Brigitte Thomasset

Comparative stabilization of biological photosystems by several immobilization procedures

SummaryLettuce thylakoids were immobilized by various methods selected to provide the chloroplast membrane with different environments. These included proteins (albumin and gelatin), polysaccharides (carrageenan and alginate gels) and synthetic polymers (photocrosslinkable resins and polyurethans). Large variations were observed in the activity yield after immobilization (ranging from 3% to 70%), in the storage stability (at 4°C in absence of light) and in the functional stability (continuous work at 20°C under illumination).

Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray

BackgroundFlax (Linum usitatissimum L.) has been cultivated for around 9,000 years and is therefore one of the oldest cultivated species. Today, flax is still grown for its oil (oil-flax or linseed cultivars) and its cellulose-rich fibres (fibre-flax cultivars) used for high-value linen garments and composite materials. Despite the wide industrial use of flax-derived products, and our actual understanding of the regulation of both wood fibre production and oil biosynthesis more information must be acquired in both domains. Recent advances in genomics are now providing opportunities to improve our fundamental knowledge of these complex processes. In this paper we report the development and validation of a high-density oligo microarray platform dedicated to gene expression analyses in flax.ResultsNine different RNA samples obtained from flax inner- and outer-stems, seeds, leaves and roots were used to generate a collection of 1,066,481 ESTs by massive parallel pyrosequencing. Sequences were assembled into 59,626 unigenes and 48,021 sequences were selected for oligo design and high-density microarray (Nimblegen 385K) fabrication with eight, non-overlapping 25-mers oligos per unigene. 18 independent experiments were used to evaluate the hybridization quality, precision, specificity and accuracy and all results confirmed the high technical quality of our microarray platform. Cross-validation of microarray data was carried out using quantitative qRT-PCR. Nine target genes were selected on the basis of microarray results and reflected the whole range of fold change (both up-regulated and down-regulated genes in different samples). A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates both in qRT-PCR and microarray results. Further experiments illustrated the capacity of our arrays to detect differential gene expression in a variety of flax tissues as well as between two contrasted flax varieties.ConclusionAll results suggest that our high-density flax oligo-microarray platform can be used as a very sensitive tool for analyzing gene expression in a large variety of tissues as well as in different cultivars. Moreover, this highly reliable platform can also be used for the quantification of mRNA transcriptional profiling in different flax tissues.

An in silico compartmentalized metabolic model of Brassica napus enables the systemic study of regulatory aspects of plant central metabolism

Biochemical network reconstructions represent valuable tools for the computational metabolic modeling of organisms that present a great biotechnological interest. An in silico multi‐compartmental model of the central metabolism of the plant Brassica napus (Rapeseed) was constructed, aiming to investigate the metabolic properties of the Brassicaceae family. This family comprises many plants with major importance for the energy and nutrition sector, including the model plant Arabidopsis thaliana. The model utilized as objective function to be subsequently optimized, the biomass production of rapeseed developing embryos, which are characterized by a very high, oil content, up to 60% of biomass weight. In order to study global network properties of seed metabolism, various methods were employed, like Flux Balance Analysis, Principal Component Analysis of the flux space and reaction deletion studies, which simulate the effect of gene knock‐out experiments. The model successfully simulated seed growth during the stage of oil accumulation and provided insight, regarding certain aspects of network plasticity, with the emphasis given in lipid biosynthesis regulation. Biotechnol. Bioeng. 2011; 108:1673–1682.

Electron microscopic evidence of an immobilized living cell system

Abstract Whole cells of Rhodopseudomonas capsulata were immobilized by entrapment in Ca 2+ alginate gel. The cell growth inside the matrix was observed by transmission electron microscopy. Colonies enlarged upon incubation, giving some evidence of an immobilized living cell system. This observation was correlated by measurements of bacteriochlorophyll, nitrogen, and protein content.

Efficiency of physical (light) or chemical (ABA, tetracycline, CuSO4 or 2-CBSU)-stimulus-dependent gus gene expression in tobacco cell suspensions

In this study, the efficiency of inducible promoters to switch on gene expression in the presence of inducer or to switch it off in its absence was evaluated in tobacco cell suspensions transformed with the gus gene coding sequence. Either plant (pats1A, pSalT, pIn2-2) or microbial (pMre, pTet) inducible promoters were used to drive gus expression. The inducers were light, abscisic acid, 2-CBSU, CuSO4, tetracycline, respectively. For each construct (inducible promoter-gus coding sequence), the optimal induction conditions were determined (inducer concentration, induction time, and age of cells in culture cycle before induction). The efficiency of the inducible promoter was then evaluated under optimal induction conditions. GUS-expression levels obtained under non-inducing and inducing conditions were systematically compared. Thirty or forty percent of the clones transformed with the pSalT-gus or pTet-gus construct, respectively, showed high induction rates (>1000) and GUS activities of the same order as those obtained with a constitutive system. However, basal GUS levels were always high for the pTet-gus cell lines. Seventy or eighty-five percent of the cell lines transformed with the pMre-gus or pln2-2-gus construct, respectively, had induction rates of 1.5 to 1000. The pats1A-gus construct gave very low induction rates-55% of cell lines had induction rates less than 1.5. Only the pSalt-gus construct gave both the highest induction rates and basal GUS-levels equivalent to the endogenous GUS background.

Highlighting the tricarboxylic acid cycle: Liquid and gas chromatography–mass spectrometry analyses of 13C-labeled organic acids

The tricarboxylic acid (TCA) cycle is involved in the complete oxidation of organic acids to carbon dioxide in aerobic cells. It not only uses the acetyl-CoA derived from glycolysis but also uses breakdown products of proteins, fatty acids, and nucleic acids. Therefore, the TCA cycle involves numerous carbon fluxes through central metabolism to produce reductant power and transfer the generated electrons to the aerobic electron transport system where energy is formed by oxidative phosphorylation. Although the TCA cycle plays a crucial role in aerobic organisms and tissues, the lack of direct isotopic labeling information in its intermediates (organic acids) makes the quantification of its metabolic fluxes rather approximate. This is the major technical gap that this study intended to fill. In this work, we established and validated liquid and gas chromatography-mass spectrometry methods to determine (13)C labeling in organic acids involved in the TCA cycle using scheduled multiple reaction monitoring and single ion monitoring modes, respectively. Labeled samples were generated using maize embryos cultured with [(13)C]glucose or [(13)C]glutamine. Once steady-state labeling was reached, (13)C-labeled organic acids were extracted and purified. When applying our mass spectrometric methods to those extracts, mass isotopomer abundances of seven major organic acids were successfully determined.

Immobilized organelles and whole cells into protein foam structures: scanning and transmission electron microscope observations.

Protein foam structures bearing cells and organelles were produced by using a co-crosslinking method with serum albumin and glutaraldehyde at sub-zero temperature. Morphological observations obtained with scanning and transmission electron microscopy indicate macroporous and homogeneous structures. The glutaraldehyde concentration was varied to reveal its effect on immobilized red cells. It was observed that the structural appearance of preatreated cells or subcellular fractions (thylakoids from lettuce ; spheroplasts and chromatophores from R. capsulata) is preserved during the immobilization process. The morphological features of foam particles are always related to the observed kinetic activities of the specimens.

Gas chromatography-mass spectrometry analysis of 13C labeling in sugars for metabolic flux analysis.

Metabolic flux analysis, using 13C labeled substrates, has become a powerful methodology for quantifying intracellular fluxes. Most often, analysis is restricted to nuclear magnetic resonance or mass spectrometry measurement of 13C label incorporation into protein amino acids. However, amino acid isotopomer distribution insufficiently covers the entire network of central metabolism, especially in plant cells with highly compartmented metabolism, and analysis of other metabolites is required. Analysis of label in saccharides provides complementary data to better define fluxes around hexose, pentose, and triose phosphate pools. Here, we propose a gas chromatography-mass spectrometry (GC-MS) method to analyze 13C labeling in glucose and fructose moieties of sucrose, free glucose, fructose, maltose, inositol, and starch. Our results show that saccharide labeling for isotopomer quantification is better analyzed by chemical ionization than by electron ionization. The structure of the generated fragments was simulated and validated using labeled standards. The method is illustrated by analysis of saccharides extracted from developing rapeseed (Brassica napus L.) embryos. It is shown that glucose 6-phosphate isomerase and plastidial glucose 6-phosphate transport reactions are not at equilibrium, and light is shed on the pathways leading to fructose, maltose, and inositol synthesis.

Different expression of an S-adenosylmethionine synthetase gene in transgenic tobacco callus modifies alkaloid biosynthesis.

Transformed callus cultures of Nicotiana tabacum were generated in which the SAM-1 gene from Arabidopsis thaliana encoding S-adenosylmethionine synthetase (SAM-S), under the control of the 35S promoter, had been integrated. The presence of the SAM-1 gene was detected in all tested transformants and the SAM-S activity correlated with the accumulation of SAM in the tobacco callus cultures. Three distinct phenotypic classes were identified among the transgenic cell lines in relation to growth of the cells, structure of the calli, and level of SAM. Transgene silencing was observed in several cultivated transgenic calli and this phenomenon was correlated directly with a low level of SAM-1 mRNA accompanied by a decrease of the SAM-S activity. The transgenic calli overexpressing the SAM-1 gene accumulated a high SAM level. The modifications in SAM-S activity were reflected in the pattern of secondary products present in the different cell lines, thereby demonstrating that the flux through the biosynthetic pathway of a plant secondary product can be modified by means of genetic engineering.

β-Glucuronidase activity in transgenic and non-transgenic tobacco cells: specific elimination of plant inhibitors and minimization of endogenous GUS background

Abstract Bacterial β-glucuronidase is often introduced into plants as a reporter gene fused to constitutive or inducible promoters. However, the presence of both endogenous inhibitors of GUS activity and endogenous GUS enzymes in transgenic plants could lead to an underestimation of GUS. In this paper, a decrease of the Vm values and a greater affinity (Km) of the GUS enzyme for its substrate (p-NPG) has been recorded when increasing amounts of protein from untransformed tobacco cells has been added to the pure β-glucuronidase. The observed inhibition is not competitive and can be completely removed when the tobacco extracts are passed through Sephadex G-25 spin columns prior to the assays. After such a treatment, the activity of E. coli GUS in transgenic tobacco cells (constitutive or inducible systems) was stimulated by a factor 1.2 or 2 for p-NPG or 4-MUG substrates, respectively. This method was also effective in suppressing the endogenous GUS or GUS-like activity which can interfere with the activity originating from the introduced GUS gene.