Chapter 1 MeRy-B, a Metabolomic Database and Knowledge Base for Exploring Plant Primary Metabolism Catherine Deborde and Daniel Jacob Abstract Plant primary metabolites are organic compounds that are common to all or most plant species and are essential for plant growth, development, and reproduction. They are intermediates and products of metabolism involved in photosynthesis and other biosynthetic processes. Primary metabolites belong to different compound families, mainly carbohydrates, organic acids, amino acids, nucleotides, fatty acids, steroids, or lipids. Until recently, unlike the Human Metabolome Database (http://www.hmdb.ca) dedicated to human metabolism, there was no centralized database or repository dedicated exclusively to the plant kingdom that contained information on metabolites and their concentrations in a detailed experimental context. MeRy-B is the first platform for plant 1H-NMR metabolomic profiles (MeRy-B, http://bit.ly/ meryb), designed to provide a knowledge base of curated plant profiles and metabolites obtained by NMR, together with the corresponding experimental and analytical metadata. MeRy-B contains lists of plant metabolites, mostly primary metabolites and unknown compounds, with information about experimental conditions, the factors studied, and metabolite concentrations for 19 different plant species (Arabidopsis, broccoli, daphne, grape, maize, barrel clover, melon, Ostreococcus tauri, palm date, palm tree, peach, pine tree, eucalyptus, plantain rice, strawberry, sugar beet, tomato, vanilla), compiled from more than 2,300 annotated NMR profiles for various organs or tissues deposited by 30 different private or public contributors in September 2013. Currently, about half of the data deposited in MeRy-B is publicly available. In this chapter, readers will be shown how to (1) navigate through and retrieve data of publicly available projects on MeRy-B website; (2) visualize lists of experimentally identified metabolites and their concentrations in all plant species present in MeRy-B; (3) get primary metabolite list for a particular plant species in MeRy-B; and for a particular tissue (4) find information on a primary metabolite regardless of the species. Key words Primary metabolism, Metabolite, Database, Concentration, Tissue, NMR
1
Introduction
1.1 What Are Plant Primary Metabolites?
Plant primary metabolites are organic compounds, common to all or most plant species. Their functions are essential for plant growth, development, and reproduction. They are intermediates and products of metabolism involved in photosynthesis and other biosynthetic processes. These primary metabolites belong to several
Ganesh Sriram (ed.), Plant Metabolism: Methods and Protocols, Methods in Molecular Biology, vol. 1083, DOI 10.1007/978-1-62703-661-0_1, © Springer Science+Business Media New York 2014
3
4
Catherine Deborde and Daniel Jacob
compound families including carbohydrates, organic acids, amino acids, nucleotides, fatty acids, steroids, or lipids. Pichersky and Lewinsohn [1] estimated the plant primary metabolite number under 10,000 compounds. Some primary metabolites are also present in animals and microorganisms, whereas others are specific to the plant kingdom. A comparative analysis of the Arabidopsis and yeast genomes placed the number of genes involved in primary metabolism at 13,250 and the number of primary metabolites at 8,000 in Arabidopsis [1]. Nevertheless, de Oliveira Dal’Molin C et al. [2] who developed AraGEM, a genome-scale compartmented metabolic network model for Arabidopsis, included only 1,748 primary metabolites. Metabolomics studies of Arabidopsis based on hydromethanolic extraction, derivatization, and gas chromatographymass spectrometric (GC-MS) detection of hydrophilic metabolites yield a few hundred analytes, mainly primary metabolites. However, only 30–40 % of these analytes can be linked to known metabolites (specifically, only 81 are identified) [3]. In many cases such GC-MS analyses do not allow an absolute quantification but usually provide relative quantification (metabolite levels relative to control samples). In metabolomics studies of Arabidopsis by NMR on hydromethanolic extracts, 40 metabolites were identified and quantified [4]. Therefore the plant primary metabolite data tightly depend on the extraction process to prepare the sample and on the analytical method chosen (see ref. 5 for practical considerations). For comparison, in September 2013 the Human Metabolome Database v3.5 (HMDB, http://www.hmdb.ca/) compiled detailed literature-derived information on 41,528 metabolites found in human body (tissues, organs, or biofluids) and an extensive collection of experimental metabolite concentration data for biofluids, namely, plasma, urine, cerebrospinal fluid, and experimentally acquired 1H and 13C NMR and MS/MS spectra of commercially available “reference” or “authentic standard” compounds [6]. Until recently, unlike HMDB, there was no centralized database or repository dedicated exclusively to the plant kingdom and containing information on metabolites and their concentrations in a detailed experimental context. 1.2
What Is MeRy-B?
MeRy-B stands for Metabolomic Repository of Bordeaux and is available from http://bit.ly/meryb. It is the first platform for plant 1 H-NMR metabolomic profiles [7]. MeRy-B is designed (1) to provide a knowledge base of curated plant profiles and metabolites obtained by NMR, together with the corresponding experimental and analytical metadata; (2) to query and visualize the data; (3) to discriminate profiles with spectrum visualization tools and statistical analysis; and (4) to facilitate compound identification. MeRy-B structure (Fig. 1) follows the steps of a metabolomics experiment. It consists of four principal components: (A) “experimental design,”
MeRy-B, a Plant Metabolomic Database
5
Administration - Users, Access rights, Project status (public or private)
E Experimental design
Analytical metadata
- Biological source - Project - Experiments - Genotype(s) - Development stage(s) - Protocols (PDF)
- Instrument - Technique - Extraction method - Protocols (PDF)
A
B
Spectra data
Compounds
- Pre-processed spectra data (JCAMP-DX) - Processed spectra data - Peak lists
- Identified compounds (KEGG) - Unknown compounds - Quantifications
C
D
Controlled vocabularies (MSI) + Ontologies (OBO - obo.sourceforge.net) Query Builder
Statistical Analysis
Fig. 1 Structure of MeRy-B database and knowledge base
(B) “analytical metadata,” (C) “spectra data,” and (D) “compounds.” There is a fifth component (E) for “administration.” MeRy-B contains lists of plant metabolites and unknown compounds, with information about experimental conditions, the factors studied, and metabolite concentrations for 19 different plant species (Arabidopsis, broccoli, daphne, grape, maize, barrel clover, melon, Ostreococcus tauri, palm date, palm tree, peach, pine tree, eucalyptus, plantain rice, strawberry, sugar beet, tomato, vanilla), compiled from more than 2,300 annotated NMR profiles for various organs (e.g., fruit, seed, leaf, root) or tissues (e.g., mesocarp, epicarp, endosperm) deposited by 30 different private or public contributors in September 2013. Currently about half of the data deposited in MeRy-B is publicly available. MeRy-B manages all the data generated by NMR-based plant metabolomics experiments, from description of the biological source to identification of the metabolites and determinations of their concentrations. It is the first database allowing the display and overlay of NMR metabolomic profiles selected through queries on data or metadata. MeRy-B contains a collection of experimental metabolite concentration data for more than 40 metabolites coming from 20 public projects (48 experiments and 1,191 NMR spectra). The metabolites are mainly primary metabolites due to the choice of the extraction processes and NMR methods used. In this chapter, readers will be shown how to navigate through and retrieve data from MeRy-B website, how to get the list of the experimentally identified metabolites (Protocol 1), how to retrieve concentrations of a particular metabolite in all plant species present in MeRy-B (Protocol 2), how to get primary metabolite list for a particular plant species in MeRy-B (Protocol 3), and for a particular tissue of a plant species (Protocol 4) how to find information on a primary metabolite regardless of species (Protocol 5).
6
2
Catherine Deborde and Daniel Jacob
Material: Necessary Resources 1. Hardware: Computer with Internet access. 2. Software: An up-to-date web browser (see Note 1 for java applet). 3. Connection to MeRy-B http://bit.ly/meryb (see Note 2).
3
Methods On the home page and most pages of MeRy-B website, there is a menu bar located at the left-hand side of the page, with ten clickable links: four under General Information, four under Data consultation, and two under Other Information. Click on the button Compounds under Data consultation. Within a few seconds a simplified map of plant metabolism pathways will be displayed with two types of metabolites (Fig. 2). Some metabolite names are spotlighted with a colored circle indicating that these metabolites are either identified or quantified in projects deposited in MeRy-B database. Other metabolite names are not
Fig. 2 A screenshot showing a simplified map of plant (mainly primary) metabolism. This figure appears in color in the online version of this chapter
MeRy-B, a Plant Metabolomic Database
7
circled, which means that these metabolites have not been identified in projects deposited in MeRy-B database, e.g., starch and oxalate. The circles are colored green for amino acids, red for sugars, blue for organic acids, and yellow for other metabolite families. For each metabolite identified in projects deposited in MeRy-B database, there is one MeRy-B card. Click on the circle near the metabolite name, and a new window will appear containing the MeRy-B card. The number of identified metabolites in MeRy-B database is summarized in the upper part of the page, above the map: Compound(s) found. Currently 77 metabolites are reported. Move the mouse over the word PlantCyc ([8], and see Chapter 13) on the left-hand side of the map to change the hyperlinks of spotlighting to the corresponding PlantCyc compounds. Click on the circle near the metabolite name, and a new window will appear containing the Plant Metabolic Network—PlantCyc Compound card. 3.1 Protocol 1: Obtaining the List of Experimentally Identified Primary Metabolites in MeRy-B
Click on List near Compounds by on the left-hand side of the page, above the pathway map. Within a few seconds a five-column table will be displayed with all the metabolites identified and/or quantified coming from all public projects deposited into MeRy-B database (see Note 3 for the list of unknown metabolites). To survey the list (Fig. 3), in alphabetical order, of the metabolites experimentally identified in MeRy-B, use the scroll bar on the right side of the browser window. This table consists of five columns. The first column displays the line number of
Fig. 3 A screenshot depicting a table with all the metabolites identified and/or quantified in the projects deposited in MeRy-B database
8
Catherine Deborde and Daniel Jacob
the table, the second column displays the name of the metabolite (see Note 4), the third column the species where the metabolite has been identified, the fourth column contains the number of experiments where the metabolite has been identified, and the fifth column contains the MeRy-B card hyperlink (a green button). 3.2 Protocol 2: MeRy-B Card Overview; Retrieving Concentrations of a Particular Metabolite in All Plant Species Present in MeRy-B
For each metabolite in MeRy-B database, there is one MeRy-B card (Fig. 4). The concept of a MeRy-B card is analogous to the MetaboCard in HMDB. Each MeRy-B card contains several sections, depending on whether information is available or not, mainly subdivided into two kinds: chemical and experimental. To survey the type of information displayed in a typical MeRy-B card, use the scroll bar on the right side of your browser window to scroll down the page. First, on the left top of the header are
Fig. 4 A MeRy-B card screenshot. The MeRy-B card displays all public data stored in the MeRy-B knowledge base for a given compound. For each species and tissue in which a given compound is found, this card displays data concerning 1H-NMR chemical shifts, multiplicity, and quantification. Data may be filtered and sorted by species and/or tissue
MeRy-B, a Plant Metabolomic Database
9
displayed (1) the name of the metabolite and the user synonyms (see Note 4) and (2) hyperlinks to the Chemical Translation Service (CTS) [9] and PlantCyc compounds [8]. On the top right of the header, a summary of experimental data linked with this compound is displayed, namely, species, tissues, and analytical techniques. The chemical information sections are laid out as follows: (1) Kyoto Encyclopedia of Genes and Genomes (KEGG) Compound [10], (2) Other Links, (3) Pathways & Reactome, (4) HMDB NMR Peak List [6, 11], and (5) NMR Spectrum. To expand a section, click on the small square icon with a “plus” sign inside. When expanded, the sign inside the icon changes itself to “minus.” The “KEGG” section contains essential information on the identity of the compound coming from the KEGG compound database. For more details, click on the KEGG Accession identifier on the right column. The “Other Links” section brings together useful hyperlinks to references and other public databases including the chemical entities of biological interest (CHEBI) [12], the KNApSAck database [13, 14], the Golm Metabolome database (MPIMP) [15], and HMDB [11]. The “Pathways & Reactome” section provides the set of biological pathways retrieved from PlantCyc database [8] (see Note 5). To view the reactions for a particular biological pathway, click on the small square icon with a “plus” sign located on the left corresponding to the pathway. To view all reactions for all pathways, click on “all” located at the top of the pathways list. For each reaction, the Enzyme Commission number is given as a hyperlink to the KEGG Enzyme website. The last two sections provide analytical information on NMR, namely, the HMDB Peak List (with compound chemical shifts when available) and an interactive NMR Spectrum Viewer in MeRy-B (see Note 6). To view the 1HNMR spectrum, scroll down to NMR Spectrum and click on the button. This launches a Java applet, and the 1H-NMR spectrum of the metabolite of interest will appear in the applet window. If not, see Notes 1 and 2. This applet allows the user to interactively zoom into the spectrum by holding down the left mouse button, selecting the desired area, and finally releasing the mouse button. To zoom out, click on the right mouse button. The “MeRy-B card” displays the list of experiments in which the metabolite was detected, and, for each experiment, additional metadata are listed (species, tissue/organ, and project name), together with a summary of the analytical results (e.g., for 1H-NMR: chemical shift, multiplicity, minimum and maximum values for quantification). This card also highlights quantitative differences between species, tissues, organs, or experiments for the compound. Users can filter the desired data by using the filters located on top of this list, by selecting a species, a tissue/ organ, or both and can also sort them by choosing a criterion: “Species” or “Tissue/Organ.”
10
Catherine Deborde and Daniel Jacob
There are several ways to display a MeRy-B card and retrieve concentrations of a particular metabolite in plant species present in MeRy-B: 1. Click on the button Compounds under Data consultation. Within a few seconds a simplified map of plant metabolism will appear. Click on the colored spot close to the chosen metabolite in order to display the MeRy-B card. Within a few seconds a new window will be displayed with this card. 2. Follow Protocol 1 and click on the MeRy-B card hyperlink of the metabolite of interest. 3. Follow Protocol 5. 3.3 Protocol 3: Obtaining the List of Primary Metabolites for a Particular Plant Species
Click on the button Compounds under Data consultation (see Fig. 2). Click in the text search box Species in the first frame named General, located in the upper part of the window, and select the species (e.g., Lycopersicon esculentum for tomato) in the dropdown list. Within a few seconds a simplified map of plant metabolism will be displayed (see Protocol 3.1 for explanation). If the selected species is also described specifically in PlantCyc, e.g., LycoCyc for tomato, two maps are proposed, one with information coming from MeRy-B database and the other from LycoCyc database (for moving from MeRy-B to LycoCyc data: move the mouse over the word LycoCyc on the left-hand side of the map to update the map with LycoCyc data). Click on List near Compounds by on the left-hand side of the page, above the pathway map. Within a few seconds a five-column table will be displayed with all the identified and/or quantified metabolites in tomato coming from the projects deposited in MeRy-B database (33 metabolites in September 2013). In this case the third column displays the tissue or the organ where the metabolite has been identified (Fig. 5). Click on the hyperlink of the fifth column of asparagine (MRB85) for example. Within a few seconds a new window will appear: the MeRy-B card for the selected metabolite (see Subheading 3.2 for the general description of MeRy-B card) (Fig. 6). To survey the minimal and maximal concentrations reported for each publicly available project deposited in MeRy-B, use the scroll bar on the right side of the browser window. Be aware of the different concentration units used (see Note 7). This card highlights quantitative differences between tissues, organs, or experiments for a given metabolite. The fourth column provides a hyperlink to the project to enable the user to understand the biological and analytical context of the reported concentration values of the metabolite of interest (click on the hyperlink [see Note 8 How to consult a project in MeRy-B]).
MeRy-B, a Plant Metabolomic Database
11
Fig. 5 A screenshot showing the table with all the metabolites identified and/or quantified in the tomato species coming from the projects deposited in MeRy-B database
Fig. 6 A screenshot showing the MeRy-B card of asparagine in tomato
12
Catherine Deborde and Daniel Jacob
3.4 Protocol 4: Obtaining the List of Primary Metabolites for a Particular Tissue of a Given Plant Species
Click on the button Compounds under Data consultation. Click in the text search box Species in the first frame named General, located in the upper part of the window, and select the species in the dropdown list, for instance tomato. Click also in the text search box Tissue/Organ, and select the term of interest in the drop-down list. Within a few seconds a simplified four-column table will be displayed with all the identified and/or quantified metabolites in the tissue/organ selected in tomato coming from the projects deposited in MeRy-B database (e.g., 21 metabolites for leaf tissue, 27 metabolites for seed, 30 metabolites for pericarp tissue in September 2013). Click on the hyperlink of the fourth column. Within a few seconds a new window will appear: the MeRy-B card for the selected metabolite (see Subheading 3.2 for the general description of MeRy-B card and Protocol 3.2).
3.5 Protocol 5: Obtaining Information on a Primary Metabolite Regardless of Species
Click on the button Compounds under Data consultation. This interface allows the user to search all the metabolites available in MeRy-B by Name or by Elemental Formula. The first way of searching metabolite within this MeRy-B interface is by Name. Click in the text search box Name or User Synonym in the second frame named Compounds, located in the upper part of the window; once the cursor appears type the first letter of the metabolite. A drop-down list will appear. Select the metabolite of interest. For example, type “a” and select Alanine; within a few seconds a five-column table will be displayed with three options: alanine, phenylalanine, and beta-alanine (Fig. 7).
Fig. 7 A screenshot of how the MeRy-B browser page will appear when searching metabolites by name. This example shows the search results for the word “alanine.” The MeRy-B card accession numbers on the right side of the table are hyperlinked
MeRy-B, a Plant Metabolomic Database
13
Fig. 8 A screenshot of how the MeRy-B browser page will appear when searching metabolites by elemental formula. This example shows the search results for the formula “C6” (containing six carbon atoms). The MeRy-B card accession numbers on the right side of the table are hyperlinked
Click on the hyperlink of the fifth column on the first line for alanine (MRB63). Within a few seconds a new window will appear: the MeRy-B card for Alanine, the selected metabolite (see Subheading 3.2 for the general description of MeRy-B card, Protocol 3.2, and Note 8 How to consult project in MeRy-B). Alanine has been reported for 10 species in MeRy-B, 8 of them belonging to publicly available projects, and for 38 experiments with 24 of them publicly available. Another method for searching metabolites within MeRy-B is by Elemental Formula. Elemental formulae follow the Hill notation in MeRy-B (see Note 9). Click in the text search box Elemental Formula and enter either the full or the partial elemental formula (i.e., only the number of carbon atoms). For example, if the user searches in MeRy-B for identified metabolites containing four carbon atoms, the number of hits displayed in the five-column table is eight (Fig. 8).
4
Notes 1. MeRy-B is a PostgreSQL relational database accessible through a web interface developed in the PHP language. The web interface is rendered dynamic by the use of JavaScript and AJAX technologies. The application is maintained on a Linux server. A Java applet has been developed for 1H-NMR spectrum visu-
14
Catherine Deborde and Daniel Jacob
alization (the self-signed certificate is available on the “About MeRy-B” page). If NMR spectrum does not appear, this likely indicates that your browser lacks the Java Virtual Machine and needs upgrading by downloading the necessary Java software at http://www.java.com/en/download/index.jsp. 2. The MeRy-B website has been tested with IE10 (Windows), Safari 5.1.7, Google Chrome v29.0, and Firefox/Mozilla23.0.1 browsers. Some pages may not work as expected if you are using older browsers. For best results, update your browser and enable JavaScript. The web browser must be capable of handling Java applets, i.e., equipped with a recent version of Java interpreter. 3. Pertains to lists of “unknown” metabolites. In the MeRy-B database, an unknown compound is a compound with an unknown structure but a known 1D 1H-NMR signature (pattern of the NMR signal: singlet, doublet, triplet, or multiplet, and their chemical shifts). A specific nomenclature is used to allocate identifiers to the unknown compounds, to link these unknown signatures in the various spectra of the database. MeRy-B contains 105 of such unknown compounds. For example, when an interesting doublet peak has been detected on a spectrum at 7.95 ppm, this unknown compound is thus named unkD7.95: with D for doublet and 7.95 for the chemical shift expressed in ppm in agreement with the recommendations of MSI [16]. A putative identification may be added as a comment and in some cases quantification in arbitrary units may be included. The unknown concentration is calculated on the assumption that the measured resonance corresponded to one proton and using an arbitrary molecular weight of 100 Da. 4. The metabolite names are based on the KEGG compound database when created, the latter serving as a reference base. User synonyms could be added by users at this stage of creation and generally correspond to the common name. (For example: “GABA” is the common name for the metabolite referenced in the KEGG database as “4-aminobutanoate.”) 5. Pathways and Reactome are given as described in the PlantCyc database. It means that the metabolite might be involved in these biological pathways and/or these reactions but in fact, it highly depends on species, tissues, and cellular compartments. 6. The experimental conditions for acquiring the NMR spectra are given in the corresponding HMDB MetaboCard when available (mostly: pH 7, 25 °C, DSS as Chemical Shift Reference, water as Solvent, NMR field 600 MHz). When metabolites are not available in HMDB, experimentally 1H-NMR spectra have been acquired with “authentic standard compounds” (pH 6, 27 °C, TSP as Chemical Shift Reference, deuterated phosphate buffer
MeRy-B, a Plant Metabolomic Database
15
solution as Solvent, NMR field 500 MHz) by Bordeaux Metabolome Facility. 7. Concentration units. Comparisons must take into account the possible use of different quantification units. Units are always provided on MeRy-B cards to prevent inappropriate comparisons. DW: Dry weight. 8. How to consult a project in MeRy-B. Once a project has been selected, a new window will appear with a short description of the project, its publication reference, DOI when available, and a global view of each experiment of the project. Click on the name of one experiment. A new window will appear with a detailed view, from which all related information, such as experimental protocols (related to growth, harvest, and storage) and the experimental data and related metadata, is accessible. Click on the name of a sample and a new window will display details about the instrument used and all analytical protocols (extraction, analytical, and processing protocols). An interactive graphical tool can be used to either view the entire spectrum or zoom in and focus on one part of the spectrum. Click on the hyperlinked button called Viewer on the upper part of the window to launch the NMR viewer Java applet. This interactive graphical tool can be used to either view the entire spectrum or zoom in and focus on one part of the spectrum. Click on the hyperlinked button called Compounds on the upper part of the window to edit the list of identified and possibly quantified metabolites and of unknown compounds. 9. Hill notation is a common way of writing elemental formula of compounds. There is a specific order for the elements: first the carbon atoms, then the hydrogen atoms followed by the other chemical element in alphabetical order. For alanine, the Hill notation is C3H7NO2.
Acknowledgements The authors thank all the contributors who deposit their data in MeRy-B and especially the Metabolome Facility members of Bordeaux Functional Genomics Center and their collaborators, the META-PHOR EU consortium (FOOD-CT-2006-036220), the Genoplante GEN036 consortium, and the FRIM EU ERASysBio+ project. The authors wish to thank INRA and IBiSA for financial support in the development and maintenance of the MeRy-B database and knowledge base and CBiB for housing the database. The authors thank Dr A. Moing for critical reading.
16
Catherine Deborde and Daniel Jacob
References 1. Pichersky E, Lewinsohn E (2011) Convergent evolution in plant specialized metabolism. Annu Rev Plant Biol 62:549–566 2. de Oliveira Dal’Molin CG, Quek L-E, Palfreyman RW et al (2010) AraGEM, a genome-scale reconstruction of the primary metabolic network in Arabidopsis. Plant Physiol 152:579–589. doi:10.1104/pp. 109.148817 3. Kaplan F, Kopka J, Haskell DW et al (2004) Exploring the temperature stress metabolome of Arabidopsis. Plant Physiol 136:4159–4168 4. Gromova M, Roby C (2010) Toward Arabidopsis thaliana hydrophilic metabolome: assessment of extraction methods and quantitative 1H NMR. Physiol Plant 140:111–127 5. Allwood JW, de Vos RC, Moing A et al (2011) Plant metabolomics and its potential for systems biology research background concepts, technology, and methodology. Methods Enzymol 500:299–336 6. Forsythe IJ, Wishart DS (2009) Exploring human metabolites using the human metabolome database. Curr Protoc Bioinformatics 14(8):1–45. doi:10.1002/0471250953.bi1408s25 7. Ferry-Dumazet H, Gil L, Deborde C et al (2011) MeRy-B: a web knowledgebase for the storage, visualization, analysis and annotation of plant NMR metabolomic profiles. BMC Plant Biol 11:104. doi:10.1186/1471-2229-11-104 8. Plant Metabolic Network (PMN) http:// www.plantcyc.org. 28 Feb 2008 9. Wohlgemuth G, Haldiya P, Willighagen E et al (2010) The chemical translation ser-
10.
11.
12.
13.
14.
15. 16.
vice—a web-based tool to improve standardization of metabolomic reports. Bioinformatics 26:2647–2648 Kanehisa M, Goto S, Hattori M et al (2006) From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res 34:D354–D357 Wishart DS, Knox C, Guo AC et al (2009) HMDB: a knowledgebase for the human metabolome. Nucleic Acids Res 37((Database issue)):D603–D610 Degtyarenko K, de Matos P, Ennis M et al (2007) ChEBI: a database and ontology for chemical entities of biological interest. Nucleic Acids Res 36(Database issue):D344–D350 Shinbo Y, Nakamura Y, Altaf-Ul-Amin M et al (2006) KNApSAcK: a comprehensive speciesmetabolite relationship database. In: Saito K, Dixon RA, Willmitzer L (eds) Biotechnology in agriculture and forestry, vol 57, Plant metabolomics. Springer, Berlin, pp 165–181 Mochamad Afendi F, Okada T, Yamazaki M et al (2012) KNApSAcK family databases: integrated metabolite-plant species databases for multifaceted plant research. Plant Cell Physiol. doi:10.1093/pcp/pcr165 Kopka J, Schauer N, Krueger S et al (2005) [email protected]: the Golm metabolome database. Bioinformatics 21:1635–1638 Sumner L, Amberg A, Barrett D et al (2007) Proposed minimum reporting standards for chemical analysis. Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI). Metabolomics 3:211–221
1
Introduction
1.1 What Are Plant Primary Metabolites?
Plant primary metabolites are organic compounds, common to all or most plant species. Their functions are essential for plant growth, development, and reproduction. They are intermediates and products of metabolism involved in photosynthesis and other biosynthetic processes. These primary metabolites belong to several
Ganesh Sriram (ed.), Plant Metabolism: Methods and Protocols, Methods in Molecular Biology, vol. 1083, DOI 10.1007/978-1-62703-661-0_1, © Springer Science+Business Media New York 2014
3
4
Catherine Deborde and Daniel Jacob
compound families including carbohydrates, organic acids, amino acids, nucleotides, fatty acids, steroids, or lipids. Pichersky and Lewinsohn [1] estimated the plant primary metabolite number under 10,000 compounds. Some primary metabolites are also present in animals and microorganisms, whereas others are specific to the plant kingdom. A comparative analysis of the Arabidopsis and yeast genomes placed the number of genes involved in primary metabolism at 13,250 and the number of primary metabolites at 8,000 in Arabidopsis [1]. Nevertheless, de Oliveira Dal’Molin C et al. [2] who developed AraGEM, a genome-scale compartmented metabolic network model for Arabidopsis, included only 1,748 primary metabolites. Metabolomics studies of Arabidopsis based on hydromethanolic extraction, derivatization, and gas chromatographymass spectrometric (GC-MS) detection of hydrophilic metabolites yield a few hundred analytes, mainly primary metabolites. However, only 30–40 % of these analytes can be linked to known metabolites (specifically, only 81 are identified) [3]. In many cases such GC-MS analyses do not allow an absolute quantification but usually provide relative quantification (metabolite levels relative to control samples). In metabolomics studies of Arabidopsis by NMR on hydromethanolic extracts, 40 metabolites were identified and quantified [4]. Therefore the plant primary metabolite data tightly depend on the extraction process to prepare the sample and on the analytical method chosen (see ref. 5 for practical considerations). For comparison, in September 2013 the Human Metabolome Database v3.5 (HMDB, http://www.hmdb.ca/) compiled detailed literature-derived information on 41,528 metabolites found in human body (tissues, organs, or biofluids) and an extensive collection of experimental metabolite concentration data for biofluids, namely, plasma, urine, cerebrospinal fluid, and experimentally acquired 1H and 13C NMR and MS/MS spectra of commercially available “reference” or “authentic standard” compounds [6]. Until recently, unlike HMDB, there was no centralized database or repository dedicated exclusively to the plant kingdom and containing information on metabolites and their concentrations in a detailed experimental context. 1.2
What Is MeRy-B?
MeRy-B stands for Metabolomic Repository of Bordeaux and is available from http://bit.ly/meryb. It is the first platform for plant 1 H-NMR metabolomic profiles [7]. MeRy-B is designed (1) to provide a knowledge base of curated plant profiles and metabolites obtained by NMR, together with the corresponding experimental and analytical metadata; (2) to query and visualize the data; (3) to discriminate profiles with spectrum visualization tools and statistical analysis; and (4) to facilitate compound identification. MeRy-B structure (Fig. 1) follows the steps of a metabolomics experiment. It consists of four principal components: (A) “experimental design,”
MeRy-B, a Plant Metabolomic Database
5
Administration - Users, Access rights, Project status (public or private)
E Experimental design
Analytical metadata
- Biological source - Project - Experiments - Genotype(s) - Development stage(s) - Protocols (PDF)
- Instrument - Technique - Extraction method - Protocols (PDF)
A
B
Spectra data
Compounds
- Pre-processed spectra data (JCAMP-DX) - Processed spectra data - Peak lists
- Identified compounds (KEGG) - Unknown compounds - Quantifications
C
D
Controlled vocabularies (MSI) + Ontologies (OBO - obo.sourceforge.net) Query Builder
Statistical Analysis
Fig. 1 Structure of MeRy-B database and knowledge base
(B) “analytical metadata,” (C) “spectra data,” and (D) “compounds.” There is a fifth component (E) for “administration.” MeRy-B contains lists of plant metabolites and unknown compounds, with information about experimental conditions, the factors studied, and metabolite concentrations for 19 different plant species (Arabidopsis, broccoli, daphne, grape, maize, barrel clover, melon, Ostreococcus tauri, palm date, palm tree, peach, pine tree, eucalyptus, plantain rice, strawberry, sugar beet, tomato, vanilla), compiled from more than 2,300 annotated NMR profiles for various organs (e.g., fruit, seed, leaf, root) or tissues (e.g., mesocarp, epicarp, endosperm) deposited by 30 different private or public contributors in September 2013. Currently about half of the data deposited in MeRy-B is publicly available. MeRy-B manages all the data generated by NMR-based plant metabolomics experiments, from description of the biological source to identification of the metabolites and determinations of their concentrations. It is the first database allowing the display and overlay of NMR metabolomic profiles selected through queries on data or metadata. MeRy-B contains a collection of experimental metabolite concentration data for more than 40 metabolites coming from 20 public projects (48 experiments and 1,191 NMR spectra). The metabolites are mainly primary metabolites due to the choice of the extraction processes and NMR methods used. In this chapter, readers will be shown how to navigate through and retrieve data from MeRy-B website, how to get the list of the experimentally identified metabolites (Protocol 1), how to retrieve concentrations of a particular metabolite in all plant species present in MeRy-B (Protocol 2), how to get primary metabolite list for a particular plant species in MeRy-B (Protocol 3), and for a particular tissue of a plant species (Protocol 4) how to find information on a primary metabolite regardless of species (Protocol 5).
6
2
Catherine Deborde and Daniel Jacob
Material: Necessary Resources 1. Hardware: Computer with Internet access. 2. Software: An up-to-date web browser (see Note 1 for java applet). 3. Connection to MeRy-B http://bit.ly/meryb (see Note 2).
3
Methods On the home page and most pages of MeRy-B website, there is a menu bar located at the left-hand side of the page, with ten clickable links: four under General Information, four under Data consultation, and two under Other Information. Click on the button Compounds under Data consultation. Within a few seconds a simplified map of plant metabolism pathways will be displayed with two types of metabolites (Fig. 2). Some metabolite names are spotlighted with a colored circle indicating that these metabolites are either identified or quantified in projects deposited in MeRy-B database. Other metabolite names are not
Fig. 2 A screenshot showing a simplified map of plant (mainly primary) metabolism. This figure appears in color in the online version of this chapter
MeRy-B, a Plant Metabolomic Database
7
circled, which means that these metabolites have not been identified in projects deposited in MeRy-B database, e.g., starch and oxalate. The circles are colored green for amino acids, red for sugars, blue for organic acids, and yellow for other metabolite families. For each metabolite identified in projects deposited in MeRy-B database, there is one MeRy-B card. Click on the circle near the metabolite name, and a new window will appear containing the MeRy-B card. The number of identified metabolites in MeRy-B database is summarized in the upper part of the page, above the map: Compound(s) found. Currently 77 metabolites are reported. Move the mouse over the word PlantCyc ([8], and see Chapter 13) on the left-hand side of the map to change the hyperlinks of spotlighting to the corresponding PlantCyc compounds. Click on the circle near the metabolite name, and a new window will appear containing the Plant Metabolic Network—PlantCyc Compound card. 3.1 Protocol 1: Obtaining the List of Experimentally Identified Primary Metabolites in MeRy-B
Click on List near Compounds by on the left-hand side of the page, above the pathway map. Within a few seconds a five-column table will be displayed with all the metabolites identified and/or quantified coming from all public projects deposited into MeRy-B database (see Note 3 for the list of unknown metabolites). To survey the list (Fig. 3), in alphabetical order, of the metabolites experimentally identified in MeRy-B, use the scroll bar on the right side of the browser window. This table consists of five columns. The first column displays the line number of
Fig. 3 A screenshot depicting a table with all the metabolites identified and/or quantified in the projects deposited in MeRy-B database
8
Catherine Deborde and Daniel Jacob
the table, the second column displays the name of the metabolite (see Note 4), the third column the species where the metabolite has been identified, the fourth column contains the number of experiments where the metabolite has been identified, and the fifth column contains the MeRy-B card hyperlink (a green button). 3.2 Protocol 2: MeRy-B Card Overview; Retrieving Concentrations of a Particular Metabolite in All Plant Species Present in MeRy-B
For each metabolite in MeRy-B database, there is one MeRy-B card (Fig. 4). The concept of a MeRy-B card is analogous to the MetaboCard in HMDB. Each MeRy-B card contains several sections, depending on whether information is available or not, mainly subdivided into two kinds: chemical and experimental. To survey the type of information displayed in a typical MeRy-B card, use the scroll bar on the right side of your browser window to scroll down the page. First, on the left top of the header are
Fig. 4 A MeRy-B card screenshot. The MeRy-B card displays all public data stored in the MeRy-B knowledge base for a given compound. For each species and tissue in which a given compound is found, this card displays data concerning 1H-NMR chemical shifts, multiplicity, and quantification. Data may be filtered and sorted by species and/or tissue
MeRy-B, a Plant Metabolomic Database
9
displayed (1) the name of the metabolite and the user synonyms (see Note 4) and (2) hyperlinks to the Chemical Translation Service (CTS) [9] and PlantCyc compounds [8]. On the top right of the header, a summary of experimental data linked with this compound is displayed, namely, species, tissues, and analytical techniques. The chemical information sections are laid out as follows: (1) Kyoto Encyclopedia of Genes and Genomes (KEGG) Compound [10], (2) Other Links, (3) Pathways & Reactome, (4) HMDB NMR Peak List [6, 11], and (5) NMR Spectrum. To expand a section, click on the small square icon with a “plus” sign inside. When expanded, the sign inside the icon changes itself to “minus.” The “KEGG” section contains essential information on the identity of the compound coming from the KEGG compound database. For more details, click on the KEGG Accession identifier on the right column. The “Other Links” section brings together useful hyperlinks to references and other public databases including the chemical entities of biological interest (CHEBI) [12], the KNApSAck database [13, 14], the Golm Metabolome database (MPIMP) [15], and HMDB [11]. The “Pathways & Reactome” section provides the set of biological pathways retrieved from PlantCyc database [8] (see Note 5). To view the reactions for a particular biological pathway, click on the small square icon with a “plus” sign located on the left corresponding to the pathway. To view all reactions for all pathways, click on “all” located at the top of the pathways list. For each reaction, the Enzyme Commission number is given as a hyperlink to the KEGG Enzyme website. The last two sections provide analytical information on NMR, namely, the HMDB Peak List (with compound chemical shifts when available) and an interactive NMR Spectrum Viewer in MeRy-B (see Note 6). To view the 1HNMR spectrum, scroll down to NMR Spectrum and click on the button. This launches a Java applet, and the 1H-NMR spectrum of the metabolite of interest will appear in the applet window. If not, see Notes 1 and 2. This applet allows the user to interactively zoom into the spectrum by holding down the left mouse button, selecting the desired area, and finally releasing the mouse button. To zoom out, click on the right mouse button. The “MeRy-B card” displays the list of experiments in which the metabolite was detected, and, for each experiment, additional metadata are listed (species, tissue/organ, and project name), together with a summary of the analytical results (e.g., for 1H-NMR: chemical shift, multiplicity, minimum and maximum values for quantification). This card also highlights quantitative differences between species, tissues, organs, or experiments for the compound. Users can filter the desired data by using the filters located on top of this list, by selecting a species, a tissue/ organ, or both and can also sort them by choosing a criterion: “Species” or “Tissue/Organ.”
10
Catherine Deborde and Daniel Jacob
There are several ways to display a MeRy-B card and retrieve concentrations of a particular metabolite in plant species present in MeRy-B: 1. Click on the button Compounds under Data consultation. Within a few seconds a simplified map of plant metabolism will appear. Click on the colored spot close to the chosen metabolite in order to display the MeRy-B card. Within a few seconds a new window will be displayed with this card. 2. Follow Protocol 1 and click on the MeRy-B card hyperlink of the metabolite of interest. 3. Follow Protocol 5. 3.3 Protocol 3: Obtaining the List of Primary Metabolites for a Particular Plant Species
Click on the button Compounds under Data consultation (see Fig. 2). Click in the text search box Species in the first frame named General, located in the upper part of the window, and select the species (e.g., Lycopersicon esculentum for tomato) in the dropdown list. Within a few seconds a simplified map of plant metabolism will be displayed (see Protocol 3.1 for explanation). If the selected species is also described specifically in PlantCyc, e.g., LycoCyc for tomato, two maps are proposed, one with information coming from MeRy-B database and the other from LycoCyc database (for moving from MeRy-B to LycoCyc data: move the mouse over the word LycoCyc on the left-hand side of the map to update the map with LycoCyc data). Click on List near Compounds by on the left-hand side of the page, above the pathway map. Within a few seconds a five-column table will be displayed with all the identified and/or quantified metabolites in tomato coming from the projects deposited in MeRy-B database (33 metabolites in September 2013). In this case the third column displays the tissue or the organ where the metabolite has been identified (Fig. 5). Click on the hyperlink of the fifth column of asparagine (MRB85) for example. Within a few seconds a new window will appear: the MeRy-B card for the selected metabolite (see Subheading 3.2 for the general description of MeRy-B card) (Fig. 6). To survey the minimal and maximal concentrations reported for each publicly available project deposited in MeRy-B, use the scroll bar on the right side of the browser window. Be aware of the different concentration units used (see Note 7). This card highlights quantitative differences between tissues, organs, or experiments for a given metabolite. The fourth column provides a hyperlink to the project to enable the user to understand the biological and analytical context of the reported concentration values of the metabolite of interest (click on the hyperlink [see Note 8 How to consult a project in MeRy-B]).
MeRy-B, a Plant Metabolomic Database
11
Fig. 5 A screenshot showing the table with all the metabolites identified and/or quantified in the tomato species coming from the projects deposited in MeRy-B database
Fig. 6 A screenshot showing the MeRy-B card of asparagine in tomato
12
Catherine Deborde and Daniel Jacob
3.4 Protocol 4: Obtaining the List of Primary Metabolites for a Particular Tissue of a Given Plant Species
Click on the button Compounds under Data consultation. Click in the text search box Species in the first frame named General, located in the upper part of the window, and select the species in the dropdown list, for instance tomato. Click also in the text search box Tissue/Organ, and select the term of interest in the drop-down list. Within a few seconds a simplified four-column table will be displayed with all the identified and/or quantified metabolites in the tissue/organ selected in tomato coming from the projects deposited in MeRy-B database (e.g., 21 metabolites for leaf tissue, 27 metabolites for seed, 30 metabolites for pericarp tissue in September 2013). Click on the hyperlink of the fourth column. Within a few seconds a new window will appear: the MeRy-B card for the selected metabolite (see Subheading 3.2 for the general description of MeRy-B card and Protocol 3.2).
3.5 Protocol 5: Obtaining Information on a Primary Metabolite Regardless of Species
Click on the button Compounds under Data consultation. This interface allows the user to search all the metabolites available in MeRy-B by Name or by Elemental Formula. The first way of searching metabolite within this MeRy-B interface is by Name. Click in the text search box Name or User Synonym in the second frame named Compounds, located in the upper part of the window; once the cursor appears type the first letter of the metabolite. A drop-down list will appear. Select the metabolite of interest. For example, type “a” and select Alanine; within a few seconds a five-column table will be displayed with three options: alanine, phenylalanine, and beta-alanine (Fig. 7).
Fig. 7 A screenshot of how the MeRy-B browser page will appear when searching metabolites by name. This example shows the search results for the word “alanine.” The MeRy-B card accession numbers on the right side of the table are hyperlinked
MeRy-B, a Plant Metabolomic Database
13
Fig. 8 A screenshot of how the MeRy-B browser page will appear when searching metabolites by elemental formula. This example shows the search results for the formula “C6” (containing six carbon atoms). The MeRy-B card accession numbers on the right side of the table are hyperlinked
Click on the hyperlink of the fifth column on the first line for alanine (MRB63). Within a few seconds a new window will appear: the MeRy-B card for Alanine, the selected metabolite (see Subheading 3.2 for the general description of MeRy-B card, Protocol 3.2, and Note 8 How to consult project in MeRy-B). Alanine has been reported for 10 species in MeRy-B, 8 of them belonging to publicly available projects, and for 38 experiments with 24 of them publicly available. Another method for searching metabolites within MeRy-B is by Elemental Formula. Elemental formulae follow the Hill notation in MeRy-B (see Note 9). Click in the text search box Elemental Formula and enter either the full or the partial elemental formula (i.e., only the number of carbon atoms). For example, if the user searches in MeRy-B for identified metabolites containing four carbon atoms, the number of hits displayed in the five-column table is eight (Fig. 8).
4
Notes 1. MeRy-B is a PostgreSQL relational database accessible through a web interface developed in the PHP language. The web interface is rendered dynamic by the use of JavaScript and AJAX technologies. The application is maintained on a Linux server. A Java applet has been developed for 1H-NMR spectrum visu-
14
Catherine Deborde and Daniel Jacob
alization (the self-signed certificate is available on the “About MeRy-B” page). If NMR spectrum does not appear, this likely indicates that your browser lacks the Java Virtual Machine and needs upgrading by downloading the necessary Java software at http://www.java.com/en/download/index.jsp. 2. The MeRy-B website has been tested with IE10 (Windows), Safari 5.1.7, Google Chrome v29.0, and Firefox/Mozilla23.0.1 browsers. Some pages may not work as expected if you are using older browsers. For best results, update your browser and enable JavaScript. The web browser must be capable of handling Java applets, i.e., equipped with a recent version of Java interpreter. 3. Pertains to lists of “unknown” metabolites. In the MeRy-B database, an unknown compound is a compound with an unknown structure but a known 1D 1H-NMR signature (pattern of the NMR signal: singlet, doublet, triplet, or multiplet, and their chemical shifts). A specific nomenclature is used to allocate identifiers to the unknown compounds, to link these unknown signatures in the various spectra of the database. MeRy-B contains 105 of such unknown compounds. For example, when an interesting doublet peak has been detected on a spectrum at 7.95 ppm, this unknown compound is thus named unkD7.95: with D for doublet and 7.95 for the chemical shift expressed in ppm in agreement with the recommendations of MSI [16]. A putative identification may be added as a comment and in some cases quantification in arbitrary units may be included. The unknown concentration is calculated on the assumption that the measured resonance corresponded to one proton and using an arbitrary molecular weight of 100 Da. 4. The metabolite names are based on the KEGG compound database when created, the latter serving as a reference base. User synonyms could be added by users at this stage of creation and generally correspond to the common name. (For example: “GABA” is the common name for the metabolite referenced in the KEGG database as “4-aminobutanoate.”) 5. Pathways and Reactome are given as described in the PlantCyc database. It means that the metabolite might be involved in these biological pathways and/or these reactions but in fact, it highly depends on species, tissues, and cellular compartments. 6. The experimental conditions for acquiring the NMR spectra are given in the corresponding HMDB MetaboCard when available (mostly: pH 7, 25 °C, DSS as Chemical Shift Reference, water as Solvent, NMR field 600 MHz). When metabolites are not available in HMDB, experimentally 1H-NMR spectra have been acquired with “authentic standard compounds” (pH 6, 27 °C, TSP as Chemical Shift Reference, deuterated phosphate buffer
MeRy-B, a Plant Metabolomic Database
15
solution as Solvent, NMR field 500 MHz) by Bordeaux Metabolome Facility. 7. Concentration units. Comparisons must take into account the possible use of different quantification units. Units are always provided on MeRy-B cards to prevent inappropriate comparisons. DW: Dry weight. 8. How to consult a project in MeRy-B. Once a project has been selected, a new window will appear with a short description of the project, its publication reference, DOI when available, and a global view of each experiment of the project. Click on the name of one experiment. A new window will appear with a detailed view, from which all related information, such as experimental protocols (related to growth, harvest, and storage) and the experimental data and related metadata, is accessible. Click on the name of a sample and a new window will display details about the instrument used and all analytical protocols (extraction, analytical, and processing protocols). An interactive graphical tool can be used to either view the entire spectrum or zoom in and focus on one part of the spectrum. Click on the hyperlinked button called Viewer on the upper part of the window to launch the NMR viewer Java applet. This interactive graphical tool can be used to either view the entire spectrum or zoom in and focus on one part of the spectrum. Click on the hyperlinked button called Compounds on the upper part of the window to edit the list of identified and possibly quantified metabolites and of unknown compounds. 9. Hill notation is a common way of writing elemental formula of compounds. There is a specific order for the elements: first the carbon atoms, then the hydrogen atoms followed by the other chemical element in alphabetical order. For alanine, the Hill notation is C3H7NO2.
Acknowledgements The authors thank all the contributors who deposit their data in MeRy-B and especially the Metabolome Facility members of Bordeaux Functional Genomics Center and their collaborators, the META-PHOR EU consortium (FOOD-CT-2006-036220), the Genoplante GEN036 consortium, and the FRIM EU ERASysBio+ project. The authors wish to thank INRA and IBiSA for financial support in the development and maintenance of the MeRy-B database and knowledge base and CBiB for housing the database. The authors thank Dr A. Moing for critical reading.
16
Catherine Deborde and Daniel Jacob
References 1. Pichersky E, Lewinsohn E (2011) Convergent evolution in plant specialized metabolism. Annu Rev Plant Biol 62:549–566 2. de Oliveira Dal’Molin CG, Quek L-E, Palfreyman RW et al (2010) AraGEM, a genome-scale reconstruction of the primary metabolic network in Arabidopsis. Plant Physiol 152:579–589. doi:10.1104/pp. 109.148817 3. Kaplan F, Kopka J, Haskell DW et al (2004) Exploring the temperature stress metabolome of Arabidopsis. Plant Physiol 136:4159–4168 4. Gromova M, Roby C (2010) Toward Arabidopsis thaliana hydrophilic metabolome: assessment of extraction methods and quantitative 1H NMR. Physiol Plant 140:111–127 5. Allwood JW, de Vos RC, Moing A et al (2011) Plant metabolomics and its potential for systems biology research background concepts, technology, and methodology. Methods Enzymol 500:299–336 6. Forsythe IJ, Wishart DS (2009) Exploring human metabolites using the human metabolome database. Curr Protoc Bioinformatics 14(8):1–45. doi:10.1002/0471250953.bi1408s25 7. Ferry-Dumazet H, Gil L, Deborde C et al (2011) MeRy-B: a web knowledgebase for the storage, visualization, analysis and annotation of plant NMR metabolomic profiles. BMC Plant Biol 11:104. doi:10.1186/1471-2229-11-104 8. Plant Metabolic Network (PMN) http:// www.plantcyc.org. 28 Feb 2008 9. Wohlgemuth G, Haldiya P, Willighagen E et al (2010) The chemical translation ser-
10.
11.
12.
13.
14.
15. 16.
vice—a web-based tool to improve standardization of metabolomic reports. Bioinformatics 26:2647–2648 Kanehisa M, Goto S, Hattori M et al (2006) From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res 34:D354–D357 Wishart DS, Knox C, Guo AC et al (2009) HMDB: a knowledgebase for the human metabolome. Nucleic Acids Res 37((Database issue)):D603–D610 Degtyarenko K, de Matos P, Ennis M et al (2007) ChEBI: a database and ontology for chemical entities of biological interest. Nucleic Acids Res 36(Database issue):D344–D350 Shinbo Y, Nakamura Y, Altaf-Ul-Amin M et al (2006) KNApSAcK: a comprehensive speciesmetabolite relationship database. In: Saito K, Dixon RA, Willmitzer L (eds) Biotechnology in agriculture and forestry, vol 57, Plant metabolomics. Springer, Berlin, pp 165–181 Mochamad Afendi F, Okada T, Yamazaki M et al (2012) KNApSAcK family databases: integrated metabolite-plant species databases for multifaceted plant research. Plant Cell Physiol. doi:10.1093/pcp/pcr165 Kopka J, Schauer N, Krueger S et al (2005) [email protected]: the Golm metabolome database. Bioinformatics 21:1635–1638 Sumner L, Amberg A, Barrett D et al (2007) Proposed minimum reporting standards for chemical analysis. Chemical Analysis Working Group (CAWG) Metabolomics Standards Initiative (MSI). Metabolomics 3:211–221
