Caractérisation de la mort cellulaire programmée induite par les UV-C chez Arabidopsis thaliana

Dans l'optique d'étudier la mort cellulaire programmée (PCD) chez Arabidopsis, nous avons mis au point un système qui couple induction et quantification de la PCD. Notre système repose sur l'induction de la PCD par les UV-C. L'irradiation de protoplastes couplée à l'utilisation de la réaction TUNEL, nous a permis de quantifier la proportion de cellules en voie de PCD. La tranfection des protoplastes a ensuite permis de mettre en évidence la conservation du rôle de suppresseur de PCD des deux gènes At-DAD (Defender against apoptotic death) d'Arabidopsis ainsi que du gène viral p35 impliqué dans l'inhibition des caspases chez les animaux. Les caspases sont des proteinases spécifiquement induites au cours de la PCD chez les animaux, et qui sont responsables de la dégradation de composants importants pour la cellule. Nous avons pu détecter une activité de type caspase induite par les UV-C et spécifiquement inhibée par des inhibiteurs synthétiques de caspase. Ces mêmes inhibiteurs sont capable de protéger les protoplastes de la PCD induite par les UV-C. Nous avons purifié cette activité de type caspase par FPLC et chromatographie d'affinité et sommes en attente d'identifier par MS/MS les bandes protéiques obtenues par électrophorèse.PERPIGNAN-BU Sciences (661362101) / SudocSudocFranceF

l-Galactono-1,4-lactone Dehydrogenase Is Required for the Accumulation of Plant Respiratory Complex I

International audienceMitochondrial NADH-ubiquinone oxidoreductase (complex I) is the largest enzyme of the oxidative phosphorylation system, with subunits located at the matrix and membrane domains. In plants, holocomplex I is composed of more than 40 subunits, 9 of which are encoded by the mitochondrial genome (NAD subunits). In Nicotiana sylvestris, a minor 800-kDa subcomplex containing subunits of both domains and displaying NADH dehydrogenase activity is detectable. The NMS1 mutant lacking the membrane arm NAD4 subunit and the CMSII mutant lacking the peripheral NAD7 subunit are both devoid of the holoenzyme. In contrast to CMSII, the 800-kDa subcomplex is present in NMS1 mitochondria, indicating that it could represent an assembly intermediate lacking the distal part of the membrane arm. L-galactono-1,4-lactone dehydrogenase (GLDH), the last enzyme in the plant ascorbate biosynthesis pathway, is associated with the 800-kDa subcomplex but not with the holocomplex. To investigate possible relationships between GLDH and complex I assembly, we characterized an Arabidopsis thaliana gldh insertion mutant. Homozygous gldh mutant plants were not viable in the absence of ascorbate supplementation. Analysis of crude membrane extracts by blue native and two-dimensional SDS-PAGE showed that complex I accumulation was strongly prevented in leaves and roots of Atgldh plants, whereas other respiratory complexes were found in normal amounts. Our results demonstrate the role of plant GLDH in both ascorbate biosynthesis and complex I accumulation

Ultraviolet-C Overexposure Induces Programmed Cell Death in Arabidopsis, Which Is Mediated by Caspase-like Activities and Which Can Be Suppressed by Caspase Inhibitors, p35 and Defender against Apoptotic Death

International audiencePlants, animals, and several branches of unicellular eukaryotes use programmed cell death (PCD) for defense or developmental mechanisms. This argues for a common ancestral apoptotic system in eukaryotes. However, at the molecular level, very few regulatory proteins or protein domains have been identified as conserved across all eukaryotic PCD forms. A very important goal is to determine which molecular components may be used in the execution of PCD in plants, which have been conserved during evolution, and which are plant-specific. Using Arabidopsis thaliana, we have shown that UV radiation can induce apoptosis-like changes at the cellular level and that a UV experimental system is relevant to the study of PCD in plants. We report here that UV induction of PCD required light and that a protease cleaving the caspase substrate Asp-Glu-Val-Asp (DEVDase activity) was induced within 30 min and peaked at 1 h. This DEVDase appears to be related to animal caspases at the biochemical level, being insensitive to broad-range cysteine protease inhibitors. In addition, caspase-1 and caspase-3 inhibitors and the pan-caspase inhibitor p35 were able to suppress DNA fragmentation and cell death. These results suggest that a YVADase activity and an inducible DEVDase activity possibly mediate DNA fragmentation during plant PCD induced by UV overexposure. We also report that At-DAD1 and At-DAD2, the two A. thaliana homologs of Defender against Apoptotic Death-1, could suppress the onset of DNA fragmentation in A. thaliana, supporting an involvement of the endoplasmic reticulum in this form of the plant PCD pathway

Characterization of soldat8, a Suppressor of Singlet Oxygen-Induced Cell Death in Arabidopsis Seedlings

ISSN:0032-0781ISSN:1471-905

Blasticidin S Deaminase: A New Efficient Selectable Marker for Chlamydomonas reinhardtii

International audienceChlamydomonas reinhardtii is a model unicellular organism for basic or biotechnological research, such as the production of high-value molecules or biofuels thanks to its photosynthetic ability. To enable rapid construction and optimization of multiple designs and strains, our team and collaborators have developed a versatile Chlamydomonas Modular Cloning toolkit comprising 119 biobricks. Having the ability to use a wide range of selectable markers is an important benefit for forward and reverse genetics in Chlamydomonas. We report here the development of a new selectable marker based on the resistance to the antibiotic blasticidin S, using the Bacillus cereus blasticidin S deaminase (BSR) gene. The optimal concentration of blasticidin S for effective selection was determined in both liquid and solid media and tested for multiple laboratory strains. In addition, we have shown that our new selectable marker does not interfere with other common antibiotic resistances: zeocin, hygromycin, kanamycin, paromomycin, and spectinomycin. The blasticidin resistance biobrick has been added to the Chlamydomonas Modular Cloning toolkit and is now available to the entire scientific community

Histochemical and biochemical characteristics of four major muscles of the ham

International audienceReduction of salt content in processed food is an important issue for both human nutrition and industry. Ham is composed of different muscles and the impact of salt reduction on each of them is unknown. To analyze and understand the effect of salting on the evolution of ham, it is essential to know the characteristics of muscles before applying any technological treatment. Muscles semi-membranosus, biceps femoris, rectus femoris and gluteus medius were selected on their physiological differences. These muscles were finely characterized in their structure and biochemical composition. Each muscle was then cured and cooked with two brines, which brought respectively 1.3% and 1.8% of sodium chloride in the meat. Cooking yield was determined. Significant differences were observed between muscles for physical, biochemical or histological parameters of non-cured muscles and between muscles and salt contents for cooking yield. Thus, muscle characteristics have to be taken into account in any study on the optimization of salting meat

Histochemical and biochemical characteristics of four major muscles of the ham

International audienceReduction of salt content in processed food is an important issue for both human nutrition and industry. Ham is composed of different muscles and the impact of salt reduction on each of them is unknown. To analyze and understand the effect of salting on the evolution of ham, it is essential to know the characteristics of muscles before applying any technological treatment. Muscles semi-membranosus, biceps femoris, rectus femoris and gluteus medius were selected on their physiological differences. These muscles were finely characterized in their structure and biochemical composition. Each muscle was then cured and cooked with two brines, which brought respectively 1.3% and 1.8% of sodium chloride in the meat. Cooking yield was determined. Significant differences were observed between muscles for physical, biochemical or histological parameters of non-cured muscles and between muscles and salt contents for cooking yield. Thus, muscle characteristics have to be taken into account in any study on the optimization of salting meat

Rapid Induction of Distinct Stress Responses after the Release of Singlet Oxygen in Arabidopsis

The conditional fluorescent (flu) mutant of Arabidopsis accumulates the photosensitizer protochlorophyllide in the dark. After a dark-to-light shift, the generation of singlet oxygen, a nonradical reactive oxygen species, starts within the first minute of illumination and was shown to be confined to plastids. Immediately after the shift, plants stopped growing and developed necrotic lesions. These early stress responses of the flu mutant do not seem to result merely from physicochemical damage. Peroxidation of chloroplast membrane lipids in these plants started rapidly and led to the transient and selective accumulation of a stereospecific and regiospecific isomer of hydroxyoctadecatrieonic acid, free (13S)-HOTE, that could be attributed almost exclusively to the enzymatic oxidation of linolenic acid. Within the first 15 min of reillumination, distinct sets of genes were activated that were different from those induced by superoxide/hydrogen peroxide. Collectively, these results demonstrate that singlet oxygen does not act primarily as a toxin but rather as a signal that activates several stress-response pathways. Its biological activity in Arabidopsis exhibits a high degree of specificity that seems to be derived from the chemical identity of this reactive oxygen species and/or the intracellular location at which it is generated