A Topological Map of the Compartmentalized Arabidopsis thaliana Leaf Metabolome

Abstract

BackgroundThe extensive subcellular compartmentalization of metabolites and metabolism in eukaryotic cells is widely acknowledged and represents a key factor of metabolic activity and functionality. In striking contrast, the knowledge of actual compartmental distribution of metabolites from experimental studies is surprisingly low. However, a precise knowledge of, possibly all, metabolites and their subcellular distributions remains a key prerequisite for the understanding of any cellular function.Methodology/Principal FindingsHere we describe results for the subcellular distribution of 1,117 polar and 2,804 lipophilic mass spectrometric features associated to known and unknown compounds from leaves of the model plant Arabidopsis thaliana. Using an optimized non-aqueous fractionation protocol in conjunction with GC/MS- and LC/MS-based metabolite profiling, 81.5% of the metabolic data could be associated to one of three subcellular compartments: the cytosol (including the mitochondria), vacuole, or plastids. Statistical analysis using a marker-‘free’ approach revealed that 18.5% of these metabolites show intermediate distributions, which can either be explained by transport processes or by additional subcellular compartments.Conclusion/SignificanceNext to a functional and conceptual workflow for the efficient, highly resolved metabolite analysis of the fractionated Arabidopsis thaliana leaf metabolome, a detailed survey of the subcellular distribution of several compounds, in the graphical format of a topological map, is provided. This complex data set therefore does not only contain a rich repository of metabolic information, but due to thorough validation and testing by statistical methods, represents an initial step in the analysis of metabolite dynamics and fluxes within and between subcellular compartments.

Metabolic Fluxes in an Illuminated Arabidopsis Rosette

“…Some metabolites, such as RuBP, sedoheptulose-1,7-bisphosphate (SBP), Fru-1,6-bisphosphate (FBP), ADPG, UDPG, malate, fumarate, and isocitrate were largely in a single compartment, while others like dihydroxyacetone phosphate (DHAP), 3PGA, hexose phosphates, hexoses, most amino acids, and organic Photosynthetic Fluxes in Arabidopsisacids were present in two or more compartments. This resembles results from earlier studies in spinach (Spinacia oleracea) (Gerhardt et al, 1987;Riens et al, 1991;Winter et al, 1994), maize (Zea mays) (Weiner and Heldt, 1992), barley (Hordeum vulgare) (Winter et al, 1993), and Arabidopsis (Krueger et al, 2011) leaves.…”

“…Neither approach pinpointed the location of an inactive pool; nevertheless, two other possibilities remain. First, bioinformatic analysis of Arabidopsis metabolite profiles revealed patterns suggestive of microcompartmentation of metabolism within organelles (Krueger et al, 2011), as previously postulated for a pool of UDPG that is involved in cellulose synthesis (Amor et al, 1995). Secondly, recent studies in Arabidopsis and potato implicate the presence of as yet unidentified G1P transporters at the plastid and plasma membranes (Fettke et al, 2011).…”

Reconstruction of Arabidopsis metabolic network models accounting for subcellular compartmentalization and tissue-specificity

“…The resulting prediction accuracy was significantly high (hyper-geometric P value < 10 −13 ), with a precision of 0.47 and recall of 0.50 (Table S2). To further validate predictions of enzyme subcellular localizations, we used recently published metabolomic data by Krueger et al (19), which describes subcellular distribution of dozen of compounds from leaves of Arabidopsis. Comparing the measured metabolites in leaves to those computationally predicted to belong to the leaves model showed a significantly high prediction accuracy (hyper-geometric P value = 0.03), with a precision of 0.62 and recall of 0.72.…”

Genome-Wide Association Mapping Reveals That Specific and Pleiotropic Regulatory Mechanisms Fine-Tune Central Metabolism and Growth in Arabidopsis

“…Alternatively, fumarase activity might be modified in a manner that our standardized assays do not detect (e.g., via a change in substrate affinity). The vast majority of the Mal and Fum in plant cells is located in the vacuole (Krueger et al, 2011;Arrivault et al, 2014). The lack of effect of the FUM1 enzyme activity QTL on the levels of these metabolites, and the association of Mal and Fum metabolite QTL with FUM2, indicate that cytosolic fumarase determines the vacuolar levels of Mal and Fum.…”