L-Galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) catalyzes the last step in the main pathway of vitamin C (L-ascorbic acid) biosynthesis in higher plants. In this study, we first characterized the spatial and temporal expression of SlGalLDH in several organs of tomato (Solanum lycopersicum) plants in parallel with the ascorbate content. P 35S :SlgalldhRNAi silenced transgenic tomato lines were then generated using an RNAi strategy to evaluate the effect of any resulting modification of the ascorbate pool on plant and fruit development. In all P 35S :SlgalldhRNAi plants with reduced SlGalLDH transcript and activity, plant growth rate was decreased. Plants displaying the most severe effects (dwarf plants with no fruit) were excluded from further analysis. The most affected lines studied exhibited up to an 80% reduction in SlGalLDH activity and showed a strong reduction in leaf and fruit size, mainly as a consequence of reduced cell expansion. This was accompanied by significant changes in mitochondrial function and altered ascorbate redox state despite the fact that the total ascorbate content remained unchanged. By using a combination of transcriptomic and metabolomic approaches, we further demonstrated that several primary, like the tricarboxylic acid cycle, as well as secondary metabolic pathways related to stress response were modified in leaves and fruit of P 35S :Slgalldh RNAi plants. When taken together, this work confirms the complexity of ascorbate regulation and its link with plant metabolism. Moreover, it strongly suggests that, in addition to ascorbate synthesis, GalLDH could play an important role in the regulation of cell growth-related processes in plants.

The purpose of this work was to analyze the contribution of the mitochondria to the optical properties, i.e., light absorption and scattering, of the blood-free rat liver. Firstly, a theoretical model of the reduced scattering coefficient of the liver was performed by using the Mie theory, the Rayleigh-Debye-Gans approximation, and the electron microscopy descriptions of the liver ultrastructure. Compared with the hepatocyte volume, the nucleus and the peroxisomes, the mitochondria compartment, accounting for 22% of the liver cell volume, seemed to be the predominant factor for the light scattering of the liver. Second, by using time-resolved spectroscopy and a sample substitution method, we have measured the absorption and reduced scattering coefficients of blood-free perfused rat livers, isolated hepatocyte suspensions, and isolated mitochondria suspensions. A subsequent extrapolation of the isolated mitochondria data to the in vivo mitochondrial content and a comparison with the whole liver measurements lead to the following conclusions: 1) the mitochondria account for about 50% of the liver absorption coefficient at 780 nm (mu a = 0.25 cm-1 extrapolated from isolated mitochondria vs. 0.53 +/- 0.05 cm-1 measured for the liver); and 2) the mitochondrial compartment is the primary factor for the light scattering in the rat liver (mu s' = 15.5 cm-1 extrapolated from the isolated mitochondria versus 15.9 +/- 2.4 cm-1 measured for the liver), demonstrating the relevancy of our preliminary theoretical study.

To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum 'Moneymaker') plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels.

Saccharomyces cerevisiae is a facultative anaerobe devoid of mitochondrial alternative oxidase. In this yeast, the structure and biogenesis of the respiratory chain, on the one hand, and the functional interactions of oxidative phosphorylation with the cellular energetic metabolism, on the other, are well documented. However, to our knowledge, the molecular aspects and the physiological roles of the non-respiratory pathways that utilize molecular oxygen have not yet been reviewed. In this paper, we review the various non-respiratory pathways in a global context of utilization of molecular oxygen in S. cerevisiae. The roles of these pathways are examined as a function of environmental conditions, using either physiological, biochemical or molecular data. Special attention is paid to the characterization of the so-called 'cyanide-resistant respiration' that is induced by respiratory deficiency, catabolic repression and oxygen limitation during growth. Finally, several aspects of oxygen sensing are discussed.

A metabolomics approach combining (1)H NMR and gas chromatography-electrospray ionization time-of-flight mass spectrometry (GC-EI-TOFMS) profiling was employed to characterize melon (Cucumis melo L.) fruit. In a first step, quantitative (1)H NMR of polar extracts and principal component analyses (PCA) of the corresponding data highlighted the major metabolites in fruit flesh, including sugars, organic acids, and amino acids. In a second step, the spatial localization of metabolites was investigated using both analytical techniques. Direct (1)H NMR profiling of juice or GC-EI-TOFMS profiling of tissue extracts collected from different locations in the fruit flesh provided information on advantages and drawbacks of each technique for the analysis of a sugar-rich matrix such as fruit. (1)H NMR and GC-EI-TOFMS data sets were compared using independently performed PCA and multiblock hierarchical PCA (HPCA), respectively. In addition a correlation-based multiblock HPCA was used for direct comparison of both analytical data sets. These data analyses revealed several gradients of metabolites in fruit flesh which can be related with differences in metabolism and indicated the suitability of multiblock HPCA for correlation of data from two (or potentially more) metabolomics platforms.

A kinetic model combining enzyme activity measurements and subcellular compartmentation was parameterized to fit the sucrose, hexose, and glucose-6-P contents of pericarp throughout tomato (Solanum lycopersicum) fruit development. The model was further validated using independent data obtained from domesticated and wild tomato species and on transgenic lines. A hierarchical clustering analysis of the calculated fluxes and enzyme capacities together revealed stage-dependent features. Cell division was characterized by a high sucrolytic activity of the vacuole, whereas sucrose cleavage during expansion was sustained by both sucrose synthase and neutral invertase, associated with minimal futile cycling. Most importantly, a tight correlation between flux rate and enzyme capacity was found for fructokinase and PPi-dependent phosphofructokinase during cell division and for sucrose synthase, UDP-glucopyrophosphorylase, and phosphoglucomutase during expansion, thus suggesting an adaptation of enzyme abundance to metabolic needs. In contrast, for most enzymes, flux rates varied irrespectively of enzyme capacities, and most enzymes functioned at <5% of their maximal catalytic capacity. One of the major findings with the model was the high accumulation of soluble sugars within the vacuole together with organic acids, thus enabling the osmotic-driven vacuole expansion that was found during cell division.

Additions of a solute/carbohydrate in tissue affect the size of tissue cells and the refractive indexes of the extra-and intracellular fluids, and thus the overall tissue scattering properties. We use both the Rayleigh-Gans and Mie theory approximation in calculating effects of the osmolarity and refractive indexes on the reduced scattering coefficient of tissue, and employ photon diffusion theory to associate the reduced scattering coefficient to the mean optical path length. The calculations show that changes of scattering in tissue depend not only on the change in extracellular refractive index but also on the change in osmolarity, and thus on the change in cell size and volume fraction. Experimentally, we have utilized time-domain and frequencydomain NIR techniques to measure the changes of optical properties caused by an addition of a solute in tissue models and in perfused rat livers. The temperature-dependent path length measurement of the perfused liver confirms the dependence of tissue scattering on the tissue cell size. The results obtained from the liver with three kinds of carbohydrate perfusion display different scattering aspects and can be well explained by changes in cell size and in extracellular as well as intracellular refractive indexes. The consistency between the theoretical and experimental results confirms the dependence of optical properties in (liver) tissue on both tissue osmolarity and relative refractive indexes between the extracellular and intracellular compartments. This study suggests that the NIR technique is a novel and useful tool for noninvasive, physiological monitoring.

The anaerobic growth of the yeast Saccharomyces cerevisiae normally requires the addition of molecular oxygen, which is used to synthesize sterols and unsaturated fatty acids (UFAs). A single oxygen pulse can stimulate enological fermentation, but the biochemical pathways involved in this phenomenon remain to be elucidated. We showed that the addition of oxygen (0.3 to 1.5 mg/g [dry mass] of yeast) to a lipid-depleted medium mainly resulted in the synthesis of the sterols and UFAs required for cell growth. However, the addition of oxygen during the stationary phase in a medium containing excess ergosterol and oleic acid increased the specific fermentation rate, increased cell viability, and shortened the fermentation period. Neither the respiratory chain nor de novo protein synthesis was required for these medium-and long-term effects. As de novo lipid synthesis may be involved in ethanol tolerance, we studied the effect of oxygen addition on sterol and UFA auxotrophs (erg1 and ole1 mutants, respectively). Both mutants exhibited normal anaerobic fermentation kinetics. However, only the ole1 mutant strain responded to the oxygen pulse during the stationary phase, suggesting that de novo sterol synthesis is required for the oxygen-induced increase of the specific fermentation rate. In conclusion, the sterol pathway appears to contribute significantly to the oxygen consumption capacities of cells under anaerobic conditions. Nevertheless, we demonstrated the existence of alternative oxygen consumption pathways that are neither linked to the respiratory chain nor linked to heme, sterol, or UFA synthesis. These pathways dissipate the oxygen added during the stationary phase, without affecting the fermentation kinetics.