Cylindrospermopsin induces biochemical changes leading to programmed cell death in plants.

In the present study we provide cytological and biochemical evidence that the cyanotoxin cylindrospermopsin (CYN) induces programmed cell death (PCD) symptoms in two model vascular plants: the dicot white mustard (Sinapis alba) and the monocot common reed (Phragmites australis). Cytological data include chromatin fragmentation and the increase of the ratio of TUNEL-positive cells in roots, the latter being detected in both model systems studied. The strongest biochemical evidence is the elevation of the activity of several single-stranded DNA preferring nucleases-among them enzymes active at both acidic and alkaline conditions and are probably directly related to DNA breaks occurring at the initial stages of plant PCD: 80 kDa nucleases and a 26 kDa nuclease, both having dual (single- and double-stranded nucleic acid) specificity. Moreover, the total protease activity and in particular, a 53-56 kDa alkaline protease activity increases. This protease could be inhibited by PMSF, thus regarded as serine protease. Serine proteases are detected in all organs of Brassicaceae (Arabidopsis) having importance in differentiation of specialized plant tissue through PCD, in protein degradation/processing during early germination and defense mechanisms induced by a variety of biotic and abiotic stresses. However, knowledge of the physiological roles of these proteases and nucleases in PCD still needs further research. It is concluded that CYN treatment induces chromatin fragmentation and PCD in plant cells by activating specific nucleases and proteases. CYN is proposed to be a suitable molecule to study the mechanism of plant apoptosis.

Quantification of main bioactive metabolites from saffron (Crocus sativus) stigmas by a micellar electrokinetic chromatographic (MEKC) method.

Saffron is an expensive spice, cultivated in many regions of the world. Its chief metabolites include crocins, which are responsible for the coloring ability, safranal, which is the main essential oil constituent, and picrocrocin which is the main bitter constituent of the spice. A simple micellar capillary electrochromatographic (MEKC) method capable of quantifying all three types of main constituents was established. The pH, sodium dodecyl sulphate (SDS) content and electrolyte concentration of the background electrolyte was optimized. A simple extraction protocol was developed which can extract all metabolites of different polarity from the saffron stigmas. Optimal background electrolyte composed of 20 mM disodium phosphate, 5mM sodium tetraborate, 100 mM SDS, pH was set 9.5. Optimal extracting solvent was the background electrolyte, incubated with the sample for 60 min. The proposed method allows quantification of picrocrocin, safranal, crocetin- Di-(ß-D-gentiobiosyl) ester and crocetin (ß-D-glycosyl)-(ß-D-gentiobiosyl) ester within 17.5 min, with limit of detection values ranging from 0.006 to 0.04 mg/ml, from a single stigma.

Observation of sward destruction caused by irrigation with toxic Microcystis morphospecies containing water in Southern Hungary.

In the summer of 2006 bloom-like phenomenon occurred in a garden pond in Szeged, Southern Hungary. After regular watering of a sward with pond water containing the algal mass, destruction of garden grass occurred. Microcystis aeruginosa, Microcystis viridis, Microcystis ichthyoblabe, and Microcystis wesenbergii were identified by light microscopy in the water sample; microcystin-FR, -LR, -RR and -YR were determined by matrix-assisted laser desorption/ionization--time-of-flight analysis. There was an 80% decrease in the green mass (87% in chlorophyll-content) of the grass in a 1 m² area of the garden irrigated with pond water.

Cylindrospermopsin induces alterations of root histology and microtubule organization in common reed (Phragmites australis) plantlets cultured in vitro.

We aimed to study the histological and cytological alterations induced by cylindrospermopsin (CYN), a protein synthesis inhibitory cyanotoxin in roots of common reed (Phragmites australis). Reed is an ecologically important emergent aquatic macrophyte, a model for studying cyanotoxin effects. We analyzed the histology and cytology of reed roots originated from tissue cultures and treated with 0.5-40 microg ml(-1) (1.2-96.4 microM) CYN. The cyanotoxin decreased root elongation at significantly lower concentrations than the elongation of shoots. As general stress responses of plants to phytotoxins, CYN increased root number and induced the formation of a callus-like tissue and necrosis in root cortex. Callus-like root cortex consisted of radially swollen cells that correlated with the reorientation of microtubules (MTs) and the decrease of MT density in the elongation zone. Concomitantly, the cyanotoxin did not decrease, rather it increased the amount of beta-tubulin in reed plantlets. CYN caused the formation of double preprophase bands; the disruption of mitotic spindles led to incomplete sister chromatid separation and disrupted phragmoplasts in root tip meristems. This work shows that CYN alters reed growth and anatomy through the alteration of MT organization.

Microcystin-LR induces abnormal root development by altering microtubule organization in tissue-cultured common reed (Phragmites australis) plantlets.

Microcystin-LR (MC-LR) is a heptapeptide cyanotoxin, known to be a potent inhibitor of type 1 and 2A protein phosphatases in eukaryotes. Our aim was to investigate the effect of MC-LR on the organization of microtubules and mitotic chromatin in relation to its possible effects on cell and whole organ morphology in roots of common reed (Phragmites australis). P. australis is a widespread freshwater and brackish water aquatic macrophyte, frequently exposed to phytotoxins in eutrophic waters. Reed plantlets regenerated from embryogenic calli were treated with 0.001-40 microg ml(-1) (0.001-40.2 microM) MC-LR for 2-20 days. At 0.5 microg ml(-1) MC-LR and at higher cyanotoxin concentrations, the inhibition of protein phosphatase activity by MC-LR induced alterations in reed root growth and morphology, including abnormal lateral root development and the radial swelling of cells in the elongation zone of primary and lateral roots. Both short-term (2-5 days) and long-term (10-20 days) of cyanotoxin treatment induced microtubule disruption in meristems and in the elongation and differentiation zones. Microtubule disruption was accompanied by root cell shape alteration. At concentrations of 0.5-5 microg ml(-1), MC-LR increased mitotic index at long-term exposure and induced the increase of the percentage of meristematic cells in prophase as well as telophase and cytokinesis of late mitosis. High cyanotoxin concentrations (10-40 microg ml(-1)) inhibited mitosis at as short as 2 days of exposure. The alteration of microtubule organization was observed in mitotic cells at all exposure periods studied, at cyanotoxin concentrations of 0.5-40 microg ml(-1). MC-LR induced spindle anomalies at the metaphase-anaphase transition, the formation of asymmetric anaphase spindles and abnormal sister chromatid separation. This paper reports for the first time that MC-LR induces cytoskeletal changes that lead to alterations of root architecture and development in common reed and generally, in plant cells. The MC-LR induced alterations in cells of an ecologically important aquatic macrophyte can reveal the importance of the effects of a cyanobacterial toxin in aquatic ecosystems.

Microcystin-LR, a cyanobacterial toxin, induces growth inhibition and histological alterations in common reed (Phragmites australis) plants regenerated from embryogenic calli.

The aim of this study was to establish the histological effects of exposure to microcystin-LR (MC-LR), a cyanotoxin, on axenic Phragmites australis plantlets. Plantlets were regenerated from embryogenic reed calli by tissue culture methods. Microcystin-LR inhibited the growth and development of embryogenic calli and the growth of reed plantlets. The 50% plantlet growth inhibitory concentration value (IC50) of MC-LR was 12 microg ml(-1) (12.07 microM) on mineral medium and 36 microg ml(-1) (36.22 microM) on Murashige-Skoog medium. In the case of roots, the IC50 value was 4.1 microg ml(-1) (4.12 microM) on both media. Microcystin-LR induced aerenchyma obturation, altered lignification of cell walls in the axial organs, root necrosis and the capture of lateral or adventitious roots in the tissues of axial organs of reed plantlets. Cyanotoxin induced the premature development of lateral roots, root coalescence and early aerenchyma formation. Our data suggest that microcystin-LR, a cyanotoxin, induced developmental and histological alterations leading to growth inhibition of reed, and the induced harms have an impact on understanding reed decay in eutrophic fresh waters.

Alteration of cylindrospermopsin production in sulfate- or phosphate-starved cyanobacterium Aphanizomenon ovalisporum.

The effect of sulfate and phosphate deprivation on cell growth and cylindrospermopsin level was studied in Aphanizomenon ovalisporum ILC-164. Sulfate starvation induced a characteristic reduction of cylindrospermopsin pool size on the basis of cell number and unit of dry mass of culture. Phosphorous starvation of A. ovalisporum cultures induced a lesser reduction of cylindrospermopsin pool size. This divergence in the pool size of cylindrospermopsin may be the consequence of different growth rate. To show the metabolic changes concomitant with reduction of cylindrospermopsin pool size were obtained by measurement of ATP sulfurylase and alkaline phosphatase activity. The present study is the first concerning the cylindrospermopsin content under sulfate starvation and discusses it in relation to phosphorous starvation.

Interlaboratory comparison trial on cylindrospermopsin measurement.

The hepatotoxin cylindrospermopsin (CYN) is a potent inhibitor of protein synthesis in mammalian cells. It is produced by freshwater cyanobacterial blooms in countries such as Australia, the United States, Israel, Thailand, and Brazil. An interlaboratory comparison was organized as a first step to evaluate the measurement of CYN in lyophilized cyanobacterial cells. Six laboratories from Europe, Israel, and Australia participated in the trial. All of the methods used for extraction of the toxin and the high-performance liquid chromatography (HPLC) analysis were satisfactory on the basis of statistical evaluation, according to ISO standards 5725-1 and -2. Further comparison of all the extraction methods by the organizer indicated that the most effective extraction procedure used 5% formic acid to prevent interference in chromatograms by contaminant compounds when analyzed using HPLC employing isocratic conditions of 5% (v/v) aqueous methanol plus 0.1% (v/v) trifluoroacetic acid as the mobile phase.

Analysis of cyanobacterial toxins (anatoxin-a, cylindrospermopsin, microcystin-LR) by capillary electrophoresis.

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were applied to the simultaneous separation of cyanobacterial toxins (anatoxin-a, microcystin-LR, cylindrospermopsin). The analytical performance data of both methods, optimized for the three toxins, were similar with a precision of migration times smaller than 0.8 RSD% and a detection limit in the range of 1-4 microg/mL, using spectrophotometric detection at 230 nm. Both methods were applied to an analysis of cyanotoxins in water bloom samples and crude cyanobacterial extracts. The results obtained indicate that, for complex matrices, the sequential application of CZE and MEKC is necessary. It is recommended to use both CE techniques for the analysis of the same sample in order to confirm the results by an orthogonal approach.

Capillary electrophoretic assay and purification of cylindrospermopsin, a cyanobacterial toxin from Aphanizomenon ovalisporum, by plant test (blue-green Sinapis test).

Toxic cyanobacteria are known to produce cyanotoxins, toxic secondary metabolites. In recent years the cylindrospermopsin (tricyclic guanidinyl hydroxymethyluracil)-producing organisms Aphanizomenon ovalisporum, Cylindrospermopsis raciborskii, and Umezakia natans have been inhabiting polluted fresh waters. Cylindrospermopsin, a potent hepatotoxic cyanotoxin, has been implicated in cases of human poisoning as well. This study describes the isolation and purification of cylindrospermopsin from A. ovalisporum with the help of a slightly modified Blue-Green Sinapis Test, a plant test suitable for determining the cyanotoxin content of chromatographic fractions besides plankton samples. The recent modification, using microtiter plates for the assay, improves the method and reduces the amount of sample needed for the assay. This approach proved that plant growth and metabolism, at least in the case of etiolated Sinapis alba seedlings, are inhibited by cylindrospermopsin. The establishment of capillary electrophoresis of cylindrospermopsin and consideration of the results reported here lead us to the expectation that capillary electrophoresis of cylindrospermopsin may be a powerful and useful analytical method for investigating cyanobacterial blooms for potential cylindrospermopsin content and toxicity. Confirmation of chemical identity of the purified compound is performed by UV spectrophotometry, NMR, and MALDI-TOF.