Johann Petit

Silencing of the mitochondrial ascorbate synthesizing enzyme L-galactono-1,4-lactone dehydrogenase affects plant and fruit development in tomato.

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. P35S: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 P35S: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 P35S:SlgalldhRNAi 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.

Changes in Transcriptional Profiles Are Associated with Early Fruit Tissue Specialization in Tomato

The cell expansion phase contributes in determining the major characteristics of a fleshy fruit and represents two-thirds of the total fruit development in tomato (Solanum lycopersicum). So far, it has received very little attention. To evaluate the interest of a genomic scale approach, we performed an initial sequencing of approximately 1,200 cell expansion stage-related sequence tags from tomato fruit at 8, 12, and 15 d post anthesis. Interestingly, up to approximately 35% of the expressed sequence tags showed no homology with available tomato expressed sequence tags and up to approximately 21% with any known gene. Microarrays spotted with expansion phase-related cDNAs and other fruit cDNAs involved in various developmental processes were used (1) to profile gene expression in developing fruit and other plant organs and (2) to compare two growing fruit tissues engaged mostly in cell division (exocarp) or in cell expansion (locular tissue surrounding the seeds). Reverse transcription-polymerase chain reaction analysis was further used to confirm microarray results and to specify expression profiles of selected genes (24) in various tissues from expanding fruit. The wide range of genes expressed in the exocarp is consistent with a protective function and with a high metabolic activity of this tissue. In addition, our data show that the expansion of locular cells is concomitant with the expression of genes controlling water flow, organic acid synthesis, sugar storage, and photosynthesis and suggest that hormones (auxin and gibberellin) regulate this process. The data presented provide a basis for tissue-specific analyses of gene function in growing tomato fruit.

Gene and Metabolite Regulatory Network Analysis of Early Developing Fruit Tissues Highlights New Candidate Genes for the Control of Tomato Fruit Composition and Development

Variations in early fruit development and composition may have major impacts on the taste and the overall quality of ripe tomato (Solanum lycopersicum) fruit. To get insights into the networks involved in these coordinated processes and to identify key regulatory genes, we explored the transcriptional and metabolic changes in expanding tomato fruit tissues using multivariate analysis and gene-metabolite correlation networks. To this end, we demonstrated and took advantage of the existence of clear structural and compositional differences between expanding mesocarp and locular tissue during fruit development (12–35 d postanthesis). Transcriptome and metabolome analyses were carried out with tomato microarrays and analytical methods including proton nuclear magnetic resonance and liquid chromatography-mass spectrometry, respectively. Pairwise comparisons of metabolite contents and gene expression profiles detected up to 37 direct gene-metabolite correlations involving regulatory genes (e.g. the correlations between glutamine, bZIP, and MYB transcription factors). Correlation network analyses revealed the existence of major hub genes correlated with 10 or more regulatory transcripts and embedded in a large regulatory network. This approach proved to be a valuable strategy for identifying specific subsets of genes implicated in key processes of fruit development and metabolism, which are therefore potential targets for genetic improvement of tomato fruit quality.

GDP‐d‐mannose 3,5‐epimerase (GME) plays a key role at the intersection of ascorbate and non‐cellulosic cell‐wall biosynthesis in tomato

The GDP-D-mannose 3,5-epimerase (GME, EC 5.1.3.18), which converts GDP-d-mannose to GDP-l-galactose, is generally considered to be a central enzyme of the major ascorbate biosynthesis pathway in higher plants, but experimental evidence for its role in planta is lacking. Using transgenic tomato lines that were RNAi-silenced for GME, we confirmed that GME does indeed play a key role in the regulation of ascorbate biosynthesis in plants. In addition, the transgenic tomato lines exhibited growth defects affecting both cell division and cell expansion. A further remarkable feature of the transgenic plants was their fragility and loss of fruit firmness. Analysis of the cell-wall composition of leaves and developing fruit revealed that the cell-wall monosaccharide content was altered in the transgenic lines, especially those directly linked to GME activity, such as mannose and galactose. In agreement with this, immunocytochemical analyses showed an increase of mannan labelling in stem and fruit walls and of rhamnogalacturonan labelling in the stem alone. The results of MALDI-TOF fingerprinting of mannanase cleavage products of the cell wall suggested synthesis of specific mannan structures with modified degrees of substitution by acetate in the transgenic lines. When considered together, these findings indicate an intimate linkage between ascorbate and non-cellulosic cell-wall polysaccharide biosynthesis in plants, a fact that helps to explain the common factors in seemingly unrelated traits such as fruit firmness and ascorbate content.

Tomato GDSL1 Is Required for Cutin Deposition in the Fruit Cuticle

This study analyzes the mechanism by which cutin is deposited. GDSL1, which belongs to the GDSL esterase/acylhydrolase family of plant proteins, is found to play a key role in cutin deposition during fruit cuticle development. The plant cuticle consists of cutin, a polyester of glycerol, hydroxyl, and epoxy fatty acids, covered and filled by waxes. While the biosynthesis of cutin building blocks is well documented, the mechanisms underlining their extracellular deposition remain unknown. Among the proteins extracted from dewaxed tomato (Solanum lycopersicum) peels, we identified GDSL1, a member of the GDSL esterase/acylhydrolase family of plant proteins. GDSL1 is strongly expressed in the epidermis of growing fruit. In GDSL1-silenced tomato lines, we observed a significant reduction in fruit cuticle thickness and a decrease in cutin monomer content proportional to the level of GDSL1 silencing. A significant decrease of wax load was observed only for cuticles of the severely silenced transgenic line. Fourier transform infrared (FTIR) analysis of isolated cutins revealed a reduction in cutin density in silenced lines. Indeed, FTIR-attenuated total reflectance spectroscopy and atomic force microscopy imaging showed that drastic GDSL1 silencing leads to a reduction in ester bond cross-links and to the appearance of nanopores in tomato cutins. Furthermore, immunolabeling experiments attested that GDSL1 is essentially entrapped in the cuticle proper and cuticle layer. These results suggest that GDSL1 is specifically involved in the extracellular deposition of the cutin polyester in the tomato fruit cuticle.

Down-regulation of a single auxin efflux transport protein in tomato induces precocious fruit development

The PIN-FORMED (PIN) auxin efflux transport protein family has been well characterized in the model plant Arabidopsis thaliana, where these proteins are crucial for auxin regulation of various aspects of plant development. Recent evidence indicates that PIN proteins may play a role in fruit set and early fruit development in tomato (Solanum lycopersicum), but functional analyses of PIN-silenced plants failed to corroborate this hypothesis. Here it is demonstrated that silencing specifically the tomato SlPIN4 gene, which is predominantly expressed in tomato flower bud and young developing fruit, leads to parthenocarpic fruits due to precocious fruit development before fertilization. This phenotype was associated with only slight modifications of auxin homeostasis at early stages of flower bud development and with minor alterations of ARF and Aux/IAA gene expression. However, microarray transcriptome analysis and large-scale quantitative RT-PCR profiling of transcription factors in developing flower bud and fruit highlighted differentially expressed regulatory genes, which are potential targets for auxin control of fruit set and development in tomato. In conclusion, this work provides clear evidence that the tomato PIN protein SlPIN4 plays a major role in auxin regulation of tomato fruit set, possibly by preventing precocious fruit development in the absence of pollination, and further gives new insights into the target genes involved in fruit set.

Psychosocial risks: acting upon the organisation by ergonomic intervention

In this paper we discuss the possibilities of acting on psychosocial risk (PSR) factors by modifying the way in which an organisation operates. On the basis of an ergonomic intervention in an insurance company, we were able to show that the health problems observed by the operators were mainly the result of their inability to produce work of quality. Next, our analyses revealed the links between poor perceived quality, production difficulties and the rigidity of the organisational structure. After setting up working groups to deal with production difficulties, we were able to identify and test an organisational form that was better adapted to managing day-to-day production constraints and which was ultimately better able to be attentive to individual difficulties which had given rise, in the long term, to intrapsychic conflicts.

Identification of differentially expressed genes during early development of tomato fruit. Characterisation of a novel cDNA coding for a RAD23 protein.

Before the onset of ripening, tomato fruit development comprises three distinct phases: fruit set, a cell division phase and a cell expansion phase. In this study, we used the method of mRNA differential display in order to isolate tomato genes specifically expressed during these early phases of fruit development. Among 40 differen-tial bands, nine cDNAs were selected for further investigations based on their identification after nucleotide sequencing. We isolated the full-length cDNA corresponding to one of these fragments, coding for RAD23, a protein involved in the excision repair system, thus providing new sequence information on a poorly characterised protein in plants. All the isolated cDNAs were mapped on the tomato genome and their expression studied by northern blot and semi-quantitative RT–PCR during early fruit development and in vegetative organs of tomato plants. The sequence data are deposited in the GenBank under the accession numbers: AJ270956 (mo5-3C11/1), AJ270957 (mo5-3G12/4), AJ270958 (mo5-3G17), AJ270959 (mo5-3T12), AJ270960 (mo1-6A1), AJ270961 (mo1-6T1), AJ270962 (mo5-10G1), AJ270963 (mo6-20G1), AJ270964 (mo6-MGT2) and AJ243875 (LeRAD23-8 full-length cDNA).

Développement de la pluridisciplinarité au sein d’un service de santé au travail : la démarche innovante du CHU de Bordeaux

En 2007, afin de renforcer et d’elargir l’action medicale, le service de sante au travail du CHU de Bordeaux s’est engage dans la pluridisciplinarite avec l’introduction d’une competence en psychologie de la sante au travail. Depuis l’hygiene et securite, l’ergonomie et l’epidemiologie sont venues elargir le champ disciplinaire et d’action de l’equipe, bien avant l’evolution recente des textes reglementaires qui prevoient cette pluridisciplinarite dans la fonction publique hospitaliere. Une posture reflexive constante, assuree avec les medecins sur l’activite de chacun, a permis de formaliser une charte sur la prevention et la gestion des RPS puis une seconde sur les missions de l’equipe pluridisciplinaire. En 2012, a la faveur de l’obtention d’un contrat local d’amelioration des conditions de travail (CLACT) sur la prevention conjointe des troubles musculo-squelettiques et des risques psychosociaux, une methodologie d’intervention en binome ergonome et psychologue en sante au travail a ete eprouvee. Dans le cadre de ce dispositif, l’equipe pluridisciplinaire a beneficie d’une analyse des pratiques animee par l’equipe d’ergonomie des systemes complexes de Bordeaux-institut polytechnique de Bordeaux (INP) au rythme d’environ une seance par bimestre. Ces seances presentaient un double enjeu. Le premier etait de mettre en debat les logiques disciplinaires, a travers les pratiques professionnelles, entre hygiene et securite, ergonomie et psychologie puis de les articuler avec celle de la responsabilite et du pilotage medical. Le second etait de consolider une organisation de travail en equipe permettant la construction d’un veritable collectif de travail pluridisciplinaire pilote par les medecins du travail. Dans ce sens, deux modalites organisationnelles ont ete posees. Tout d’abord, la mise en place de staffs mensuels dans lesquels les intervenants en prevention des risques professionnels (IPRP) et medecins du travail de secteur debattent des demandes et s’entendent sur une conduite a tenir. Puis a ete creee une mission de coordination de l’equipe IPRP assuree par le membre le plus ancien de l’equipe pluridisciplinaire. Enfin, institutionnellement, la mise en place d’une cellule sante travail animee par la Direction des ressources humaines et impliquant les instances representatives du personnel, a permis a la reflexion–action du service de sante au travail de s’inscrire dans un contexte favorable au developpement et a la reussite de cette pluridisciplinarite. Ce travail d’equipe permet au service de sante au travail de mieux repondre a ses missions, par le renforcement de l’expertise medicale et le developpement des actions sur le terrain tant sur le plan individuel que collectif.