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. 2017 Jun;174(2):922-934.
doi: 10.1104/pp.17.00242. Epub 2017 Apr 25.

ChloroKB: A Web Application for the Integration of Knowledge Related to Chloroplast Metabolic Network

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Free PMC article

ChloroKB: A Web Application for the Integration of Knowledge Related to Chloroplast Metabolic Network

Pauline Gloaguen et al. Plant Physiol. .
Free PMC article

Abstract

Higher plants, as autotrophic organisms, are effective sources of molecules. They hold great promise for metabolic engineering, but the behavior of plant metabolism at the network level is still incompletely described. Although structural models (stoichiometry matrices) and pathway databases are extremely useful, they cannot describe the complexity of the metabolic context, and new tools are required to visually represent integrated biocurated knowledge for use by both humans and computers. Here, we describe ChloroKB, a Web application (http://chlorokb.fr/) for visual exploration and analysis of the Arabidopsis (Arabidopsis thaliana) metabolic network in the chloroplast and related cellular pathways. The network was manually reconstructed through extensive biocuration to provide transparent traceability of experimental data. Proteins and metabolites were placed in their biological context (spatial distribution within cells, connectivity in the network, participation in supramolecular complexes, and regulatory interactions) using CellDesigner software. The network contains 1,147 reviewed proteins (559 localized exclusively in plastids, 68 in at least one additional compartment, and 520 outside the plastid), 122 proteins awaiting biochemical/genetic characterization, and 228 proteins for which genes have not yet been identified. The visual presentation is intuitive and browsing is fluid, providing instant access to the graphical representation of integrated processes and to a wealth of refined qualitative and quantitative data. ChloroKB will be a significant support for structural and quantitative kinetic modeling, for biological reasoning, when comparing novel data with established knowledge, for computer analyses, and for educational purposes. ChloroKB will be enhanced by continuous updates following contributions from plant researchers.

Figures

Figure 1.
Figure 1.
Reconstructed network visible on the ChloroKB home page. The network includes N and S assimilation, synthesis of carbon precursors, amino acids, nucleotides, vitamins, cofactors, pigments, some lipids, hormones, glucosinolates, and plastidial protein degradation apparatus along with their major connections. Each purple icon represents a metabolic map that can be accessed by clicking on the icon.
Figure 2.
Figure 2.
How ChloroKB represents a multicompartmentalized process (vitamin B3 NAD synthesis). The chloroplast is represented surrounded by its double membrane, or envelope (in yellow), the peroxisome is in cyan, and the mitochondrion is in red. Cytosolic proteins and metabolites are represented in white.
Figure 3.
Figure 3.
Overview of the subcellular distribution of proteins represented in the reconstructed network. Proteins with multiple localizations were counted only once and are represented with additional color boxes indicating alternative subcellular localization. This counting does not refer to a specific cell type; hence, we use the term plastid rather than chloroplast. For more detailed counting, see Supplemental Table S1, and for gene/localization correspondence, see Supplemental Table S2.
Figure 4.
Figure 4.
Querying and browsing in ChloroKB.
Figure 5.
Figure 5.
How incomplete knowledge, prediction, and hypotheses are indicated in ChloroKB. The codes used to represent reactions or catalytic processes differ depending on whether they are supported by experimental data or represent hypothetical reactions (or transports) or predictions based on sequence similarity. Unknown subcellular localizations also can be distinguished from experimentally established localizations. See legends in the figure.
Figure 6.
Figure 6.
Exploration of chorismate and isochorismate metabolism. The so-called shikimate pathway, with the input carbon precursors (erythrose 4-phosphate and phosphoenolpyruvate) and the links toward erythrose 4-phosphate and phosphoenolpyruvate metabolisms (purple hexagons). The six outputs from chorismate and isochorismate (salicylate, phylloquinone, vitamin B9, and the three aromatic amino acids) are represented at the bottom. The transformation of erythrose 4-phosphate and phosphoenolpyruvate into chorismate has been characterized extensively, and all the catalytic conversions (arrows) are thus represented in black. In contrast, the connections between shikimate and shikimate/l-quinate metabolism in the cytosol are only poorly described, as represented by the pale gray color of the arrows. Shikimate O-hydroxycinnamoyltransferase in the cytosol uses shikimate (or l-quinate) as a substrate; therefore, shikimate and/or l-quinate must be exported from the chloroplast. This export step has yet to be characterized at the molecular level, and the nature of the molecule exported (shikimate, 3-dehydroshimikate, or quinate) is currently unclear. This uncertainty is indicated by the gray color of the symbols.
Figure 7.
Figure 7.
Chorismate commitment to aromatic amino acid synthesis: passing from a high-level view of metabolism to detailed representation of regulatory processes. A, Zoom on the chorismate and isochorismate map showing the link toward the chorismate HUB. B, The chorismate HUB map is at the crossroads for 11 metabolic maps. Note the minus and plus signs above the metabolites in the protein complexes. The minus sign appended to l-Trp in the anthranilate synthase subunit 1 complex indicates that binding of l-Trp to this subunit in the complex inhibits the protein’s activity. The red dot indicates that text-based information is available for the complex. C, Detailed mechanistic description of how the activity of chorismate mutase1 (CM1) and CM3 are controlled, based on 3D data. The model shows that CM3 is activated only by l-Trp and that CM1 and CM2 are inhibited by l-Tyr and l-Phe and activated by l-Trp. l-Trp is a nonessential activator; therefore, CM1 and CM2 are active in the absence of l-Trp (a catalysis symbol links the protein and the reaction arrow). The presence of l-Trp renders the two enzymes more active, as symbolized by the thicker catalysis symbol connecting the protein and the reaction. Binding of l-Trp, l-Tyr, and l-Phe to CM1 is mutually exclusive (the protein has a single regulatory site); this is represented by three different complexes, each binding one of the three amino acids. When in complex with l-Tyr or l-Phe, the CM1 dimer is inactive, as represented by the absence of the catalysis symbol connecting these complexes to the reaction. CM2 is unregulated, and its function in the cytosol is unclear. Note that the box representing CM3 is surrounded by a thin line to indicate that it was not experimentally detected in leaf.

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