Degreening of the unicellular alga Euglena gracilis: thylakoid composition, room temperature fluorescence spectra and chloroplast morphology.

Thylakoid dismantling is one of the most relevant processes occurring when chloroplasts are converted to non-photosynthetically active plastids. The process is well characterised in senescing leaves, but other systems could present different features. In this study, thylakoid dismantling has been analysed in dividing cells of the unicellular alga, Euglena gracilis, cultured in darkness. Changes in photosynthetic pigments and in the abundance of LHC and PSII core proteins (D2 and CP43) showed that: (i) during the 0-24 h interval, the decline in LHCII was faster than that in the PSII core; (ii) during the 24-48 h interval, PSII and LHCII were strongly degraded to nearly the same extent; (iii) in the 48-72 h interval, the PSII core proteins declined markedly, while LHCII was maintained. These changes were accompanied by variations in room temperature fluorescence emission spectra recorded from single living cells with a microspectrofluorimeter (excitation, 436 nm; range 620-780 nm). Emission in the 700-715 nm range was proposed to derive from LHCI-II assemblages; changes in emission at 678 nm relative to PSII matched PSII core degradation phases. Overall, the results suggest that, in degreening E. gracilis, thylakoid dismantling is somewhat different from that associated with senescence, because of the early loss of LHCII. Moreover, it is proposed that, in this alga, disruption of the correct LHCI-II stoichiometry alters the energy transfer to photosystems and destabilises membrane appression leading to the thylakoid destacking observed using transmission electron microscopy.

Responses of Trapa natans L. floating laminae to high concentrations of manganese.

The present study focuses on the responses of floating laminae of the Mn-tolerant hydrophyte Trapa natans L. to 1 mM Mn and their ability to accumulate the metal. Studies were carried out first on young floating laminae belonging to the second verticil of 30-day-old plants which originated from fruits that had been maintained in a 1 mM Mn-treated environment and again on the young floating laminae after 10 days of further treatment with 1 mM Mn. Mn storing was observed from the first days after germination, but only 10-day-treated laminae showed the capability to hyperaccumulate the element inside specialised cells (>20000 microg/g [dry weight]). Electron microscopy and the Folin-Ciocalteu reaction for phenolics revealed deposits of chelated material inside vacuoles of the first palisade layer and of idioblasts in the spongy tissue. X-ray microanalysis indicated that the deposits were Mn chelated with phenolic compounds. Numerous trichomes were observed at the lower epidermis of 10-day-treated laminae. They were rich in phenolics and characterised by Mn concretions at their base. As they are associated with a high concentration of the metal in culture water and sediments, trichomes may constitute a morphological differentiation for the secretion of Mn-chelating molecules into the culture water, as a probable "avoidance" mechanism. Finally, monitoring of the photosynthetic apparatus showed that photosynthetic function was not impaired, though differences in development occurred.

Modulations of the thylakoid system in snow xanthophycean alga cultured in the dark for two months: comparison between microspectrofluorimetric responses and morphological aspects.

The response of the plastid was studied, with a special emphasis on thylakoid structure and function, in a snow filamentous xanthophycean alga (Xanthonema sp.) incubated in darkness for two months. Microspectrofluorimetric analyses were performed on single living cells to study the variations in the assembly of the chlorophyll-protein complexes of photosystem II, in comparison with cells grown in light. In parallel, changes in micro- and submicroscopic plastid morphology and in photosynthetic pigment content were monitored. Throughout the experiment, the lamellar architecture of thylakoids in the alga was relatively well preserved, whereas photosystem II underwent disassembly and degradation triggered by prolonged darkness. Conversely, the light-harvesting complex of photosystem II proved to be relatively stable for long periods in darkness. Moreover, a role of the peripheral antennae in determining thylakoid arrangement in xanthophycean algae is implied. Although the responses observed in Xanthonema sp. can be considered in terms of acclimation to darkness, the progressive destabilisation of the light-harvesting complex of photosystem II testifies to incipient ageing of the cells after 35 days.

Adaptive modifications of the photosynthetic apparatus in Euglena gracilis Klebs exposed to manganese excess.

Asynchronous cultures of wild-type Euglena gracilis were tested for their morphophysiological response to 10 mM MnSO4. Growth was only moderately slowed (15%), while oxygen evolution was never compromised. Inductively coupled plasma analyses indicated that the Mn cell content doubled with respect to controls, but no signs of localised accumulation were detected with X-ray microanalysis. Evident morphological alterations were found at the plastid level with transmission electron microscopy and confocal laser scanning microscopy. An increase in the plastid mass, accompanied by frequent aberrations of chloroplast shape and of the organisation of the thylakoid system, was observed. These aspects paralleled a decrease in the molar ratio of chlorophyll a to b and an increase in the fluorescence emission ratio of light-harvesting complex II to photosystem II, the latter evaluated by in vivo single-cell microspectrofluorimetry. These changes were observed between 24 and 72 h of treatment. However, the alterations in the pigment pattern and photosystem II fluorescence were no longer observed after 96 h of Mn exposure, notwithstanding the maintenance of the large plastid mass. The response of the photosynthetic apparatus probably allows the alga to limit the photooxidative damage linked to the inappropriately large peripheral antennae of photosystem II. On the whole, the resistance of Euglena gracilis to Mn may be due to an exclusion-tolerance mechanism since most Mn is excluded from the cell, and the small amount entering the organism is tolerated by means of morphophysiological adaptation strategies, mainly acting at the plastid level.

Specific intra-tissue responses to manganese in the floating lamina of Trapa natans L.

Plant tolerance to heavy metals requires morpho-physiological mechanisms that are still poorly understood, especially in hydrophytes. This study focuses on the young floating lamina of the rhyzophyte Trapa natans exposed for 10 d to 130 microM Mn. The lamina has the ability to bioaccumulate Mn (> 3000 microg g(-1)). X-ray microanalysis of Mn cellular distribution revealed accumulation in the upper epidermis, in the first palisade layer, and in the idioblasts of the spongy tissue, which were shown with electron microscopy to contain osmiophilic vacuolar deposits, also observed to a minor extent in the control leaves. On the basis of biochemical and histochemical tests, these deposits were attributed to phenolic compounds that were probably able to chelate Mn. Net photosynthesis, photosynthetic pigments, room temperature microspectrofluorimetric analyses, and ultrastructural studies of plastids were performed to evaluate the status of the photosynthetic apparatus. A greater development of thylakoid membranes was observed in plastids of the second palisade and spongy tissue, which, however, did not accumulate Mn. Only the spongy tissue experienced inadequate assembly of PS II, but this did not significantly influence the photosynthetic yield of the whole lamina. It was concluded that T. natans can optimise productivity in the presence of Mn by means of specific intra-tissue responses within the framework of the floating lamina.

Carotenoid and ultrastructure variations in plastids of Arum italicum Miller fruit during maturation and ripening.

The changes in the pigment pattern and composition occurring in the Arum italicum berry during the various steps of maturation (ivory to deep-green stages) and ripening (yellow and red-orange stages) were studied and correlated to the ultrastructural modifications of plastids. Transmission electron microscopy showed that each stage was characterized by a specific plastidial type following the unusual sequence amyloplast-->chloroplast-->chromoplast. Plastidial transitions were accompanied by profound modifications in the pigmental composition, in particular, in the pattern of carotenoids and their precursors. The HPLC analysis of the carotenoids showed that, besides the two usual all-trans metabolic pathways leading to lutein through alpha-carotene and to auroxanthin through beta-carotene, an additional cis-isomeric biosynthetic pathway leading to cis-neoxanthin through cis-beta-carotene exists. During the pre-ripening stages, the three pathways were present even if with qualitative and quantitative variations. When complete ripening was reached, a block occurred at the cyclization level causing the accumulation of both all-trans (i.e. gamma-carotene and neurosporene) and cis-isomer (i.e. lycopene and zeta-carotene) carotene precursors. Because of the occurrence of unusual pigments and the presence of the three main plastidial types, the fruit of A. italicum may constitute a most instructive model for the study of carotenogenesis.

Morphogenetic effects induced by spermine and ruthenium red in yeasts.

Spermine (Sp) produces growth inhibition and wall malformation in Saccharomyces cerevisiae in response to oversynthesis of beta-glucans and chitin. The effect is related to the polycation nature of the molecule. In the present work, to verify this hypothesis, the yeast was treated with the abiogenic polycation ruthenium red (RR). The strict analogy observed between the RR- and Sp-induced alterations reinforced our earlier assumption that Sp interacted with the anionic sites of the plasmalemma determining a spurious activation of the two inserted enzymes beta-glucan and chitin synthases. This view was further confirmed by the aberrant accumulation of beta-glucans in Schizosaccharomyces pombe and of chitin in Rhodotorula glutinis treated with Sp and RR. In these micro-organisms spermidine, which bears three amino groups instead of the four encountered in Sp, was ineffective. It is inferred that at least four cation sites must be present in a compound in order to affect wall morphogenesis in yeasts.

Interaction of monensin on dimorphic transition and hyphal growth in Candida albicans.

Monensin, an exocytosis perturber, was assayed on the dimorphic fungus Candida albicans in order to study its effects on wall synthesis and morphogenesis. In the presence of the drug, both germ-tube formation and hyphal elongation were markedly inhibited according to the dose and exposure time. The effect was accompanied by morphogenetic malformations which could be detected under UV light and by transmission electron microscopy. Enormous wall and septa thickenings revealed by cytochemical tests indicated an abnormal deposition of chitin. Since chitin synthase is inserted in the plasma membrane in a latent state and its activation depends on regulatory factors reaching the cell periphery through an orderly exocytosis, it is assumed that monensin affects the vesicular traffic leading to a prevalence of activators over inhibitors of the enzyme.

Stimulation of the autophagic activity in blastospores of Candida albicans exposed in vitro to fluconazole.

Blastospores of Candida albicans were exposed in vitro to fluconazole, a bis-triazole which inhibits ergosterol biosynthesis in fungi by interfering with the cytochrome P-450 dependent 14 alpha-demethylase. Electron microscope examination revealed that a low dose (3 micrograms/ml), short treatment (1-3 h) with this compound greatly stimulated autophagic activity not accompanied by alterations of the cell organelles. Only rarely, wall thickenings and some damage to the membrane system, except for the plasma membrane, was noted. This unusual phenomenon suggests that in the presence of fluconazole, due to the depletion of ergosterol and the consequent accumulation of 14 alpha-methylsterols, changes are induced in the properties of the tonoplast. It seems that in the presence of the molecule the membrane is not able to distinguish what is to be degraded from what is still useful for intracellular metabolism with the consequent disintegration of cell compartmentation. Fluconazole may be useful for indicating the mechanism for controlling lytic activity and the homeostatic role of the vacuole in yeasts.

Effects of spermine and spermidine on cell division and wall morphogenesis in Saccharomyces cerevisiae.

The effect of polyamines (PA) on the synthesis and deposition of wall constituents in Saccharomyces cerevisiae was investigated. Spermidine (Spd) in various doses was ineffective whereas spermine (Sp) in the same concentrations caused a marked inhibition (25-60%) of cell proliferation accompanied by evident morphogenetic malformations. Sp-treated cells were elongated, grouped in small clusters, and showed malformed septa and aberrant wall thickenings. The PATAg technique revealed that the aberrations consisted of an abnormal accumulation of both reactive materials, like 1,3-beta-glucans, and unstained chitin components. Since 1,3-beta-glucan synthases and chitin synthases are inserted in the plasma membrane, whose anionic sites interacted with the cation groups of Sp, it is assumed that the molecule determines a condition resulting in an unregulated activation of the two enzymes. The fact that Spd, which contains three cationic groups instead of the four contained in Sp, is without effect suggests that a compound must have at least four cation sites in order to affect the cell division and wall morphogenesis of S. cerevisiae.

Ultrastructural alterations in Epidermophyton floccosum and Trichophyton mentagrophytes exposed in vitro to protoanemonin.

Protoanemonin (PrA) is an antimicrobial agent whose mechanism of action has not been clearly described. Some evidence exists which indicates that the compound inhibits growth by interacting with microtubules (MTs). In order to confirm this hypothesis, an ultrastructural study was undertaken in vitro on Epidermophyton floccosum and Trichophyton mentagrophytes, two dermatophytes which proved to be sensitive to the drug. A concentration of 1.25 x 10(-4) M (half-MIC dose) for 48 h, caused a series of ultrastructural alterations which were detected by scanning and transmission electron microscopy. Wave-like hyphae with distorted apical tips were frequently observed. Wall formation was variously affected as revealed by the deposition of incomplete septa and the accumulation of lomasome-like infoldings. An extrusion of extraparietal material binding adjacent filaments of the mycelia together was also produced. The nucleoplasm was characteristically crossed by MT-like structures. The possibility exists that PrA exerts its antifungal activity by MT interaction.

Ultraviolet-mediated antimycotic activity of alpha-terthienyl on Microsporum cookei.

Alpha-terthienyl (alpha-T) in the presence of UV-A irradiation reduced the growth rate of Microsporum cookei. In the dark, alpha-T accumulated in small diffuse vacuoles within the hyphae. After UV-A treatment, alpha-T caused damage to the membranes of the nucleus, mitochondria and endoplasmic reticulum. Plasmolytic and autolytic changes occurred resulting in plasma membrane breakage and cell wall aberrations. UV-A activated alpha-T would appear to target membrane proteins.

5-Azacytidine accelerates yeast-mycelium conversion in Candida albicans.

The effect of the DNA demethylating agent 5-azacytidine (5-azaC) on the morphological development of Candida albicans blastospores has been investigated by microscopic observations. It was found that this compound does not produce any morphogenetic effect when the cells are not induced to mycelial form. By contrast, on the induced cells, 5-azaC markedly accelerates the process of germ tube formation. In addition in the treated cells, yeast-mycelium conversion develops synchronously, whereas it is asynchronous and slow in the normal cells. These data indicate that, together with phenotypic modifications, modulation of gene activity by DNA demethylation occurs in Candida albicans during morphogenetic changes.

Antipyretic and antibacterial actions of Teucrium polium (L.).

The antipyretic and antibacterial activities of the ethanolic extract of the flowering tops of Teucrium polium (L.) were been studied. The extract was effective against both yeast and carrageenin pyrexia in rats. It also exhibited a marked antibacterial action against both gram positive and gram negative organisms and was found to be non toxic in acute studies.

Effects of erythromycin on Euglena gracilis as a model for testing the toxicity of antibacterial drugs.

Based on the main physiological and ultrastructural effects induced in Euglena gracilis by erythromycin, one of the least toxic of the commonly used antibiotics that specifically inhibit protein synthesis on 70S ribosomes of prokaryotic organisms, a symptomatologic picture is outlined which could be useful for the preliminary evaluation of the toxicity of antibacterial agents.

Electron microscopic evidence of Phosfon D-induced alterations in the membranes of the dermatophytic fungus Microsporum cookei.

The dermatophytic fungus Microsporum cookei was cultivated for 24 h in the presence of subinhibitory and inhibitory concentrations (50 and 100 micrograms/ml) of Phosfon D, a growth retardant for higher plants also affecting fungal development, and its toxic effects were examined at the ultrastructural level. In both treatments, Phosfon D attacked the membranes, whose structural integrity was clearly compromised with damage of particular severity to mitochondria, nuclei and endoplasmic reticulum. In the instance of fungal growth suppression, the compound also caused plasmolytic and autolytic phenomena, sometimes accompanied by plasma membrane breakages. The submicroscopic effects observed confirm that Prosfon D is an antifungal compound which displays its toxic effects in the area of lipid metabolism, probably preventing the synthesis of fundamental components of the cellular membranes, such as unsaturated fatty acids and sterols.

Submicroscopic morphology of Trichophyton mentagrophytes grown at different temperatures.

Several modifications were observed in Trichophyton mentagrophytes cultivated at 19 degrees and 37 degrees C, i.e. nine degrees below and above the optimum of 28 degrees C. The phenomena included inhibition of the growth rate, changes in the gross aspects of the cultures as well as of the microscopic and submicroscopic morphology of the hyphal cells. At the ultrastructural level, in particular, it was shown that, at the suboptimal temperature, although the organelle structure in both young and aged hyphal cells remained nearly unchanged, unusual bodies of probable storage significance and plasmalemmasomes were formed. At the supraoptimal temperature, the youngest cells showed a normal organization but were richer in glycogen clusters and enveloped by a cell wall thicker than the ones at the optimal condition. In the cells far from the apex, the endomembrane integrity was lost and consequently an autolytic activity occurred. Degradation phenomena were detectable also at cell wall level. The cytological changes observed were tentatively correlated with a possible different sensitivity of the membrane system at the experimented temperature conditions.

Fluorogenic detection of primary amines in plant histochemistry with fluorescamine: a comparative study on the effects of coagulant and non-coagulant fixatives.

The new highly sensitive method of fluorescamine reaction for the topochemical detection of primary amino groups was studied as a substitude of ninhydrin-Schiff's reaction for the localisation of total proteins in plant tissues. The influence of various coagulant and non-coagulant fixatives on the induction of fluorescamine fluorescence was examined: ethanol, formaldehyde gas and solution, glutaraldehyde, acrolein, osmium tetroxide, Bouin, Rossman, Clarke and Zenker's fluids and FMA were employed. It was found that the use of the fluorogenic method is conditioned by the fixative ability to keep the amino groups disposable and by its capability to reduce the natural autofluorescence of plant material. A detailed account of the fixation methodology demonstrated that non-coagulant acrolein and coagulant mercuric chloride are the most promising fixatives for the use of the fluorescamine reaction in plant histochemistry.