Sporangiospores of the Fungus Mucor lusitanicus 12M: Correlation between Lipid Composition, Viability, and Morphology of Growth upon Germination

We studied viability of sporangiospores from a surface culture of the fungus Mucor lusitanicus 12M grown on wheat bran. With culture aging, the sporangiospores exhibited a tendency toward dimorphic growth upon germination and then lost the ability to germinate. This correlated with changes in the sporangiospore lipid composition, which involved a reduction in the total lipid pool and in the levels of reserve lipids and phospholipids in particular. We suggest that lipid catabolism in sporangiospores causes their defectiveness in the senescent culture.     

Effects of conditions for obtaining sporangiospores of the inoculum on the morphology and productivity of the fungus Mucor circinelloides var. lusitanicus 12 M, a producer of gamma-linolenic acid

Effects of lipid composition of sporangiospores of the fungus Mucor circinelloides var. lusitanicus 12 M, obtained within diverse time frames using distinct nutritive media, on the morphology of the fungus in submerged cultures, the yield of the biomass, and its content of gamma-linolenic acid have been studied. The levels of base phospholipids and individual fractions of neutral lipids in sporangiospores were correlated with the character of their germination. The spores characterized by a high rate of germination and giving rise to a well-developed mycelium contained more phosphatidylcholine and phosphatidylserine, but the level of diacylglycerols was low. An increase in diacylglycerols, free fatty acids, and sterols in lipids of sporangiospores of the inoculate was associated with deterioration of the mycelium development, dimorphism, and lowering of the yield of the biomass of the fungus.     

Characteristics of lipid composition in arthrospores, budding cells and mycelium of the fungus Mucor hiemalis

The fungus Mucor hiemalis F-1156, which is believed to be monomorphic, was found to be able to grow dimorphically in a liquid medium that is free of chemical agents influencing morphogenesis. The growing mycelium produced arthrospores in large amounts. The lipids of the mycelium, yeastlike budding cells, and arthrospores differed in the contents of saturated and unsaturated fatty acids and in the proportion of polar and neutral lipids. The arthrospores contained more monoenoic fatty acids in the total lipids, more triacylglycerides and sterol esters in the neutral lipids, and more phosphatidylcholine and phosphatidylethanolamine in the polar lipids than the yeastlike cells. These differences in the lipid composition of different types of fungal cells should be taken into account in the studies of the lipogenesis of M. hiemalis.     

Impact of morphogenetic effectors on the growth pattern and the lipid composition of the mycelium and the yeastlike cells of the fungus Mucor hiemalis

We investigated the growth and the cell lipid composition of the mycelium and of the yeast-like form of Mucor hiemalis VKMF-1431 obtained under aerobic conditions by treatment with the morphogenetic agents itraconazole, exogenous triacylglycerols (TAGs), and trehalose. The sporangiospores of a 20-day culture were inoculated on the medium with glucose. Under these conditions, the fungus produced both mycelium and yeast-like cells. It was established that, upon the germination of old (20-day) sporangiospores, the fungus predominantly used the mycelium development strategy in the presence of trehalose and TAGs. It was characterized by a low ratio between the two bulk membrane lipids (PEA/PC) and increased levels of PC and polyunsaturated fatty acids (FA). Compared to the mycelium, the yeast cell morphotype obtained on the medium with glucose was distinguished by an elevated PEA/PC ratio, lowered TAG, free sterol (FS) and esterified sterol (ES) levels, a decreased ES/FS ratio that correlated with the reserve sterol pool size, and a lowered content of unsaturated fatty acids (the linoleic and the ??-linolenic acid). These peculiarities of the lipid composition of yeastlike cells correlated with the intensity of yeastlike growth. Light and electron microscopy revealed differences between the above cell morphotypes. With itraconazole, yeast-like cells were characterized by the destruction of the endoplasmic reticulum membranes and formation of a large number of vacuoles. The suggestion was confirmed that the state/age of inoculum sporangiospores exerts an influence on the capacity for dimorphism in mucorous fungi such as M. hiemalis. The data obtained testify to an involvement of lipids in the process of adaptation to environmental factors and to their regulatory role in morphogenetic processes associated with the formation of alternative morphotypes of the mucorous fungus.     

Effect of active dry wine yeast storage upon viability and lipid composition

Alterations in the plasma membrane of Active Dry Wine Yeast (ADWY) produce deleterious effects upon yeast fermentation activity. The aim of this study was to evaluate the changes in viability mediated by plasma membrane alterations induced by poor ADWY storage conditions. Yeast recovery was analyzed after growth in optimal medium. The effects upon vitality were measured by impedance variation. Cell membrane functionality was measured by anisotropy and cell lipid composition was also determined. The results showed a reduction in viability of up to 57–87% in the stored yeast. The storage effect increased saturated fatty acids, and reduced unsaturated fatty acids and phospholipid contents. The stored yeast recovery was related to membrane functionality and significant increases in unsaturated fatty acid, sterol and phospholipid concentrations. Good vitality was positively correlated to high unsaturated fatty acid, phosphatidylserine and phosphatidylethanolamine concentrations and negatively to high saturated fatty acid and phosphatidylcholine contents.     

Relationship between spore germination kinetics and lag time during growth of Mucor racemosus

AIMS: Spore germination requires microscopic observation whereas fungal growth results in a macroscopic examination. This paper aims at establishing a relationship between the percentage of germinated spores and parameters easily available from visible development. METHODS AND RESULTS: About 225 spores of Mucor racemosus were inoculated on PDA medium and incubated at 15 degrees and 25 degrees C. Germination kinetics were modelled by a logistic function. Fungal development provided two parameters, a growth rate, micro, and a lag period, lambda, defined as the slope of the straight line of the graph radius (mm) vs time (h) and the intercept of this line with the X-axis, respectively. CONCLUSIONS: It was found that the lag period coincided with the completion of the germination process, although the number of spores inoculated should be controlled carefully. SIGNIFICANCE AND IMPACT OF THE STUDY: Providing that this result can be generalized, this procedure would constitute a significant breakthrough for predicting food spoilage by moulds.     

Metabolic characteristics and lipid composition of yeastlike cells and mycelium of Mucor circinelloides var. lusitanicus INMI grown at a high glucose content in the medium

It is shown that the fungus Mucor circinelloides var. lusitanicus INMI grown under aerobic conditions in a medium with a high glucose concentration (20%) is capable of both yeastlike and mycelial growth. In the mycelium, the activity of NAD-dependent isocitrate dehydrogenase was more than twice as high as in yeastlike cells, whereas the isocitrate lyase activity was lower. A number of significant differences were found in the lipid composition of the cells of two different morphological variants. Yeastlike cells contained more polar lipids and free fatty acids and less principal reserve lipids (triacylglycerides) than mycelial cells; the content of gamma-linolenic acid and the degree of lipid unsaturation were significantly lower in these cells than in the mycelium. In yeastlike cells, glycolipids composed the bulk of polar lipids; the proportion of phospholipids (primarily phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) was lower. The relationship between cellular metabolism and the lipid composition of fungal cells of different morphotypes grown at high concentrations of glucose, one of the main inducers of dimorphic growth, is discussed.     

Lipid composition of the mycelium of the fungus Mucor hiemalis cultivated with trehalose, triacylglycerols, and itraconazole

We investigated the growth and cell lipid composition of the fungus Mucor hiemalis VKMF-1431 cultivated under aerobic conditions in the presence of the morphogenetic agents itraconazole, exogenous triacylglycerols, and trehalose. The sporangiospores of a 6-day culture were used as inocula. Under these conditions, the fungus produced mycelium; nevertheless, solitary yeastlike cells also developed on the glucose-containing medium and in the presence of itraconazole and sterilized triacylglycerols (sTAGs). No yeastlike growth occurred in the system with trehalose and with unsterilized (native) TAGs (nTAGs). With trehalose and nTAGs in the cultivation medium, the ratio between PEA and PC, the two main types of membrane lipids, was low. This testified to a relatively high PC percentage and, accordingly, a stable structure and a highly functional state of the membranes. Moreover, if the development of the fungus occurred exclusively as mycelium formation, the level of polyunsaturated fatty acids (??-linolenic and arachidonic acid) increased in the presence of trehalose and that of linoleic acid increased in the presence of nTAGs. These results may suggest that unsaturated fatty acids and membrane lipids are related to the cell wall formation and the implementation of morphogenetic programs in mucorous fungi.     

Effect of salinity on composition, viability and germination of seeds of Chenopodium quinoa Willd

Salinity influences plant growth, seed yield and seed quality even of halophytic crops such as Chenopodium quinoa. Plant growth, total seed yield, number of seeds, fresh weight and dry weight of seeds, were all significantly reduced in the presence of salinity. Only at high salinity did the content of proteins (as well as total N) increase significantly in the seeds whereas the content of total carbohydrates (as well as total C) decrease. Aside from that the capacity for germination was diminished by a reduced seed size and a disproportionate reduction of the volume of the perisperm. However, the reduced capacity seemed to be compensated by an accelerated germination owing to high Na and Cl concentrations leading to a low water potential in the walls of the plant ovary. At high salinity the passage of NaCl to the seed interior was hindered by the seed cover. There was an obvious gradient between potentially toxic (Na and Cl) and essentially needed elements (K, Mg, Ca, P and S) across the seed coat of salt treated plants and also a significant change of the distribution of elements in the embryo. The results indicate a highly protected seed interior leading to a high salinity resistance of quinoa seeds.     

Pollenkitt of some monocotyledons: lipid composition and implications for pollen germination

• The composition of pollenkitt and its role in the progamic phase of reproduction are poorly understood. With the aim of extending knowledge on these topics, we chose to study two monocotyledons rich in pollenkitt, with bi‐celled and long‐lived pollen and dry‐type stigma: Crocus vernus Hill subsp. vernus and Narcissus poeticus L.
• Fatty acids of pollenkitt were assayed with gas chromatography. Germination tests were performed in vivo by pollinating the stigmas with a beard hair under a stereomicroscope, and in vitro in liquid culture medium using pollen, either treated or not, with carbon disulphide to remove pollenkitt. The pollen tube percentages were evaluated using fluorescence microscopy techniques. Scanning electron microscopy was used to examine pollen and to follow the early post‐pollination stages.
• Pollenkitt forms bridges between pollen grains but not between grains and stigma papillae. It consists of a mixture of 25 fatty acids, most with long and unsaturated chains, among which are some omega acids. The same acids with different percentages persist on the peritapetal membrane. After its removal, the pollen loses adhesiveness and dries quickly, but retains full capacity for germination on the papillae and can even trigger germination in contiguous pollen grains that do not touch the papillae.
• The results, while confirming the key role of pollenkitt in protecting pollen and favouring pollination, suggest secondary roles in the progamic phase, and highlight the interactive ability of the pollen regardless of lipid shell. The predominance of fatty acids with 18:3 and 16:0, as already noted in Brassica napus pollenkitt, suggests their hierarchy independent of plant species.

     

Regulatory‐associated protein of TOR (RAPTOR) alters the hormonal and metabolic composition of Arabidopsis seeds,controlling seed morphology,viability and germination potential

Target of Rapamycin (TOR) is a positive regulator of growth and development in all eukaryotes, which positively regulates anabolic processes like protein synthesis, while repressing catabolic processes, including autophagy. To better understand TOR function we decided to analyze its role in seed development and germination. We therefore performed a detailed phenotypic analysis using mutants of the REGULATORY‐ASSOCIATED PROTEIN OF TOR 1B (RAPTOR1B), a conserved TOR interactor, acting as a scaffold protein, which recruits substrates for the TOR kinase. Our results show that raptor1b plants produced seeds that were delayed in germination and less resistant to stresses, leading to decreased viability. These physiological phenotypes were accompanied by morphological changes including decreased seed‐coat pigmentation and reduced production of seed‐coat mucilage. A detailed molecular analysis revealed that many of these morphological changes were associated with significant changes of the metabolic content of raptor1b seeds, including elevated levels of free amino acids, as well as reduced levels of protective secondary metabolites and storage proteins. Most of these observed changes were accompanied by significantly altered phytohormone levels in the raptor1b seeds, with increases in abscisic acid, auxin and jasmonic acid, which are known to inhibit germination. Delayed germination and seedling growth, observed in the raptor1b seeds, could be partially restored by the exogenous supply of gibberellic acid, indicating that TOR is at the center of a regulatory hub controlling seed metabolism, maturation and germination.     

Arsonoliposomes: effect of lipid composition on their stability and morphology

The influence of the lipid composition of arsonoliposomes on their membrane integrity was investigated to evaluate whether it is possible to combine their action with drugs that can be encapsulated in their aqueous interior. This was investigated by measuring the retention of vesicle-encapsulated calcein (100 mM) during incubation, in the absence and presence of serum proteins. Liposomes containing various concentrations of arsonolipid (with the palmitoyl side chain) as well as egg-lecithin (phosphatidylcholine, PC) and cholesterol (lipid/chol 2:1 mol:mol) were prepared. In some experiments, PC was replaced by the synthetic phospholipid DSPC. All PC/arsonoliposomes tested are stable after 24 h of incubation in buffer at 37 degrees C. After incubation in the presence of serum proteins, arsonoliposomes that contain low amounts of arsonolipid (up to 5 mol% of the lipid content without cholesterol) are stable, whereas increased release of calcein is observed when vesicle arsonolipid concentration is raised (from 5 to 15 mol%). Further increase of arsonolipid content results in immediate decrease of calcein latency while the remaining calcein is rapidly released during incubation. DSPC/arsonoliposomes are comparably more stable, and membrane integrity is independent of the vesicle arsonolipid content, in the range investigated (15-40 mol% of the lipid content without cholesterol). Thereby, we conclude that more stable arsonoliposomes that incorporate high arsonolipid concentrations may be produced when PC is replaced by DSPC. The latter arsonoliposomes provide a system that may be used for combining arsonolipid activity with the activity of other drugs.     

Liposome/DNA systems: correlation between association, hydrophobicity and cell viability

Small unilamellar vesicles associated with plasmid DNA showed maximum association efficiency for a cationic mixture of egg phosphatidylcholine (EPC):1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE):di-1,2-dioleoyl-3-trimethyl ammonium propane (DOTAP) (16:8:1 molar ratio) [65%], followed by neutral lipids EPC:1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):cholesterol (Chol) (2:2:1 molar ratio) [30%], and a polymerized formulation 1,2-bis(10,12-tricosadiynoyl)sn-glycero-3-phosphocholine (DC8,9PC):DMPE:Chol (2:2:1 molar ratio) [11%]. The hydrophobicity factor (HF) for these formulations followed the trend DC8,9PC:DMPE:CHOL < EPC:DMPE:Chol < EPC:DOPE DOTAP, and DNA association did not alter this trend. Results suggest that the higher the HF value, the more fluid the membrane and the higher the efficiency of DNA association. On the other hand, no differences were observed in cell toxicity with lipids up to 1 mg/ml in VERO cells.     

Lack of correlation between membrane lipid composition and thermotolerance in Chinese hamster ovary cells

Membrane composition and fluidity, and survival of Chinese hamster ovary fibroblasts have been examined following various thermal exposures. It has been found that enhanced thermal resistance following brief exposure to 43°C is not accompanied by detectable membrane lipid alterations. This is in contrast to membrane alterations that occur following adaptation to elevated temperatures compatible with growth (39°C and 41°C).