Gábor Vasas

Microcystin-LR alters the growth, anthocyanin content and single-stranded DNase enzyme activities in Sinapis alba L. seedlings

Seedlings of the white mustard (Sinapis alba L.) are sensitive to the cell-free extracts of a toxigenic strain of Microcystis aeruginosa and to microcystin-LR. Fresh mass of plants, plant length, including hypocotyl and root length and lateral root formation is inhibited in microcystin-LR treated seedlings. The decrease of anthocyanin content is obtained in microcystin treated mustard cotyledons. The tissue necrosis of cotyledons is a characteristic consequence of microcystin treatment. Microcystin-LR induces an increase in single stranded deoxyribonucleases (ssDNases) activity of S. alba seedlings as shown by spectrophotometric assays and by ssDNase activity polyacrylamide gels. The significance of this phenomenon is discussed in relation to general stress responses in plants. We conclude that microcystin-LR affects the whole physiology and the growth of plants.

Comparative study of cyanotoxins affecting cytoskeletal and chromatin structures in CHO-K1 cells

In this study we compared the effects of the two frequently occuring and most dangerous cyanobacterial toxins on the cellular organization of microfilaments, microtubules and on the chromatin structure in Chinese hamster ovary (CHO-K1) cells. These compounds are the widely known microcystin-LR (MC-LR) and cylindrospermopsin (CYN) classified as the highest-priority cyanotoxin. Toxic effects were tested in a concentration and time dependent manner. The hepatotoxic MC-LR did not cause significant cytotoxicity on CHO-K1 cells under 20 microM, but caused apoptotic changes at higher concentrations. Apoptotic shrinkage was associated with the shortening and loss of actin filaments and with a concentration dependent depolymerization of microtubules. No necrosis was observed over the concentration range (1-50 microM MC-LR) tested. Cylindrospermopsin did cause apoptosis at low concentrations (1-2 microM) and over short exposure periods (12h). Necrosis was observed at higher concentrations (5-10 microM) and following longer exposure periods (24 or 48h). Cyanotoxins also affected the chromatin structure. The condensation process was inhibited by MC-LR at a later stage and manifested as broken elongated prechromosomes. CYN inhibited chromatin condensation at the early fibrillary stage leading to blurred fluorescent images of apoptotic bodies and preventing the formation of metaphase chromosomes. Cylindrospermopsin exhibited a more pronounced toxic effect causing cytoskeletal and nuclear changes as well as apoptotic and necrotic alterations.

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.

Phycogeography of freshwater phytoplankton: traditional knowledge and new molecular tools

Abstract“Everything is everywhere, but environments selects.” Is this true? The cosmopolitan nature of algae, including phytoplankton, has been highlighted in many textbooks and burnt into the minds of biologists during their studies. However, the accumulating knowledge on the occurrence of individual phytoplankton species in habitats where they have not been seen before, reports on invasive phytoplankton species, and the increasing number of papers with phylogenetic trees and tracing secondary metabolites, especially cyanotoxins, contradict. Phytoplankton species, with rare exceptions, are neither cosmopolitan, nor ubiquists. In this review paper, we provide an overview of the basic patterns and the processes relevant for biogeography of freshwater phytoplankton. The following topics are considered: dispersal agents and distances; survival strategies of species; geographic distribution of different types; patterns of invasions; tools of molecular genetics; and metabolomics to explore dispersal patterns, island biogeography, and associated species–area relationships for algae.

Microcystin-LR and Cylindrospermopsin Induced Alterations in Chromatin Organization of Plant Cells

Cyanobacteria produce metabolites with diverse bioactivities, structures and pharmacological properties. The effects of microcystins (MCYs), a family of peptide type protein-phosphatase inhibitors and cylindrospermopsin (CYN), an alkaloid type of protein synthesis blocker will be discussed in this review. We are focusing mainly on cyanotoxin-induced changes of chromatin organization and their possible cellular mechanisms. The particularities of plant cells explain the importance of such studies. Preprophase bands (PPBs) are premitotic cytoskeletal structures important in the determination of plant cell division plane. Phragmoplasts are cytoskeletal structures involved in plant cytokinesis. Both cyanotoxins induce the formation of multipolar spindles and disrupted phragmoplasts, leading to abnormal sister chromatid segregation during mitosis. Thus, MCY and CYN are probably inducing alterations of chromosome number. MCY induces programmed cell death: chromatin condensation, nucleus fragmentation, necrosis, alterations of nuclease and protease enzyme activities and patterns. The above effects may be related to elevated reactive oxygen species (ROS) and/or disfunctioning of microtubule associated proteins. Specific effects: MCY-LR induces histone H3 hyperphosphorylation leading to incomplete chromatid segregation and the formation of micronuclei. CYN induces the formation of split or double PPB directly related to protein synthesis inhibition. Cyanotoxins are powerful tools in the study of plant cell organization.

Cylindrospermopsin and microcystin-LR alter the growth, development and peroxidase enzyme activity of white mustard (Sinapis alba L.) seedlings, a comparative analysis

This work focuses on the comparative analysis of the effects of two cyanobacterial toxins of different chemical structure cylindrospermopsin (CYN) and microcystin-LR (MC-LR) on the white mustard (Sinapis alba L.) seedlings. Both cyanotoxins reduced significantly the fresh mass and the length of cotyledons, hypocotyls and main roots of seedlings in a concentration dependent manner. For various mustard organs the 50% inhibitory concentration values (IC50) of growth were between 3-5 μg ml(-1) for MC-LR and between 5-10 μg ml-1 for CYN, respectively. Cyanotoxins altered the development of cotyledons, the accumulation of photosynthetically active pigments and anthocyanins. Low MC-LR concentrations (0.01 and 0.1 μg ml(-1)) stimulated anthocyanin formation in the cotyledons but higher than 1 μg ml(-1) MC-LR concentrations strongly inhibited it. The CYN treated chlorotic cotyledons were violet coloured in consequence of high level of anthocyanins, while MC-LR induced chlorosis was accompanied by the appearance of necrotic patches. Necrosis and increases of peroxidase enzyme activity (POD) are general stress responses but these alterations were characteristic only for MC-LR treated mustard plants. These findings provide experimental evidences of developmental alterations induced by protein synthesis and protein phosphatase inhibitory cyanotoxins (CYN and MC-LR) in a model dicotyledonous plant.

Appearance of Planktothrix rubescens Bloom with [D-Asp3, Mdha7]MC–RR in Gravel Pit Pond of a Shallow Lake-Dominated Area

Blooms of toxic cyanobacteria are well-known phenomena in many regions of the world. Microcystin (MC), the most frequent cyanobacterial toxin, is produced by entirely different cyanobacteria, including unicellular, multicellular filamentous, heterocytic, and non-heterocytic bloom-forming species. Planktothrix is one of the most important MC-producing genera in temperate lakes. The reddish color of cyanobacterial blooms viewed in a gravel pit pond with the appearance of a dense 3 cm thick layer (biovolume: 28.4 mm3 L−1) was an unexpected observation in the shallow lake-dominated alluvial region of the Carpathian Basin. [d-Asp3, Mdha7]MC–RR was identified from the blooms sample by MALDI-TOF and NMR. Concentrations of [d-Asp3, Mdha7]MC–RR were measured by capillary electrophoresis to compare the microcystin content of the field samples and the isolated, laboratory-maintained P. rubescens strain. In analyzing the MC gene cluster of the isolated P. rubescens strain, a deletion in the spacer region between mcyE and mcyG and an insertion were located in the spacer region between mcyT and mcyD. The insertion elements were sequenced and partly identified. Although some invasive tropical cyanobacterial species have been given a great deal of attention in many recent studies, our results draw attention to the spread of the alpine organism P. rubescens as a MC-producing, bloom-forming species.

Effect of High Relative Humidity on Dried Plantago lanceolata L. Leaves during Long-term Storage: Effects on Chemical Composition, Colour and Microbiological Quality

INTRODUCTION Modern phytotherapy and quality assurance requires stability data on bioactive metabolites to identify and minimise decomposing factors during processing and storage. A compounds stability in a complex matrix can be different from the stability of the purified compound. OBJECTIVE To test the stability of iridoids and acteoside and quantify changes in colour and microbiological quality in a common herbal tea, dried P. lanceolata leaves during exposure to high-humidity air. To test the contribution of fungi to metabolite decomposition. METHODOLOGY Dried P. lanceolata leaves were exposed to atmospheres of different relative humidity (75, 45 and 0%) for 24 weeks. Changes in aucubin and catalpol concentration were determined by CE-MEKC, and those in acteoside on TLC. Colour and chlorophyll-like pigments were measured by different spectrophotometric methods. The number of fungi was monitored; 10 strains were isolated from the plant drug, and their ability to decompose the analytes of interest was tested. RESULTS During incubation at 75% relative humidity (RH), aucubin, catalpol and acteoside concentrations decreased by 95.7, 97.0 and 70.5%, respectively. Strong shifts were detected in CIELAB parameters a* and b* (browning) as a result of conversion of chlorophyll to pheophytin. Intensive microbial proliferation was also observed. Changes at 45 or 0% RH were typically insignificant. Seven of the 10 isolated fungal strains could decompose both iridoids, and five could decompose acteoside in vitro. CONCLUSION It was shown that exposure to water results in loss of bioactive molecules of P. lanceolata dried leaves, and that colonising fungi are the key contributors to this loss.

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-(β-D-gentiobiosyl) ester and crocetin (β-D-glycosyl)-(β-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.