Régis Calvayrac

EFFECTS OF CARBOHYDRATE SOURCE, POLYETHYLENE GLYCOL AND GELLAN GUM CONCENTRATION ON EMBRYONAL-SUSPENSOR MASS (ESM) PROLIFERATION AND MATURATION OF MARITIME PINE SOMATIC EMBRYOS

SummaryThe influence of carbon sources and polyethylene glycol combined with 0.45 and 0.9% (w/v) of gellan gum on the maturation of maritime pine somatic embryos was tested. The effect of the carbon source and polyethylene glycol varied widely between lines. One out of the five lines tested showed a striking response to polyethylene glycol (PEG) treatment; the addition of this osmoticum limited the embryonal-suspensor mass (ESM) proliferation while it enhanced the maturation rate. Conversely, the ESM proliferation was stimulated by PEG in the other lines without subsequent improvement of the maturation rate. The use of a high concentration of gellan gum (0.9%) improved the maturation of the five ESM lines. It was concluded that the most efficient culture medium to recover cotyledonary embryos from all lines is one supplemented with sucrose at 6% (w/v) and gellan gum at 0.9% (w/v) without PEG. The determining factor in the maturation of maritime pine somatic embryos is the genotype and/or the quality of ESM. The possible relationship between maturation performances and ESM morphology, particularly the suspensor organization, is discussed.

Lipid composition of Euglena gracilis in relation to carbon-nitrogen balance

Abstract The lipid class and fatty acid compositions of Euglena gracilis were analysed after growth under various conditions of autotrophy or photoheterotrophy, in order to identify the contribution of lactate (a carbon source) and ammonium phosphate (a nitrogen source) to the metabolism of these compounds. When ammonium and lactate were both present in the medium, in concentrations that allowed growth, the lipid composition of algae appeared to be independent of the ammonium concentration. The effects of increasing ammonium phosphate concentration on lipid metabolism were observed only when lactate was depleted. With increasing nitrogen concentration in the medium, an increase in the content of galactolipids rich in polyunsaturated C 16 and C 18 fatty acids (FA) was noted, as well as an increase in the monogalactosyldiacylglycerol: digalactosyldiacylglycerol ratio; these changes could be related to an increase in the number of the stacked lamellae present in chloroplasts. Nitrogen concentration had no influence on the C 12 and C 14 FA but induced a loss of C 22 FA. In the absence of ammonium phosphate in the culture medium, increasing lactate concentrations were accompanied by a decrease in all plastid lipids, whereas the content of storage lipids (enriched in 14 : 0 and 16 : 0) increased. Partial inhibition of the polydesaturation of C 18 FA was observed and was accompanied by an accumulation of 18 : 1.

Influence of the embryonal-suspensor mass (ESM) sampling on development and proliferation of maritime pine somatic embryos

Two maturation media with high and low concentration of gellan gum were used to evaluate the maturation performances of four maritime pine ESM (embryonal-suspensor mass) lines. The maturation performance is influenced by sampling modalities; the outer part of the ESM yielded more cotyledonary embryos than the inner part or the whole colony. ESM lines showing several stage 1 embryos at the periphery (spiky) were more productive than those for which stage 1 embryos were rarely visible (smooth). This latter group develop preferably stage 3 embryos on the maturation medium containing high concentration of gellan gum. Biomass production is higher on a medium containing low concentration of gellan gum. However, sampling modalities did not affect the biomass production, and no relation was found between the biomass production and the maturation performance of each line. Stage 3 embryos developed on the medium with low concentration of gellan gum (0.45%, w/v) were shorter than those developed on medium with 0.9% of gellan gum. These short embryos were not able to germinate whereas about 48% of germination was reached with the longest embryos. The ability to develop primary root is dependent on the genotype while epicotyl elongation was observed among all lines.

Photosynthetic properties of a DCMU resistant strain of Euglena gracilis Z.

Abstract When present during the photoheterotrophic growth of Euglena gracilis Z; DCMU, in addition to the usual inhibition of photosynthetic electron transport, induces important losses of chlorophyll and severe disorganization of chloroplast structure. After two months of growth under these conditions, however, these parameters recover their normal properties and the cells become resistant to the dose of DCMU to which they were exposed. The sensitivity of photosynthetic electron transport to increasing concentrations of DCMU was analyzed in whole cells of the wild-type and resistant strains as well as in their respective isolated chloroplasts, using oxygen evolution and fluorescence induction to measure the DCMU effects. Another photosynthetic electron transport inhibitor, ortho-1, 10-phenanthrolene (ORPHE), believed to act at the same site as DCMU, was also used to obtain dose-response curves as for DCMU, and similar results were obtained. The results suggest that the resistance, which is a function of the dose, is due neither to the acquired ability of the cells to transform DCMU into an inactive form nor to the acquisition of a permeability barrier to DCMU. It appears that DCMU resistance is caused at the level of intracellular DCMU-sensitive reactions, particularly of photosynthetic electron transport.

Retroviral-mediated correction of DNA repair defect in xeroderma pigmentosum cells is associated with recovery of catalase activity

Xeroderma pigmentosum (XP) is a rare inherited disease associated with photosensitivity, a very high susceptibility to develop neoplasm on sun-exposed skin and neurological abnormalities for some patients. We previously reported that diploid cell lines established from XP skin biopsies present an abnormal low level of catalase activity, which is involved in the defense against oxygen free radicals. This biochemical dysfunction, probably involved in the skin cancer formation, has been difficult to be directly related to the nucleotide excision repair (NER) defect in XP. In this paper we report that the retroviral-mediated transduction of XP diploid cells by the XPC and XPD/ERCC2 cDNAs fully and stably corrects the NER defect in terms of survival and unscheduled DNA synthesis (UDS) after ultraviolet (UV) irradiation. The catalase activity in transduced cells was recovered up to normal levels only in cells transduced with repair genes correcting the repair defect. These results imply that: (i) the reduced catalase activity in XP, which might result from cellular depletion of its NADPH cofactor, is directly related to impaired DNA repair, and (ii) this depletion might be one of the multiple cellular consequences of XP inborn defect.

Evolution of carboxylating enzymes involved in paramylon synthesis (phosphoenolpyruvate carboxylase and carboxykinase) in heterotrophically grown Euglena gracilis

Heterotrophically grown Euglena synthesize grains of paramylon, its reserve carbohydrate, in a vesicular complex of mitochondrial origin. A CO2 fixation activity in dark grown Euglena was demonstrated in the mitochondria via paramylon. At the beginning of the exponential phase of growth, the activity of phosphoenolpyruvate carboxykinase increases before the augmentation of paramylon.At the end of the exponential phase, the activity of this enzyme decreases, and low residual levels persist in the transition and stationary phases of growth. The activity of phosphoenolpyruvate carboxylase evolves inversely during the heterotrophic growth of the algae in succinate- or a lactate-containing medium. A compartmentalized scheme of carbon metabolism in mitochondria is presented.

PARAMYLON SYNTHESIS IN HETEROTROPHIC AND PHOTOHETEROTROPHIC EUGLENA (EUGLENOPHYCEAE)1

Growth of Euglena gracilis Klebs in medium containing DL‐lactate as sole carbon source results in the synthesis of large quantities of paramylon at the beginning of exponential growth. The accumulation of paramylon grains in the cytoplasm is observable in both dark‐grown and light‐grown cells. Furthermore, the cells fix 14CO2 in darkness. Incorporated CO2 is found in paramylon which is function of the physiological state of the cells, being most intense in exponential phase heterotrophic Euglena. The mitochondria undergo structural modifications in older cultures, i.e., when the exogenous carbon source becomes exhausted. At the beginning of exponential growth, they are swollen and vesiculated with an electrontranslucent matrix containing an extensive internal membrane system. This vesiculated mitochondrial system is correlated with paramylon synthesis, since forming paramylon grains are seen within the vesiculated complex. Stationary phase cells present a compact chondriome with a dense matrix devoid of vesiculation. Membrane‐bound paramylon grains are found in the cytoplasm of these cells.

Paramylon synthesis by Euglena gracilis photoheterotrophically grown under low O2 pressure : Description of a mitochloroplast complex.

Special culture conditions for Euglena gracilis Z and ZR are described. They induce interactions between the chloroplast and mitochondrial metabolisms leading to paramylon synthesis. When grown in continuous light under pure nitrogen and in the presence of lactate as the sole carbon source, sugar synthesis occurs during the first 24 h of culture with the participation of both mitochondria (using lactate) and of chloroplasts (fixing CO2 from lactate decarboxylation). The activities of ribulose bisphosphate carboxylase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase are very high and mitochondria and chloroplasts develop then a common network of vesicles in which paramylon grains can be seen. Electron micrographs demonstrate membrane continuity between the two types of organelles. Occasionally the mitochondrial matrix and the chloroplast stroma are separated by only a unit membrane.

Metabolic studies on eel (Anguilla anguilla L.) hepatocytes in primary culture: effect of 17β-estradiol and growth hormone

Previous studies demonstrated that native and recombinant growth hormone from mammalian and fish species potentiate the estrogenic induction of vitellogenin synthesis by cultured eel hepatocytes. In the present study, the metabolic competence (respiratory activity and estradiol catabolism) of cultured hepatocytes and their functional capacity to synthesize a specific protein, vitellogenin, in the presence of estradiol and/or bovine growth hormone was investigated. In addition, we examined the possible role of insulin-like growth factors as mediators of growth hormone. Hepatocytes retain a high level of metabolic activity under the primary culture conditions applied. Estradiol has a half life of several hours in the hepatocyte culture, and is metabolized into conjugated forms. Estradiol and/or growth hormone had no effects on respiratory activity of the cultured hepatocytes. Moreover, the estradiol catabolic parameters were not affected by growth hormone. Finally, human and trout recombinant insulin-like growth factors do not potentiate vitellogenin synthesis induced by estradiol.

Biosynthesis of polyamines in Euglena gracilis

Abstract In Euglena gracilis Z the biosynthesis of spermidine and spermine closely resembles the pathways occurring in mammalian tissues and in most microorganisms. l -Ornithine and not l -arginine, as is the case in most plants, is the main precursor of putrescine, and S-adenosylmethionine donates the propylamino moiety for the biosynthesis of spermidine and spermine. Cell-free extracts of Euglena synthesized sym-norspermidine and sym-norspermine from 1,3-diaminopropane and labelled S-adenosylmenthionine. The synthases for the biosynthesis of these two polyamines have a pH optimum of 7.6, like that of spermidine and spermine synthases. Ion exchange chromatography showed two peaks corresponding to the retention times of 2,4-diaminobutyric acid and 1,3-diaminopropane, lower homologues of ornithine and putrescine, respectively. Experiments with dl -2,4-diaminobutyric acid-[4-14C] did not result in significant incorporation of the label into 1,3-diaminopropane.