Jean Masson

Involvement of Arabidopsis thaliana ribosomal protein S27 in mRNA degradation triggered by genotoxic stress

A recessive Arabidopsis mutant with elevated sensitivity to DNA damaging treatments was identified in one out of 800 families generated by T‐DNA insertion mutagenesis. The T‐DNA generated a chromosomal deletion of 1287 bp in the promoter of one of three S27 ribosomal protein genes (ARS27A) preventing its expression. Seedlings of ars27A developed normally under standard growth conditions, suggesting wild‐type proficiency of translation. However, growth was strongly inhibited in media supplemented with methyl methane sulfate (MMS) at a concentration not affecting the wild type. This inhibition was accompanied by the formation of tumor–like structures instead of auxiliary roots. Wild‐type seedlings treated with increasing concentrations of MMS up to a lethal dose never displayed such a trait, neither was this phenotype observed in ars27A plants in the absence of MMS or under other stress conditions. Thus, the hypersensitivity and tumorous growth are mutant‐specific responses to the genotoxic MMS treatment. Another important feature of the mutant is its inability to perform rapid degradation of transcripts after UV treatment, as seen in wild‐type plants. Therefore, we propose that the ARS27A protein is dispensable for protein synthesis under standard conditions but is required for the elimination of possibly damaged mRNA after UV irradiation.

In vivo import of a normal or mutagenized heterologous transfer RNA into the mitochondria of transgenic plants: towards novel ways of influencing mitochondrial gene expression?

Evidence that nuclear‐encoded RNAs are present inside mitochondria has been reported from a wide variety of organisms, and is presumed to be due to import of specific cytosolic RNAs. In plants, the first examples were the mitochondrial leucine transfer RNAs of bean. In all cases, the evidence is circumstantial, based on hybridization of the mitochondrial RNAs to nuclear and not mitochondrial DNA. Here we show that transgenic potato plants carrying a leucine tRNA gene from bean nuclear DNA contain RNA transcribed from the introduced gene both in the cytosol and inside mitochondria, providing proof that the mitochondrial leucine tRNA is derived from a nuclear gene and imported into the mitochondria. The same bean gene carrying a 4 bp insertion in the anticodon loop was also expressed in transgenic potato plants and the transcript found to be present inside mitochondria, suggesting that this natural RNA import system could eventually be used to introduce foreign RNA sequences into mitochondria.

Selection of somatic hybrids between diploid clones of potato (Solanum tuberosum L.) transformed by direct gene transfer

SummaryFive diploid potato clones have been transformed by electroporation of protoplasts with different selectable markers. The resulting diploid regenerated plants have been used in somatic hybridization. It has been shown that hybrid cell selection on the basis of antibiotic or herbicide resistances brought by the two parents of fusion is an efficient method for the recovery of tetraploid somatic hybrids.

Medium-dependent response of grapevine somatic embryogenic cells

A set of novel media, combining significant changes in KNO3, NH4Cl, KH2PO4, CaCl2, MnSO4 and ZnSO4 concentrations have been designed for somatic embryogenesis and plant regeneration in grapevine. Friable and white embryogenic calli, consisting of viable cells, were obtained for Cabernet Sauvignon, Chardonnay, Chasselas, Gamay, Gewurztraminer, Grenache, Merlot, Muscat, Pinot Noir, Portan, Riesling, Syrah and the root-stocks 110 Richter, 3309 Couderc and SO4. The sole ammonium content was shown to drive somatic grape cells either in a proliferation phase or in a differentiation pathway, i.e. the transition from a type I callus to type II. For 14 genotypes, numerous torpedo could be initiated which developed within ∼30 days. The maximal torpedo conversion efficiencies reached 100% in Cabernet Sauvignon and Syrah. Plant regeneration efficiencies ranging from 4.75±4.92%, for Riesling to 79.25±20.84%, for Syrah were reproducibly reached within 50–100 days.

Plant regeneration from protoplasts of diploid potato derived from crosses of Solanum tuberosum with wild solanum species

Abstract A new method has been developed for the obtention of protoplasts of potato ( Solanum tuberosum L.) diploid clones and for the regeneration of whole plants. This procedure determined specific conditions of in vitro culture of shoots: the propagation medium, light intensity and length of illumination period. Four successive media from the protoplast level to the shoot formation have been optimized. This method is suitable for the obtention of calli from 13 diploid potato lines, among 15 tested. Regeneration of plants has been obtained for 10 clones with efficiencies from 0.5 to 4 plants regenerated from 100 plated protoplasts.

Specificity of Ocimum basilicum geraniol synthase modified by its expression in different heterologous systems

Numerous aromatic plant species produce high levels of monoterpenols, using geranyl diphosphate (GPP) as a precursor. Sweet basil (Ocimum basilicum) geraniol synthase (GES) was used to evaluate the monoterpenol profiles arising from heterologous expressions in various plant models. Grapevine (Vitis vinifera) calli were transformed using Agrobacterium tumefasciens and the plants were regenerated. Thale cress (Arabidopsis thaliana) was transformed using the floral dip method. Tobacco (Nicotiana benthamiana) leaves were agro-infiltrated for transient expression. Although, as expected, geraniol was the main product detected in the leaves, different minor products were observed in these plants (V. vinifera: citronellol and nerol; N. benthamiana: linalool and nerol; A. thaliana: none). O. basilicum GES expression was also carried out with microbial system yeasts (Saccharomyces cerevisiae) and Escherichia coli. These results suggest that the functional properties of a monoterpenol synthase depend not only on the enzymes amino-acidic sequence, but also on the cellular background. They also suggest that some plant species or microbial expression systems could induce the simultaneous formation of several carbocations, and could thus have a natural tendency to produce a wider spectrum of monoterpenols.

Transposable elements as plant transformation vectors for long stretches of foreign DNA

The production of transgenic plants is now routine for most crops. However, using currently available transformation methods it is still difficult and time-consuming to obtain a collection of transformed individuals containing single or low-copy-number, intact transgenic inserts. Here we describe a set of broad-hostrange transformation vectors based on the Ac/Ds transposition system that improve both transformation efficiency and the quality of transgenic loci. These vectors efficiently deliver long stretches of foreign DNA into the genome, leading to transgenic strains containing an intact single-copy insert of 10kb. This type of vector could be an important additional tool for the production of transgenic plants with the well-defined, foreign DNA inserts required for biosafety approval and commercialisation.

Contrasting Susceptibilities to Flavescence Dorée in Vitis vinifera, Rootstocks and Wild Vitis Species

Flavescence dorée (FD) is a quarantine disease of grapevine, involving interactions between the plants, leafhopper vectors, and FD phytoplasma. Characterizing the susceptibility of vine varieties could limit disease propagation. After extensive surveys in vineyards, we showed that Cabernet Sauvignon (CS) is highly susceptible, with a high proportion of symptomatic branches and phytoplasma titers, in contrast to Merlot (M). Localized insect transmissions and grafting showed that phytoplasma circulate in the whole plant in the CS cultivar, but in M they are restricted to the transmission point. Insect-mediated transmission under high confinement mimicking natural conditions confirmed these phenotypes and allowed the classification of 28 Vitis accessions into three distinct categories, according to the percentage of infected plants and their phytoplasma titers. Reduced symptoms, low phytoplasma titers, and low percentages of infected plants were found to be associated in the Vitis vinifera cultivars tested. Interestingly, the low susceptibility of M was observed for one of its parents, i.e., Magdeleine Noire des Charentes. Rootstocks and their Vitis parents, although having high percentages of infected plants and intermediate to high phytoplasma titers, shared a symptomless response. This is troubling, because rootstocks can constitute a silent reservoir of contamination in mother plants or when they grow wild nearby vineyards. Altogether, data suggest distribution of genetic traits within the Vitis genus involved in insect-mediated phytoplasma transmission, multiplication, circulation, and symptom development.

The Plant Mitochondrial Transfer RNA Population: A Mosaic of Species with Different Genetic Origins

Plant mitochondria still contain a large (from 200 to over 2000 kbp), autonomously replicating genome, although a massive transfer of genes to the nucleus has probably occurred since their endosymbiotic formation (Palmer, 1990). The mitochondrial DNA encodes some of the polypeptides involved in the enzymatic complexes of the respiratory chain, but these organelles depend on the nuclear and cytosolic compartments for most of their proteins, including the aminoacyl-transfer RNA (tRNA) synthetases and the majority of the ribosomal proteins. Plant mitochondria also do not retain all the tRNA genes of the ancestral endosymbiotic genome (called “native” or “genuine” genes). The higher plant mitochondrial tRNA population is therefore of particular complexity. First, a number of chloroplast DNA sequences, some of them comprising tRNA genes, have been integrated into the plant mitochondrial DNA during evolution. Some of these chloroplast-originating tRNA genes are expressed in mitochondria and produce mature, functional “chloroplast-like” species. Second, even including the “chloroplast-like” tRNA genes, plant mitochondria do not contain a complete set of tRNA genes and import some species from the cytosol. The plant mitochondrial tRNA population appears therefore to be a mosaic of species with nuclear, chloroplast and genuine mitochondrial origins.

Introduction of selectable markers into different diploid potato (Solanum tuberosum L.) genotypes by direct gene transfer

Introduction of selectable markers into different diploid potato (Solanum tuberosum L.) genotypes by direct gene transfer