Subcellular Flux Analysis of Central Metabolism in a Heterotrophic Arabidopsis Cell Suspension Using Steady-State Stable Isotope Labeling    

Abstract

The presence of cytosolic and plastidic pathways of carbohydrate oxidation is a characteristic feature of plant cell metabolism. Ideally, steady-state metabolic flux analysis, an emerging tool for creating flux maps of heterotrophic plant metabolism, would capture this feature of the metabolic phenotype, but the extent to which this can be achieved is uncertain. To address this question, fluxes through the pathways of central metabolism in a heterotrophic Arabidopsis (Arabidopsis thaliana) cell suspension culture were deduced from the redistribution of label in steady-state 13 C-labeling experiments using [1-13 C]-, [2-13 C]-, and [U- 13C 6 ]glucose. Focusing on the pentose phosphate pathway (PPP), multiple data sets were fitted simultaneously to models in which the subcellular compartmentation of the PPP was altered. The observed redistribution of the label could be explained by any one of three models of the subcellular compartmentation of the oxidative PPP, but other biochemical evidence favored the model in which the oxidative steps of the PPP were duplicated in the cytosol and plastids, with flux through these reactions occurring largely in the cytosol. The analysis emphasizes the inherent difficulty of analyzing the PPP without predefining the extent of its compartmentation and the importance of obtaining high-quality data that report directly on specific subcellular processes. The Arabidopsis flux map also shows that the potential ATP yield of respiration in heterotrophic plant cells can greatly exceed the direct metabolic requirements for biosynthesis, highlighting the need for caution when predicting flux through metabolic networks using assumptions based on the energetics of resource utilization.Extensive subcellular compartmentation, with unique locations for many steps and pathways, as well as the duplication of other steps and pathways in different compartments, adds greatly to the structural complexity of the plant metabolic network (Lunn, 2007;. The need to consider discrete pools of metabolites in specific compartments, and the transporters that link them, complicates the quest for a detailed, predictive understanding of the regulation of plant metabolism; as a result, it remains difficult to manipulate flows of material through the central metabolic network in a predictable way (Carrari et al., 2003;Sweetlove et al., 2008).The emergence of steady-state metabolic flux analysis (MFA) as a practicable systems biology tool for generating flux maps of the central metabolic pathways in plants offers new opportunities for analyzing plant metabolic phenotypes (Ratcliffe and ShacharHill, 2006;Libourel and Shachar-Hill, 2008;Schwender, 2008;Kruger and Ratcliffe, 2009). In this approach, substrates labeled with stable isotopes are introduced into the network, and fluxes are determined by measuring the labeling of the system after it has reached an isotopic and metabolic steady state. Subcellular compartmentation of steps and pathways can be incorporated into the model that describes the redistribution of t...

Carbon and Nitrogen Provisions Alter the Metabolic Flux in Developing Soybean Embryos

“…The coordinated modeling of multiple independent labeling experiments has been done previously (Schwender et al, 2006;Allen et al, 2009b;Masakapalli et al, 2010) and has resulted in confident flux estimates. We combined different labeling data sets into our simulations for central metabolism.…”

“…The flux models establish network function (Ratcliffe and Shachar-Hill, 2006) and can occasionally identify unique roles for enzymes . Most MFA studies in plants have focused on seeds (Troufflard et al, 2007;Iyer et al, 2008;Allen et al, 2009b;Lonien and Schwender, 2009;Alonso et al, 2010Alonso et al, , 2011 because of their pseudo-steady-state metabolism and economic importance, although plant cell suspensions have been used (Rontein et al, 2002;Baxter et al, 2007;Williams et al, 2008;Masakapalli et al, 2010) because of their experimental versatility. Together, these studies have quantified roles for metabolic pathways, such as the use of the tricarboxylic acid cycle (Schwender, 2008;Sweetlove et al, 2008Sweetlove et al, , 2010Allen et al, 2009b;Kruger and Ratcliffe, 2009) to produce ATP (Alonso et al, 2007a) or to supply citrate for cytosolic acetyl-CoA when operating as an incomplete cycle (Schwender et al, 2006).…”

Experimental Definition and Validation of Protein Coding Transcripts in Chlamydomonas reinhardtii

“…One of the fundamental difficulties with complex multicompartmental models is determining the compartments to which specific metabolic reactions are localized, as duplication of portions of biochemical pathways occurs. A recent study employing three pentose phosphate model alternatives in A. thaliana was not able to distinguish between the possibilities using steady-state isotope labeling data [66].This result emphasizes the need for additional biochemical evidence to develop accurate metabolic models, especially if a metabolic design situation requires manipulating compartmentspecific reaction fluxes. However, it is worth noting that networks as large as that of the human [67] and A. thaliana [68] have been modeled with less accounting for compartmentalization.…”

Capturing Metabolite Channeling in Metabolic Flux Phenotypes

“…High-quality labeling data, coupled with the simultaneous analysis of experiments with different labeled precursors (Schwender et al, 2006;Libourel et al, 2007;Masakapalli et al, 2010), is improving the definition of the flux maps produced by steady-state MFA, and it is imperative to base these maps on realistic metabolic models that capture the likely features of the network. Second, steady-state MFA now offers a general noninvasive strategy for the detection of metabolite channeling in the central metabolic network that complements the existing methods, many of which are invasive.…”