Michel Briquet

Microanalysis of Plant Mitochondrial Protein-synthesis Products - Detection of Variant Polypeptides Associated With Cytoplasmic Male-sterility

SummaryA mitochondrial fraction obtained from 0.5 g of leaves was purified on a 0.75 ml Percoll gradient and used for an in vitro mitochondrial protein synthesis assay in the presence of [35S] methionine. A set of 15 to 20 labeled polypeptides were revealed by autoradiography after sodium dodecylsulfate polyacrylamide gel electrophoresis. This could be applied at an early growth stage by using a few leaves from individual seedlings. It revealed the presence of variant mitochondrially translated polypeptides in green leaves of cytoplasmic male sterile lines from various cultivated plants of large economic importance: maize, wheat, sugar beet, tobacco and faba bean. This non-destructive microanalysis is thus of general use and opens new possibilities for rapid and large mass screening of mitochondrial parameters such as male sterility.

Mitochondrial DNA modifications associated with cytoplasmic male sterility in rice

SummaryMitochondrial DNA was isolated from fertile and cytoplasmic male sterile lines of rice. Restriction analysis showed specific modifications in the male sterile cytoplasm. In addition to the major mitochondrial DNA, three small plasmid-like DNA molecules were detected by agarose gel electrophoresis in both cytoplasms. An additional molecule was specifically found in the sterile cytoplasm. These mitochondrial DNA modifications support the hypothesis of the mitochondrial inheritance of the cytoplasmic male sterility in rice.

Migration barriers protect indigenous brown trout (Salmo trutta) populations from introgression with stocked hatchery fish

Brown trout populations in the Belgian rivers Scheldt and Meuse have been intensively stocked in the past decades, often with material of uncertain origin. Moreover, the species’ habitat has become increasingly fragmented, preventing gene flow between neighboring populations. We assessed how this impacted genetic diversity and population structure by analyzing 12 wild populations (total n=309) and seven hatchery stocks (n=200) at the mitochondrial control region with SSCP and at 27 RAPD loci. Historical records indicate that brown trout from distant locations have been used to supplement hatchery stocks; nevertheless we detected non-Atlantic mitochondrial genomes in only one population of the Scheldt basin and in one hatchery. In general, the hatchery samples displayed a higher genetic diversity and differentiated less among each other (global FST(mtDNA)=0.311/FST(RAPD)=0.029) compared to the wild populations (global FST(mtDNA)=0.477/FST(RAPD)=0.204). This is due to frequent exchanges between hatcheries and regular supplementation from several indigenous populations. Gene pools present in most downstream sections from tributaries of the Meuse were similar to each other and to the hatchery samples, despite the presence of migration barriers. Assignment analyses indicated that the contribution of hatchery material to the upstream parts was limited or even completely absent in populations separated by a physical barrier. Intensive stocking and exchange between hatcheries has homogenized the downstream sections of the Meuse River, whereas the migration barriers preserved the indigenous upstream populations. As such, uncontrolled removal of barriers might result in an irreversible loss of the remnant indigenous gene pools.

Regulation of mitochondrial biogenesis : enzymatic changes in cytochrome-deficient yeast mutants requiring delta-aminolevulinic acid.

Yeast cells almost completely deficient in all cytochromes were obtained by introducing two defective nuclear genes, cyd1 and cyc4, into the same haploid strain. The action of the two mutant genes is synergistic, since either gene acting singly results in only partial cytochrome deficiency. Normal synthesis of all cytochromes can be restored in the double mutant by adding delta-aminolevulinic acid to the growth medium. The optimum concentration of delta-aminolevulinate for restoration of cytochrome synthesis is about 40 muM; when higher concentrations are used, synthesis of cytochromes is partially suppressed, particularly that of cytochrome a.a3. Growth yield of the double mutant is stimulated by ergosterol and Tween 80, a source of unsaturated fatty acid. Methionine stimulates further. None of these nutrients is required for growth when sufficient delta-aminolevulinic acid is present in the growth medium. With respect to nutritional responses, the single-gene, cytochrome-deficient mutant, ole3, behaves like the double mutant. The frequency of the p-mutation in the double mutant grown in the absence of ergosterol, Tween 80, and delta-aminolevulinic acid is at least 15%. The frequency can be reduced to less than 1% by either delta-aminolevulinic acid or Tween 80. Ergosterol alone does not decrease the p- frequency. The ole3 mutant does not exhibit increased p-frequency under similar conditions of unsaturated fatty acid deficiency.

Use of RAPD patterns for clone verification and in studying provenance relationships in Norway spruce (Picea abies)

Abstract We have used the RAPD technique to analyse samples of Picea abies obtained from an improvement forestry station. Two types of plant material were harvested, the first being clones and the second provenances from various regions. We first checked the clonal identity of elite tree cuttings and clones; some differences in the RAPD patterns resulting from mis-planting or mis-labelling of cuttings were found. We also established a reference library of RAPD fingerprints for 96 clones, which will serve as a reference source in cases of litigation concerning clone identity. The RAPD technique was also used to study the genetic relationship between nine European provenances of Norway spruce. A dendogram was obtained by individual pairwise comparison of 42 RAPD bands, which separated the nine provenances into two major groups, one containing the Nordic provenances (Sweden and Bielorussia) and another the Alpine provenances (France, Austria, Germany and Belgium). The Belgian provenance, which is not indigenous, is most closely related to the German provenance. We conclude that the RAPD technique is a useful tool for forestry stations in managing propagation operations.

Immobilization of Yeast-cells By Entrapment and Adhesion Using Siliceous Materials

Abstract Yeast cells ( Saccharomyces cerevisiae ) have been immobilized by entrapment in silica hydrogel, without significantly changing their biological activity; a simple model describes the rate of oxygen uptake by a film of immobilized cells. The cells have also been immobilized by direct adhesion to a glass surface; this is achieved by a well-controlled drying procedure, sufficient to bring the cells into close contact with the support, but without cell dehydration. The immobilized cells consume glucose at a rate which is about half of the rate obtained in suspension and they are resistant to strong mechanical strains.

Transport of pyruvate and lactate in yeast mitochondria.

Evidence for the existence of mediated transport of pyruvate and lactate in isolated mitochondria of Saccharomyces cerevisiae is presented. 1. The mitochondrial oxidation of pyruvate is specifically inhibited by the monocarboxylic oxoacids alpha-ketoisocaproate and by alpha-cyano-3-hydroxycinnamate, while pyruvate and malate dehydrogenases activities are not inhibited. 2. The stimulation of the mitochondrial oxidations of succinate, alpha-ketoglutarate and citrate by pyruvate are also inhibited by alpha-cyano-3-hydroxycinnamate. 3. The [14C]pyruvate uptake by yeast mitochondria follows saturation kinetics and is completely inhibited by alpha-cyano-3-hydroxycinnamate. 4. Large amplitude passive swellings of mitochondria of the wild type and of cytoplasmic rho- and rho-n mutants are induced by isoosmotic ammonium pyruvate and lactate. These pH-dependent swellings are inhibited by alpha-cyano-3-hydroxycinnamate suggesting that the carrier system is not coded by mitochondrial DNA.

In vitro propagation of Vicia faba L. by micro-cutting and multiple shoot induction

The influences of nitrogen sources, culture temperature and activated charcoal supplements were studied in relation to the rooting ability of V. faba cuttings. The interaction of these factors led to quantitative and qualitative modifications of the culture responses. Low temperatures (14–18°C) were suitable for in vitro culture, limiting the formation of phenolics in plant material and making activated charcoal supplement unnecessary. Nitrogen supplements contributed in modifying the different plant responses, in accordance with temperature. Multiple shoot formation was obtained from the cotyledonary node and from the stem nodes cultivated in the presence of 6-benzylaminopurine (BAP). BAP at 4 mg l-1 was the most effective concentration in promoting high rates of shoot development. The original position of stem nodes was found to determine the explant response to plant growth regulator treatments, possibly due to the effect of residual apical dominance.

Plant cell membranes as biochemical targets of the phytotoxin helminthosporol.

Helminthosporol is one of the natural sesquiterpenoid toxins isolated and identified in the culture medium of the phytopathogenic ascomycete fungus Cochliobolus sativus. The effect of this phytotoxin was investigated on enzymatic activities, electron and ion transport in mitochondria, chloroplasts, and microsomes of plant. The results indicate that helminthosporol drastically affects the membrane permeability of these organelles to protons and subtrate anions, inhibiting the mitochondrial oxidative phosphorylation, the photophosphorylation in chloroplasts, and the proton pumping across the cell plasma membrane. The 1,3-β-glucan synthase activity, involved in defense mechanisms of plant cells against stress and damage, e.g., during pathogen attack, was also strongly inhibited by the toxin.

Identification of larch species (Larix decidua, Larix kaempferi and Larix X eurolepis) and estimation of hybrid fraction in seed lots by RAPD fingerprints

Abstract Species-specific RAPD markers were used to identify the different larch species (Larix decidua and Larix kaempferi) and their interspecific hybrid (Larix X eurolepis). Although morphological differences between pure species and the hybrids exist, differentiation is not always possible, especially at an early stage (seed or plantlet). Eleven RAPD markers differentiated the two larch species, and 4 species-specific markers were sufficient to estimate the F1 hybrid fraction in a seed lot. The species-specific markers were tested on individual trees of European and Japanese larches of diverse geographic origins and on several seed lots of different origins (F1, F2 hybrids and pure species). The 4 specific markers found for the European larch and the Japanese larch were monomorphic and present in all provenances and in all F1 hybrid trees tested. Polymorphic SCAR fragments were obtained for 3 of the 11 fragments originally selected for the RAPD screening phase. For 2 of them, the sequence had some homology with the mitochondrial genome of other organisms and is thus mitochondrial. The two mitochondrial fragments and the OPF-131000 fragment exhibited one polymorphic band, thereby maintaining its species-specific identity: OPF-131000 is specific to the European larch. The 4 RAPD primers selected in this study offer a reliable, quick and cheap tool for the identification of different larch species (Larix decidua and Larix kaempferi) and their interspecific hybrid (Larix X eurolepis).