Analysis of subcellular metabolite levels of potato tubers (Solanum tuberosum) displaying alterations in cellular or extracellular sucrose metabolism

The expression of a heterologous invertase in potato tubers (Solanum tuberosum) in either the cytosol or apoplast leads to a decrease in total sucrose content and to an increase in glucose. Depending on the targeting of the enzyme different changes in phenotype and metabolism of the tubers occur: the cytosolic invertase expressing tubers show an increase in the glycolytic flux, accumulation of amino acids and organic acids, and the appearance of novel disaccharides; however, these changes are not observed when the enzyme is expressed in the apoplast [Roessner et al. (2001). Plant Cell, 13, 11-29]. The analysis of these lines raised several questions concerning the regulation of compartmentation of metabolites in potato tubers. In the current study we addressed these questions by performing comparative subcellular metabolite profiling. We demonstrate that: (i) hexoses accumulate in the vacuole independently of their site of production, but that the cytosolic invertase expression led to a strong increase in the cytosolic glucose concentration and decrease in cytosolic sucrose, whereas these effects were more moderate in the apoplastic expressors; (ii) three out of four of the novel compounds found in the cytosolic overexpressors accumulate in the same compartment; (iii) despite changes in absolute cellular content the subcellular distribution of amino acids was invariant in the invertase overexpressing tubers. These results are discussed in the context of current models of the compartmentation of primary metabolism in heterotrophic plant tissues

Metabolic recovery of Arabidopsis thaliana roots following cessation of oxidative stress

To cope with the various environmental stresses resulting in reactive oxygen species (ROS) production plant metabolism is known to be altered specifically under different stresses. After overcoming the stress the metabolism should be reconfigured to recover basal operation however knowledge concerning how this is achieved is cursory. To investigate the metabolic recovery of roots following oxidative stress, changes in metabolite abundance and carbon flow were analysed. Arabidopsis roots were treated by menadione to elicit oxidative stress. Roots were fed with 13C labelled glucose and the redistribution of isotope was determined in order to study carbon flow. The label redistribution through many pathways such as glycolysis, the tricarboxylic acid (TCA) cycle and amino acid metabolism were reduced under oxidative stress. After menadione removal many of the stress-related changes reverted back to basal levels. Decreases in amounts of hexose phosphates, malate, 2-oxoglutarate, glutamate and aspartate were fully recovered or even increased to above the control level. However, some metabolites such as pentose phosphates and citrate did not recover but maintained their levels or even increased further. The alteration in label redistribution largely correlated with that in metabolite abundance. Glycolytic carbon flow reverted to the control level only 18 h after menadione removal although the TCA cycle and some amino acids such as aspartate and glutamate took longer to recover. Taken together, plant root metabolism was demonstrated to be able to overcome menadione-induced oxidative stress with the differential time period required by independent pathways suggestive of the involvement of pathway specific regulatory processes

Metabolic Profiling of a Mapping Population Exposes New Insights in the Regulation of Seed Metabolism and Seed, Fruit, and Plant Relations

To investigate the regulation of seed metabolism and to estimate the degree of metabolic natural variability, metabolite profiling and network analysis were applied to a collection of 76 different homozygous tomato introgression lines (ILs) grown in the field in two consecutive harvest seasons. Factorial ANOVA confirmed the presence of 30 metabolite quantitative trait loci (mQTL). Amino acid contents displayed a high degree of variability across the population, with similar patterns across the two seasons, while sugars exhibited significant seasonal fluctuations. Upon integration of data for tomato pericarp metabolite profiling, factorial ANOVA identified the main factor for metabolic polymorphism to be the genotypic background rather than the environment or the tissue. Analysis of the coefficient of variance indicated greater phenotypic plasticity in the ILs than in the M82 tomato cultivar. Broad-sense estimate of heritability suggested that the mode of inheritance of metabolite traits in the seed differed from that in the fruit. Correlation-based metabolic network analysis comparing metabolite data for the seed with that for the pericarp showed that the seed network displayed tighter interdependence of metabolic processes than the fruit. Amino acids in the seed metabolic network were shown to play a central hub-like role in the topology of the network, maintaining high interactions with other metabolite categories, i.e., sugars and organic acids. Network analysis identified six exceptionally highly co-regulated amino acids, Gly, Ser, Thr, Ile, Val, and Pro. The strong interdependence of this group was confirmed by the mQTL mapping. Taken together these results (i) reflect the extensive redundancy of the regulation underlying seed metabolism, (ii) demonstrate the tight co-ordination of seed metabolism with respect to fruit metabolism, and (iii) emphasize the centrality of the amino acid module in the seed metabolic network. Finally, the study highlights the added value of integrating metabolic network analysis with mQTL mapping

Mild reductions in cytosolic NADP-dependent isocitrate dehydrogenase activity result in lower amino acid contents and pigmentation without impacting growth

Transgenic tomato (Solanum lycopersicum) plants were generated targeting the cytosolic NADP-dependent isocitrate dehydrogenase gene (SlICDH1) via the RNA interference approach. The resultant transformants displayed a relatively mild reduction in the expression and activity of the target enzyme in the leaves. However, biochemical analyses revealed that the transgenic lines displayed a considerable shift in metabolism, being characterized by decreases in the levels of the TCA cycle intermediates, total amino acids, photosynthetic pigments, starch and NAD(P)H. The plants showed little change in photosynthesis with the exception of a minor decrease in maximum photosynthetic efficiency (Fv/Fm), and a small decrease in growth compared to the wild type. These results reveal that even small changes in cytosolic NADP-dependent isocitrate dehydrogenase activity lead to noticeable alterations in the activities of enzymes involved in primary nitrate assimilation and in the synthesis of 2-oxoglutarate derived amino acids. These data are discussed within the context of current models for the role of the various isoforms of isocitrate dehydrogenase within plant amino acid metabolism

Kinetics of labelling of organic and amino acids in potato tubers by gas chromatography-mass spectrometry following incubation in 13C labelled isotopes

Metabolic pathways of primary metabolism of discs isolated from potato tubers were evaluated by the use of a gas chromatography-mass spectrometry (GC-MS) method generated specifically for this purpose. After testing several possible methods including chemical ionization, it was decided for reasons of sensitivity, reproducibility and speed to use electron impact ionization-based GC-MS analysis. The specific labelling and label accumulation of over 30 metabolites including a broad number of sugars, organic and amino acids was analysed following the incubation of tuber discs in [U-13C]glucose. The reproducibility of this method was similar to that found for other GC-MS-based analyses and comparison of flux estimates from this method with those obtained from parallel, yet less comprehensive, radiolabel experiments revealed close agreement. Therefore, the novel method allows quantitatively evaluation of a broad range of metabolic pathways without the need for laborious (and potentially inaccurate), chemical fractionation procedures commonly used in the estimation of fluxes following incubation in radiolabelled substrates. As a first experiment the GC-MS method has been applied to compare the metabolism of wild type and wel-characterized transgenic potato tubers exhibiting an enhanced sucrose mobilization. The fact that this method is able to rapidly yield further comprehensive information into primary metabolism illustrates its power as a further phenotyping tool for the analysis of plant metabolism

Expression of a bacterial xylose isomerase in potato tubers results in an altered hexose composition and a consequent induction of metabolism

Here we investigate the role of hexoses in the metabolism of the developing potato (Solanum tuberosum) tuber by the expression of a bacterial xylose isomerase which catalyzes the interconversion of glucose and fructose. Previously, we found that glycolysis was induced in transgenic tubers expressing a yeast invertase in the cytosol and postulated that this was due either to the decreased levels of sucrose or to effects downstream of the sucrose cleavage. In the present study xylose isomerase was expressed under the control of the tuber-specific patatin promoter. Selected transformants exhibited minor changes in the levels of tuber glucose and fructose but not in sucrose. Analysis of the enzyme activities of the glycolytic pathway revealed minor yet significant increases in the maximal catalytic activities of aldolase and glyceraldehyde 3-phosphate dehydrogenase but no increase in the activities of other enzymes of glycolysis. These lines were also characterized by an elevated tuber number, glycolytic and sucrose synthetic fluxes and in some metabolite levels downstream of glycolysis. When considered together these data suggest that the perturbation of hexose levels can result in increased glycolytic and sucrose (re)synthetic fluxes in the potato tuber even in the absence of changes in the level of sucrose. The consequences of altering hexose levels in the tuber are, however, not as severe as those observed following perturbation of the level of tuber sucrose. [References: 52] 5