Csaba Máthé

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.

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.

Epidermal Pavement Cells of Arabidopsis Have Chloroplasts.

Plastids are multifunctional, pleomorphic organelles of purported endosymbiotic origin that in plants and green algae display a characteristic double membrane envelope ([Wise, 2007][1]). All plastids originate from colorless proplastids, and a simple pigmentation-based classification distinguishes

Determination of phenylethanoid glycosides and iridoid glycosides from therapeutically used Plantago species by CE-MEKC

CE methods are valuable tools for medicinal plant quality management, screening, and analysis. Therefore, the aim of the current study was to optimize and validate a CE‐MEKC method for simultaneous quantification of four chief bioactive metabolites from Plantago species. The two most important secondary metabolite groups were aimed to be separated. Different electrolyte and surfactant types were tested. Surfactant concentration, BGE pH, electrolyte concentration, and buffering capacity were optimized. The final BGE consisted of 15 mM sodium tetraborate, 20 mM TAPS, and 250 mM DOC at pH 8.50. Acceptable precision, good stability, and accuracy were achieved, with high resolution for phenylethanoid glycosides. Analytes were separated within 20 min. The method was shown to be suitable for the quantification of the iridoid glycosides aucubin and catalpol, and the phenylethanoid glycosides acteoside (verbascoside) and plantamajoside from water extracts of different samples. The method was shown to be applicable to leaf extracts of Plantago lanceolata, Plantago major, and Plantago asiatica, the main species with therapeutic applications, and a biotechnological product, plant tissue cultures (calli) of P. lanceolata. Baseline separation of the main constituents from minor peaks was achieved, regardless of the matrix type.

Microcystin-LR induces chromatin alterations and modulates neutral single-strand-preferring nuclease activity in Phragmites australis

Microcystin-LR (MCY-LR), a toxin produced mainly by freshwater cyanobacteria, is a potent inhibitor of type 1 and 2A protein phosphatases. As such, it induces biochemical, cellular and tissue alterations in vascular plants, including cell death. The aim of this study was the analysis of MCY-LR induced changes in the activity of single-strand preferring nuclease (SSP nuclease) isoenzymes that are possibly involved in programmed cell death (PCD) of Phragmites australis (common reed, an aquatic macrophyte) cells. We analyzed both single-stranded DNA (ssDNase) and double-stranded DNA (dsDNase) cleaving activities. Activity gels revealed a number of seven isoenzymes named bands A-G in control reed shoots and roots. Their activity was organ- and age-dependent. We stained nuclei of root tip meristematic cells and found total and marginal chromatin condensations at relatively short-term (2-10 days) cyanotoxin exposure. At 10-20 days of cyanotoxin treatment, the number of cells with condensed chromatin decreased, which coincided with the occurrence of necrotic cell death. In parallel, overall ssDNase activity increased in the short term (five days) and gradually decreased at 10-20 days of MCY-LR treatment. In this context, the most important changes occurred for isoenzyme G of 28-32kDa in roots and isoenzyme F of 35-38kDa in shoots. dsDNase activity of isoenzyme E was decreased by MCY-LR in shoots, but increased in roots at 10 days of exposure. We conclude that the early induction of chromatin condensation and increase of SSP nuclease activities is related to PCD that will lead to necrosis with the cease of all cellular activities, including a decrease in nuclease activity.

Isolation of viable cell mass from frozen Microcystis viridis bloom containing microcystin-RR.

Cyanobacterial species commonly occur in the phytoplankton of freshwater lakes and sometimes develop as toxin-producing blooms. Microcystis is one of the most common genera of freshwater cyanobacteria and is often the dominating phytoplankton of eutrophic lakes all over the world. In eutrophic lakes, large amounts of Microcystis may overwinter in the sediment and re-inoculate the water column in spring. In most cases, the overwintering pelagic population—if it exists—is small, and its role in re-inoculation has not been clear yet. In December 2005, we found large amounts of Microcystis on the surface, frozen in the ice cover in a eutrophic pond (Pond Hármashegy, Hungary). We identified the Microcystis species and investigated the viability and the toxicity of the frozen cells. The dominant species in the bloom samples was Microcystis viridis. Viability tests showed that the colonies isolated from the ice cover were composed of living cells. The isolated strain was found toxic, we analyzed the microcystin composition in the frozen planktonic Microcystis mass; in the investigated samples microcystin-RR was the main cyanotoxin.