A reliable system for the transformation of cantaloupe charentais melon (Cucumis melo L. var. cantalupensis) leading to a majority of diploid regenerants

An efficient system of transformation leading to a majority of transformed diploid plants from leaf explants of Cucumis melo L. var. Cantalupensis (cv. Védrantais) was developed. Several regeneration protocols using cotyledon or leaf explants were analysed with particular emphasis on the regeneration efficiency and the ploidy level of the regenerated melon plants. The use of leaf explants excised from 10 day-old seedlings, cultured in Murashige and Skoog's medium supplemented with 1 mM 6-benzylaminopurine (BAP) and 1 mM 6-(g,g-dimethylallylamino)-purine (2iP), resulted in a high regeneration frequency (73%). In these conditions, more than 84% of the regenerated plants were found to be diploid. Addition of an Agrobacterium-mediated transformation step did not significantly change the percentage (81.8%) of diploid plants regenerated. This protocol was successfully used to produce diploid transgenic melon plants expressing the antisense ACC oxidase gene, encoding ACC oxidase which catalyses the last step of ethylene biosynthesis. Ethylene production and ACC oxidase activity of the leaf explants from transgenic plants was reduced by more than 80% as compared to the control untransformed tissues. This transformation/ regeneration method could be routinely used for the introduction of other genes of interest in melon

Ethylene regulation of fruit softening and cell wall disassembly in Charentais melon

Cell wall disassembly in ripening fruit is highly complex, involving the dismantling of multiple polysaccharide networks by diverse families of wall-modifying proteins. While it has been reported in several species that multiple members of each such family are expressed in the same fruit tissue, it is not clear whether this reflects functional redundancy, with protein isozymes from a single enzyme class performing similar roles and contributing equally to wall degradation, or whether they have discrete functions, with some isoforms playing a predominant role. Experiments reported here sought to distinguish between cell wall-related processes in ripening melon that were softening-associated and softening-independent. Cell wall polysaccharide depolymerization and the expression of wall metabolism-related genes were examined in transgenic melon (Cucumis melo var. cantalupensis Naud.) fruit with suppressed expression of the 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene and fruits treated with ethylene and 1-methylcyclopropene (1-MCP). Softening was completely inhibited in the transgenic fruit but was restored by treatment with exogenous ethylene. Moreover, post-harvest application of 1-MCP after the onset of ripening completely halted subsequent softening, suggesting that melon fruit softening is ethylene-dependent. Size exclusion chromatography of cell wall polysaccharides, from the transgenic fruits, with or without exogenous ethylene, indicated that the depolymerization of both pectins and xyloglucans was also ethylene dependent. However, northern analyses of a diverse range of cell wallrelated genes, including those for polygalacturonases, xyloglucan endotransglucosylase/hydrolases, expansin, and b-galactosidases, identified specific genes within single families that could be categorized as ethylene-dependent, ethylene-independent, or partially ethylene-dependent. These results support the hypothesis that while individual cell wall-modifying proteins from each family contribute to cell wall disassembly that accompanies fruit softening, other closely related family members are regulated in an ethylene-independent manner and apparently do not directly participate in fruit softening

Characterization of Ripening-Regulated cDNAs and Their Expression in Ethylene-Suppressed Charentais Melon Fruit

Charentais melons (Cucumis melo cv Reticulatus) are climacteric and undergo extremely rapid ripening. Sixteen cDNAs corresponding to mRNAs whose abundance is ripening regulated were isolated to characterize the changes in gene expression that accompany this very rapid ripening process. Sequence comparisons indicated that eight of these cDNA clones encoded proteins that have been previously characterized, with one corresponding to ACC (1-aminocyclopropane-1-carboxylic acid) oxidase, three to proteins associated with pathogen responses, two to proteins involved in sulfur amino acid biosynthesis, and two having significant homology to a seed storage protein or a yeast secretory protein. The remaining eight cDNA sequences did not reveal significant sequence similarities to previously characterized proteins. The majority of the 16 ripening-regulated cDNAs corresponded to mRNAs that were fruit specific, although three were expressed at low levels in vegetative tissues. When examined in transgenic antisense ACC oxidase melon fruit, three distinct patterns of mRNA accumulation were observed. One group of cDNAs corresponded to mRNAs whose abundance was reduced in transgenic fruit but inducible by ethylene treatment, indicating that these genes are directly regulated by ethylene. A second group of mRNAs was not significantly altered in the transgenic fruit and was unaffected by treatment with ethylene, indicating that these genes are regulated by ethylene-independent developmental cues. The third and largest group of cDNAs showed an unexpected pattern of expression, with levels of mRNA reduced in transgenic fruit and remaining low after exposure to ethylene. Regulation of this third group of genes thus appears to ethylene independent, but may be regulated by developmental cues that require ethylene at a certain stage in fruit development. The results confirm that both ethylene-dependent and ethylene-independent pathways of gene regulation coexist in climacteric fruit

Purification and Characterization of a NADPH-Dependent Aldehyde Reductase from Mung Bean That Detoxifies Eutypine, a Toxin from Eutypa lata

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a K(m) value of 6.3 μm for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification

Correlative analysis of vertebral trabecular bone microarchitecture and mechanical properties: a combined ultra-high field (7 Tesla) MRI and biomechanical investigation

International audienc

Exploration non invasive du système ostéo-articulaire : que nous apporte l’IRM à très haut champ ? Microarchitecture osseuse, tendons, cartilage du genou chez des volontaires

National audienc

Les nouvelles techniques d’imagerie à haut et très haut champ : leur intérêt dans les rhumatismes inflammatoires : PR, SpA et autres

National audienc

Assessment of proximal femur microarchitecture using ultra-high field MRI at 7 Tesla

International audienc

AB1144 ULTRA HIGH FIELD MRI MICROARCHITECTURE ANALYSIS IMPROVES THE PREDICTION OF PROXIMAL FEMUR FRACTURE: A COMBINED STUDY WITH EX VIVO BIOMECHANICAL TESTS

International audienceThe purpose of this study was to investigate bone microarchitecture of cadaveric proximal femurs using ultra-high field (UHF) 7-Tesla magnetic resonance imaging (MRI) and to compare the corresponding metrics with failure load assessed during mechanical compression test and areal bone mineral density (ABDM) measured using dual-energy X-ray absorptiometry.Materials and methods: ABDM of ten proximal femurs from five cadavers (5 women; mean age= 86.2 ± 3.8 (SD) years; range : 82.5-90 years) were investigated using dual-energy X-ray absorptiometry and the bone volume fraction, trabecular thickness, trabecular spacing, fractal dimension, Euler characteristics, Connectivity density and degree of anisotropy of each femur was quantified using UHF MRI. The whole set of specimens underwent mechanical compression tests to failure. Associations were searched using correlation tests and multiple regression analysis