Systematic Structural Characterization of Metabolites in Arabidopsis via Candidate Substrate-Product Pair Networks  

Abstract

Plant metabolomics is increasingly used for pathway discovery and to elucidate gene function. However, the main bottleneck is the identification of the detected compounds. This is more pronounced for secondary metabolites as many of their pathways are still underexplored. Here, an algorithm is presented in which liquid chromatography-mass spectrometry profiles are searched for pairs of peaks that have mass and retention time differences corresponding with those of substrates and products from well-known enzymatic reactions. Concatenating the latter peak pairs, called candidate substrate-product pairs (CSPP), into a network displays tentative (bio)synthetic routes. Starting from known peaks, propagating the network along these routes allows the characterization of adjacent peaks leading to their structure prediction. As a proof-of-principle, this high-throughput cheminformatics procedure was applied to the Arabidopsis thaliana leaf metabolome where it allowed the characterization of the structures of 60% of the profiled compounds. Moreover, based on searches in the Chemical Abstract Service database, the algorithm led to the characterization of 61 compounds that had never been described in plants before. The CSPP-based annotation was confirmed by independent MS n experiments. In addition to being high throughput, this method allows the annotation of low-abundance compounds that are otherwise not amenable to isolation and purification. This method will greatly advance the value of metabolomics in systems biology.

Small Glycosylated Lignin Oligomers Are Stored in Arabidopsis Leaf Vacuoles

“…Recently, oligolignol derivatives have been found in the stems of flax (Huis et al, 2012) and in the stems and rosette leaves of Arabidopsis (Vanholme et al, 2012b;Morreel et al, 2014). To …”

“…In the trimer, only the product from 8-O-4-coupling is shown. Shorthand naming of the oligolignols is based on the nomenclature described by Morreel et al (2014). In brief, the linkage type is noted between parentheses, whereas the units are denoted in bold: guaiacyl (G) and syringyl (S) units are derived from coniferyl and sinapyl alcohol.…”

A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism

“…Profiling of the cells and the medium yielded 968 and 551 m/z features that could be reduced further to 430 and 225 m/z peak groups, respectively. However, 75 and 53 m/z features could not be grouped and remained as singletons (Broeckling et al, 2014;Morreel et al, 2014). Statistical analyses were performed on the pseudomolecular ion from each peak group and on the singleton ions (i.e.…”

“…In this specific tissue culture experiment, watertreated cells remained alive even when they produced extracellular lignin; hence, the lignin-forming cultures were cultured with sampling up to 20 d. The samples were stored at 280°C before the analyses. Phenolic compounds were extracted following the procedure described by Morreel et al (2014). Two replicate samples of cells and the medium of the lignin-forming and non-ligninforming cultures were analyzed at time points 0, 5, 10, 15, and 20 d, and two replicates of the lignin-reforming cultures were analyzed at time points 20 + 4, 20 + 8, 20 + 12, 20 + 16, and 20 + 20 d, making a total of 60 samples.…”

“…Metabolic conversion types on which the edges were based, and their corresponding number of CSPPs, are shown in Supplemental Table S1. Metabolic conversion types were selected from the list given by Morreel et al (2014) based on their prevalence in CSPP networks and presumed presence in gymnosperms. Structural elucidation of the multiplestage mass spectrometry fragmentation MS n spectra was based on Eklund et al (2008) and Morreel et al (2004Morreel et al ( , 2010aMorreel et al ( , 2010b for the (neo)lignans/oligolignols, on Cuyckens and Claeys (2004), Fabre et al (2001), Hughes et al (2001), Morreel et al (2006), and Hvattum and Ekeberg (2003) for the flavonoids, on Zhou et al (2013) and Abu-Reidah et al (2014) for the C-glycosylated flavanones, on Morreel et al (2014) for the phenylpropanoids/benzenoids, and on Gu et al (2003) and Jaiswal et al (2012) for the condensed tannins.…”