved with on-line PDA detection.
Keywords: Metabolic profile; Carotenoid; Tocopherol; Chlorophyll;

An approach to metabolite fingerprinting of crude plant extracts that utilizes 1H nuclear magnetic resonance (NMR) spectroscopy and multivariate statistics has been tested. Using ecotypes of Arabidopsis thaliana as experimental material, a method has been developed for the rapid analysis of unfractionated polar plant extracts, enabling the creation of reproducible metabolite fingerprints. These fingerprints could be readily stored and compared by a variety of chemometric methods. Comparison by principal component analysis using SIMCA-P allowed the generation of residual NMR spectra of the compounds that contributed significantly to the differences between samples. From these plots, conclusions were drawn with respect to the identity and relative levels of metabolites differing between samples.An approach to the metabolomic analysis of crude plant extracts has been developed using 1H nuclear magnetic resonance spectroscopy and multivariate statistics. Using a set of ecotypes of Arabidopsis thaliana, a method has been developed for the rapid analysis of unfractionated polar plant extracts, enabling the creation of metabolite fingerprints. Comparison of datasets using chemometric methods led to conclusions regarding the structures of the compounds contributing to differences between spectra.
Keywords: Arabidopsis thaliana; NMR spectroscopy; Metabolomics; Multivariate analysis; Principal component analysis; Metabolite fingerprints;

Transgenic potato plants overexpressing and repressing enzymes of flavonoids biosynthesis were created and analyzed. The selected plants clearly showed the expected changes in anthocyanins synthesis level. Overexpression of a DNA encoding dihydroflavonol 4-reductase (DFR) in sense orientation resulted in an increase in tuber anthocyanins, a 4-fold increase in petunidin and pelargonidin derivatives. A significant decrease in anthocyanin level was observed when the plant was transformed with a corresponding antisense construct. The transformation of potato plants was also accompanied by significant changes in steroid alkaloid glycosides (SAG) level in transgenic potato tuber. The changes in SAGs content was not dependent on flavonoid composition in transgenic potato. However, in an extreme situation where the highest (DFR11) or the lowest (DFRa3) anthocyanin level was detected the positive correlation with steroid alkaloid content was clearly visible. It is suggested that the changes in SAGs content resulted from chromatin stressed upon transformation. A liquid chromatography/mass spectrometry (LC/MS) system with electrospray ionization was applied for profiling qualitative and quantitative changes of steroid alkaloid glycosides in tubers of twelve lines of transgenic potato plants. Except α-chaconine and α-solanine, in the extracts from dried tuber skin α-solamargine and α-solasonine, triglycosides of solasonine, were identified in minor amounts, triglycosides of solanidine dehydrodimers were also recognized.Changes in qualitative and quantitative composition of steroid alkaloid glycosides in tubers of genetically modified plants (Solanum tuberosum) overexpressing and repressing enzymes of flavonoid synthesis are reported.
Keywords: Solanum tuberosum; Steroid alkaloid glycosides; Anthocyanins; Transgenic potato; Liquid chromatography/mass spectrometry; Electrospray ionization;

The biochemical mode-of-action (MOA) for herbicides and other bioactive compounds can be rapidly and simultaneously classified by automated pattern recognition of the metabonome that is embodied in the 1H NMR spectrum of a crude plant extract. The ca. 300 herbicides that are used in agriculture today affect less than 30 different biochemical pathways. In this report, 19 of the most interesting MOAs were automatically classified. Corn (Zea mays) plants were treated with various herbicides such as imazethapyr, glyphosate, sethoxydim, and diuron, which represent various biochemical modes-of-action such as inhibition of specific enzymes (acetohydroxy acid synthase [AHAS], protoporphyrin IX oxidase [PROTOX], 5-enolpyruvylshikimate-3-phosphate synthase [EPSPS], acetyl CoA carboxylase [ACC-ase], etc.), or protein complexes (photosystems I and II), or major biological process such as oxidative phosphorylation, auxin transport, microtubule growth, and mitosis. Crude isolates from the treated plants were subjected to 1H NMR spectroscopy, and the spectra were classified by artificial neural network analysis to discriminate the herbicide modes-of-action. We demonstrate the use and refinement of the method, and present cross-validated assignments for the metabolite NMR profiles of over 400 plant isolates. The MOA screen also recognizes when a new mode-of-action is present, which is considered extremely important for the herbicide discovery process, and can be used to study deviations in the metabolism of compounds from a chemical synthesis program. The combination of NMR metabolite profiling and neural network classification is expected to be similarly relevant to other metabonomic profiling applications, such as in drug discovery.Neural network analysis of NMR spectra extracts from plants treated with different herbicides identifies biochemical mode-of-action.
Keywords: Acetochlor; Amitrole; Artificial intelligence; Benzisothiazole; Chlorsulfuron; Corn; Dinoseb; Diuron; Glyphosate; Imazamethabenz; Imazapyr; Imazethapyr; Metabolic profiling; Metabonomics; Naptalam; Neural network; NMR; Quinclorac; Sethoxydim; Sulcotrione; Sulfometuron; Zea mays;

Metabolomics and differential gene expression in anthocyanin chemo-varietal forms of Perilla frutescens by Mami Yamazaki; Jun-ichiro Nakajima; Mutsuki Yamanashi; Mitsuyo Sugiyama; Yukiko Makita; Karin Springob; Motoko Awazuhara; Kazuki Saito (987-995).
We have investigated metabolite profiles and gene expression in two chemo-varietal forms, red and green forms, of Perilla frutescens var. crispa. Striking difference in anthocyanin content was observed between the red and green forms. Anthocyanin, mainly malonylshisonin, was highly accumulated in the leaves of the red form but not in the green form. Less obvious differences were also observed in the stems. However, there was no remarkable difference in the contents and patterns of flavones and primary metabolites such as inorganic anions, organic anions and amino acids. These results suggest that only the regulation of anthocyanin production, but not that of other metabolites, differs in red and green forms. Microscopic observation and immunohistochemical studies indicated that the epidermal cells of leaves and stems are the sites of accumulation of anthocyanins and localization of anthocyanidin synthase protein. By differential display of mRNA from the leaves of red and green forms, we could identify several genes encoding anthocyanin-biosynthetic enzymes and presumptive regulatory proteins. The possible regulatory network leading to differential anthocyanin accumulation in a form-specific manner is discussed.The metabolites and gene expression in anthocyanin chemo-varietal forms of Perilla frutescens were profiled. The localization of anthocyanin accumulation and anthocyanidin synthase protein was also investigated. The linkage of metabolites and expression of genes and proteins are described.
Keywords: Metabolomics; Metabolic profiling; Flavonoids; Differential display; Anthocyanidin synthase; Perilla frutescens;

Regulation of floral scent production in petunia revealed by targeted metabolomics by Julian C Verdonk; C.H Ric de Vos; Harrie A Verhoeven; Michel A Haring; Arjen J van Tunen; Robert C Schuurink (997-1008).
Petunia hybrida line W115 (Mitchell) has large white flowers that produce a pleasant fragrance. By applying solid phase micro extraction (SPME) techniques coupled to GC–MS analysis, volatile emission was monitored in vivo using a targeted metabolomics approach. Mature flowers released predominantly benzenoid compounds of which benzaldehyde, phenylacetaldehyde, methylbenzoate, phenylethylalcohol, iso-eugenol and benzylbenzoate were most abundant. This emission had a circadian rhythm reaching its maximum at dusk. During petal limb expansion two sesquiterpenes were emitted by the petunia flowers, tentatively identified as germacrene D and cadina-3,9-diene. In vitro analysis showed that the petal limbs and stigma were the main producers of the benzenoids and sesquiterpenes, respectively. Moreover, comparison of in vivo and in vitro analysis indicated that volatiles were not stored during periods of low emission but rather were synthesized de novo. DNA-microarray analysis revealed that genes of the pathways leading to the production of volatile benzenoids were upregulated late during the day, preceding the increase of volatile emission. RNA-gel blot analyses confirmed that the levels of phenylalanine ammonia lyase (PAL) and S-adenosyl methionine (SAM) synthase transcripts increased towards the evening. Our results suggest that the circadian production of volatile benzenoids in petunia W115 is, at least partly, regulated at the transcript level.By applying solid phase micro extraction techniques, the composition and temporal emission of floral scent by petunia W115 was determined. The floral scent was dominated by volatile benzenoids. Microarray analysis showed that synthesis of the precursors of these benzenoids was regulated at the transcript level.
Keywords: Petunia hybrida; Headspace; Floral scent; Solid phase micro extraction; Volatile benzenoids; Flower development; Microarray; Phenylalanine ammonia lyase;

1H NMR spectroscopy is one of the techniques whose potential is currently being explored in the emerging field of metabolomics. It is a non-targeted method, producing signals for all proton-containing chemical species. For crude plant materials the spectra are always complex, with many signals overlapping. Hence a most suitable approach for analysing them is ‘metabolite fingerprinting’, which is aimed at highlighting compositional similarities and exploring the overall natural variability in a population of samples. The most commonly used method for this is principal component analysis (PCA), as it allows the whole spectral trace to be analysed and the vast quantity of information to be simplified. In this paper we investigate whether there are factors which may affect the NMR spectra in a way that subsequently decreases the robustness of the metabolite fingerprinting by PCA. Imperfections in the signal registration (i.e. inconsistency of the peak position) are generally detrimental to analysing whole traces by multivariate methods. The sources of such problems are illustrated through specially designed repeatability studies using potato and tomato samples, and the analysis of a tea dataset containing many samples. Careful sample preparation can help to limit peak shifts; for instance here by attempting to control the pH of the extracts. In addition, some compounds are susceptible to interactions affecting their chemical shifts and mathematical alignment of peaks may be necessary. Lastly factors such as resolution can also affect analyses and must be carefully adjusted. Our choice of examples aims to raise awareness of potential problems. We do not question the validity of the NMR approach, but point out those areas where special care may need to be taken.The link between spectral quality and analysis by PCA is examined, highlighting the factors that may adversely affect the rigorousness of the approach.
Keywords: Chemometrics; Data registration; Exploratory data analysis; Instrumental factors; Metabolite profiling; Metabolomics; Multivariate; NMR; Non-targeted analysis; Principal component analysis;