The effect of natamycin on the transcriptome of conidia of Aspergillus niger.

The impact of natamycin on Aspergillus niger was analysed during the first 8 h of germination of conidia. Polarisation, germ tube formation, and mitosis were inhibited in the presence of 3 and 10 µM of the anti-fungal compound, while at 10 µM also isotropic growth was affected. Natamycin did not have an effect on the decrease of microviscosity during germination and the concomitant reduction in mannitol and trehalose levels. However, it did abolish the increase of intracellular levels of glycerol and glucose during the 8 h period of germination.Natamycin hardly affected the changes that occur in the RNA profile during the first 2 h of germination. During this time period, genes related to transcription, protein synthesis, energy and cell cycle and DNA processing were particularly up-regulated. Differential expression of 280 and 2586 genes was observed when 8 h old germlings were compared with conidia that had been exposed to 3 µM and 10 µM natamycin, respectively. For instance, genes involved in ergosterol biosynthesis were down-regulated. On the other hand, genes involved in endocytosis and the metabolism of compatible solutes, and genes encoding protective proteins were up-regulated in natamycin treated conidia.

Water- and air-distributed conidia differ in sterol content and cytoplasmic microviscosity.

Airborne and waterborne fungal spores were compared with respect to cytoplasmic viscosity and the presence of ergosterol. These parameters differed markedly between the two spore types and correlated with spore survival. This suggests that the mode of spore dispersal has a bearing on cellular composition, which is relevant for the eradication of industrially relevant fungal propagules.

Water replacement hypothesis in atomic details: effect of trehalose on the structure of single dehydrated POPC bilayers.

We present molecular dynamics (MD) simulations to study the plausibility of the water replacement hypothesis (WRH) from the viewpoint of structural chemistry. A total of 256 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) lipids were modeled for 400 ns at 11.7 or 5.4 waters/lipid. To obtain a single dehydrated bilayer relevant to the WRH, simulations were performed in the NP(xy)h(z)T ensemble with h(z) > 8 nm, allowing interactions between lipids in the membrane plane and preventing interactions between neighboring membranes via periodic boundary conditions. This setup resulted in a stable single bilayer in (or near) the gel state. Trehalose caused a concentration-dependent increase of the area per lipid (APL) accompanied by fluidizing the bilayer core. This mechanism has been suggested by the WRH. However, dehydrated bilayers in the presence of trehalose were not structurally identical to fully hydrated bilayers. The headgroup vector was in a more parallel orientation in dehydrated bilayers with respect to the bilayer plane and maintained this orientation in the presence of trehalose in spite of APL increase. The total dipole potential changed sign in dehydrated bilayers and remained slightly positive in the presence of trehalose. The model of a dehydrated bilayer presented here allows the study of the mechanisms of membrane protection against desiccation by different compounds.

The polyene antimycotics nystatin and filipin disrupt the plasma membrane, whereas natamycin inhibits endocytosis in germinating conidia of Penicillium discolor.

AIMS: To investigate the differences in membrane permeability and the effect on endocytosis of the polyene antimycotics nystatin, filipin and natamycin on germinating fungal conidia. METHODS AND RESULTS: The model system was Penicillium discolor, a food spoilage fungus. Filipin resulted in permeabilization of germinating conidia for the fluorescent probes TOTO-1 and FM4-64, but not for ferricyanide ions. Nystatin caused influx of all these compounds while natamycin did not. Untreated germinating conidia internalize the endocytic marker FM4-64. Pretreatment of germinating conidia with natamycin showed a dose and time dependent inhibition of endocytosis as judged by the lack of formation of early endosomal compartments. CONCLUSIONS: The results obtained from this study indicated that, unlike nystatin and filipin, natamycin is unable to permeabilize germinating conidia, but interferes with endocytosis in a dose and time dependent manner. SIGNIFICANCE AND IMPACT OF THE STUDY: Natamycin acts via a different mode of action than other polyene antimycotics. These results offer useful information for new strategies to prevent fungal spoilage on food products and infection on agricultural crops. For laboratory use, natamycin can be used as a specific inhibitor of early endocytosis in fungal cells.

Mechanisms of plant desiccation tolerance.

Anhydrobiosis ("life without water") is the remarkable ability of certain organisms to survive almost total dehydration. It requires a coordinated series of events during dehydration that are associated with preventing oxidative damage and maintaining the native structure of macromolecules and membranes. The preferential hydration of macromolecules is essential when there is still bulk water present, but replacement by sugars becomes important upon further drying. Recent advances in our understanding of the mechanism of anhydrobiosis include the downregulation of metabolism, dehydration-induced partitioning of amphiphilic compounds into membranes and immobilization of the cytoplasm in a stable multicomponent glassy matrix.

The structural differences between the embryos of viable and nonviable wheat seeds as studied with the EPR spectroscopy of lipid-soluble spin labels.

Dried and hydrated embryos of wheat seeds (viable and nonviable, harvested in 1992 and 1976, respectively) were studied by the EPR method with the use of the spin-labeling technique. Spin label Tempone was used for testing the plasmalemma integrity. It has been demonstrated that the loss of seed viability correlates with the loss of external membrane integrity. Spin-labeled derivatives of stearic acids, 5-doxylstearate I(12.3) and 16-doxylstearate I(1.14), were used to monitor the changes in structural characteristics of embryo cell membranes. The EPR spectra of these spin labels represent the superpositions of at least two signals from the molecules located in domains characterized by different fluidity. The comparison of the EPR spectra from I(12.3) in embryo cells and model systems (total fraction of lipids and purified seed oil) indicates that the majority of spin label molecules is located in the lipid surroundings, while the minor portion of I(12.3) is localized in so-called lipid bodies which contain seed oil. The embryo cells of viable and nonviable seeds differ in the sizes of these 'solid' and 'fluid' intracellular domains. The environment of spin label molecules located in cell membranes of nonviable seeds is more rigid, as compared with that in the membranes of the viable cells. The study of dehydration-rehydration effects has demonstrated that the loss of water causes the restriction of spin label mobility in embryo cells from both kinds of seeds.

Spin-labeling study of membranes in wheat embryo axes. 1. Partitioning of doxyl stearates into the lipid domains.

The interaction of lipid soluble spin labels with wheat embryo axes has been investigated to obtain insight into the structural organization of lipid domains in embryo cell membranes, using conventional electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopy. Stearic acid spin labels (n-SASL) and their methylated derivatives (n-MeSASL), labelled at different positions of their doxyl group (n=5, 12 and 16), were used to probe the ordering and molecular mobility in different regions of the lipid moiety of axis cell membranes. The ordering and local polarity in relation to the position of the doxyl group along the hydrocarbon chain of SASL, determined over the temperature range from -50 to +20 degrees C, are typical for biological and model lipid membranes, but essentially differ from those in seed oil droplets. Positional profiles for ST-EPR spectra show that the flexibility profile along the lipid hydrocarbon chain does exist even at low temperatures, when most of the membrane lipids are in solid state (gel phase). The ordering of the SASL nitroxide radical in the membrane surface region is essentially higher than that in the depth of the membrane. The doxyl groups of MeSASLs are less ordered (even at low temperatures) than those of the corresponding SASLs, indicating that the MeSASLs are located in the bulk of membrane lipids rather than in the protein boundary lipids. The analysis of the profiles of EPR and ST-EPR spectral parameters allows us to conclude that the vast majority of SASL and MeSASL molecules accumulated in embryo axes is located in the cell membranes rather than in the interior of the oil bodies. The preferential partitioning of the doxyl stearates into membranes demonstrates the potential of the EPR spin-labelling technique for the in situ study of membrane behavior in seeds of different hydration levels.

An Electron Paramagnetic Resonance Spin-Probe Study of Membrane-Permeability Changes with Seed Aging.

We developed an electron paramagnetic resonance spin-probe technique to study changes in the barrier properties of plasma membranes in wheat (Triticum aestivum L.) seeds during aging under dry storage. The estimation of these barrier properties was based on the differential permeability of membranes for the stable free radical 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy and the broadening agent ferricyanide. The line-height ratio between the water and lipid components in the electron paramagnetic resonance spectra of 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (R value) allowed for the quantitative assessment of the plasma membrane permeability in small samples, enabling separate studies of the axis, scutellum, aleurone layer, and starchy endosperm tissue. High R values corresponded to low permeability and vice versa. Starchy endosperm cells had completely permeable plasma membranes even in mature, viable seeds. The loss of germinability with aging coincided with a considerably increased plasma membrane permeability of the embryo axis cells, but not of the scutellum and aleurone layer cells. The threshold R value for the individual axes associated with viability loss was established at 5 to 6, with the total ranging from 0 to more than 12. We suggest that the R value of an individual axis is the result of contributions from all individual cells, each of them characterized by a different permeability. The loss of viability, therefore, corresponds to the accumulation of cells having permeability above a critical level.

High viscosity and anisotropy characterize the cytoplasm of fungal dormant stress-resistant spores.

Ascospores of the fungus Talaromyces macrosporus are dormant and extremely stress resistant, whereas fungal conidia--the main airborne vehicles of distribution--are not. Here, physical parameters of the cytoplasm of these types of spores were compared. Cytoplasmic viscosity and level of anisotropy as judged by spin probe studies (electron spin resonance) were extremely high in dormant ascospores and during early germination and decreased only partly after trehalose degradation and glucose efflux. Upon prosilition (ejection of the spore), these parameters fell sharply to values characteristic of vegetative cells. These changes occurred without major volume changes that suggest dramatic changes in cytoplasmic organization. Azide reversibly inhibited prosilition as well as the decline in cytoplasmic parameters. No organelle structures were observed in etched, cryoplaned specimens of ascospores by low-temperature scanning electron microscopy (LTSEM), confirming the high cytoplasmic viscosity. However, cell structures became visible upon prosilition, indicating reduced viscosity. The viscosity of fresh conidia of different Penicillium species was lower, namely, 3.5 to 4.8 cP, than that of ascospores, near 15 cP. In addition the level of anisotropic motion was markedly lower in these cells (h(0)/h(+1) = 1.16 versus 1.4). This was confirmed by LTSEM images showing cell structures. The decline of cytoplasmic viscosity in conidia during germination was linked with a gradual increase in cell volume. These data show that mechanisms of cytoplasm conservation during germination differ markedly between ascospores and conidia.

Induction of desiccation tolerance in plant somatic embryos: how exclusive is the protective role of sugars?

Plant somatic embryos usually lack desiccation tolerance. They may acquire such a tolerance upon preculture in the presence of abscisic acid (ABA), followed by slow drying, but not fast drying. ABA causes torpedo-shaped somatic embryos to lose their chlorophyll, suspend growth, exhibit low rates of respiration, and maintain elevated sucrose contents. The subsequent slow drying leads to a partial conversion of sucrose into oligosaccharides and the expression of dehydrin transcripts. Slow-dried, desiccation-tolerant somatic embryos have stable membranes, retain their native protein secondary structure, and have a densely packed cytoplasmic glassy matrix. Fast-dried, desiccation-sensitive somatic embryos experience some loss of phospholipids and an increase in free fatty acids. Their proteins show signs of denaturation and aggregation, and the glassy matrix has reduced hydrogen bonding. The reduced conversion of sucrose into oligosaccharides appears not to underlie dehydration injury. Proteins in slow-dried somatic embryos, not pretreated with ABA, also show signs of denaturation, which might be attributed to low sugar contents. We conclude that by reducing cellular metabolism, ABA maintains high sugar contents. These sugars contribute to the stability of membranes, proteins, and the cytoplasmic glassy matrix, whereas slow drying permits a further fine tuning of this stability. Partitioning of endogenous amphiphiles from the cytoplasm into membranes during drying may cause membrane perturbance, although it might confer protection to membranes in the case of amphiphilic antioxidants. The perturbance appears to be effectively controlled in desiccation-tolerant systems but not in sensitive systems, for which we suggest dehydrins are responsible. In this context, the low desiccation tolerance in the presence of ample sugars is discussed.

Viability loss of neem (Azadirachta indica) seeds associated with membrane phase behaviour.

Storage of neem (Azadirachta indica) seeds is difficult because of their sensitivity to chilling stress at moisture contents (MC) > or =10% or imbibitional stress below 10% MC. The hypothesis was tested that an elevated gel-to-liquid crystalline phase transition temperature (Tm) of membranes is responsible for this storage behaviour. To this end a spin probe technique, Fourier transform infrared microspectroscopy, and electron microscopy were used. The in situ Tm of hydrated membranes was between 10 degrees C and 15 degrees C, coinciding with the critical minimum temperature for germination. During storage, viability of fresh embryos was lost within two weeks at 5 degrees C, but remained high at 25 degrees C. The loss of viability coincided with an increased leakage of K+ from the embryos upon imbibition and with an increased proportion of cells with injured plasma membranes. Freeze-fracture replicas of plasma membranes from chilled, hydrated axes showed lateral phase separation and signs of the inverted hexagonal phase. Dehydrated embryos were sensitive to soaking in water, particularly at low temperatures, but fresh embryos were not. After soaking dry embryos at 5 degrees C (4 h) plus 1 d of further incubation at 25 degrees C, the axis cells were structurally disorganized and did not become turgid. In contrast, cells had a healthy appearance and were turgid after soaking at 35 degrees C. Imbibitional stress was associated with the loss of plasma membrane integrity in a limited number of cells, which expanded during further incubation of the embryos at 25 degrees C. It is suggested that the injuries brought about by storage or imbibition at sub-optimal temperatures in tropical seeds whose membranes have a high intrinsic Tm (10-15 degrees C), are caused by gel phase formation.

The competence to acquire cellular desiccation tolerance is independent of seed morphological development.

Acquisition of desiccation tolerance and the related changes at the cellular level in wheat (Triticum aestivum cv. Priokskaya) kernels during normal development and premature drying on the ear were studied using a spin probe technique and low temperature scanning electron microscopy. During normal development, the ability of embryos to germinate after rapid drying and rehydration was acquired after completion of morphological development, which is a few days before mass maturity. The acquisition of desiccation tolerance, as assessed by germination, was associated with an upsurge in cytoplasmic viscosity, the onset of accumulation of protein and oil bodies, and the retention of membrane integrity upon dehydration/rehydration. These features were also used to assess cellular desiccation tolerance in the cases when germination could not occur. Slow premature drying was used to decouple the acquisition of cellular desiccation tolerance from morphogenesis. Upon premature drying of kernels on the ears of plants cut at 5 d after anthesis, desiccation-tolerant dwarf embryos were formed that were able to germinate. When plants were cut at earlier stages poorly developed embryos were formed that were unable to germinate, but cellular desiccation tolerance was nevertheless acquired. In such prematurely dried kernels, peripheral meristematic endosperm cells had already passed through similar physiological and ultrastructural changes associated with the acquisition of cellular desiccation tolerance. It is concluded that despite the apparent strong integration in seed development, desiccation tolerance can be acquired by the meristematic cells in the developing embryo and cambial layer of endosperm, independently of morphological development.