Márta M-Hamvas

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.

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.

PepJ is a new extracellular proteinase of Aspergillus nidulans

Under carbon starvation, Aspergillus nidulans released a metallo-proteinase with activities comparable to those of PrtA, the major extracellular serine proteinase of the fungus. The relative molar mass of the enzyme was 19 kDa as determined with both denaturing and renaturing SDS PAGE, while its isoelectric point and pH and temperature optima were 8.6, 5.5 and 65 °C, respectively. The enzyme was stable at pH 3.5–10.5 and was still active at 95 °C in the presence of azocasein substrate. MALDI-TOF MS analysis demonstrated that the proteinase was encoded by the pepJ gene (locus ID AN7962.3), and showed high similarity to deuterolysin from Aspergillus oryzae. The size of the mature enzyme, its EDTA sensitivity and heat stability also supported the view that A. nidulans PepJ is a deuterolysin-type metallo-proteinase.

Extracellular proteinase formation in carbon starving Aspergillus nidulans cultures – physiological function and regulation

Extracellular proteinase formation in carbon depleted cultures of the model filamentous fungus Aspergillus nidulans was studied to elucidate its regulation and possible physiological function. As demonstrated by gene deletion, culture optimization, microbial physiological and enzymological experiments, the PrtA and PepJ proteinases of A. nidulans did not appear to play a decisive role in the autolytic decomposition of fungal cells under the conditions we tested. However, carbon starvation induced formation of the proteinases observable in autolytic cultures. Similar to other degradative enzymes, production of proteinase was regulated by FluG‐BrlA asexual developmental signaling and modulated by PacC‐dependent pH‐responsive signaling. Under the same carbon starved culture conditions, alterations of CreA, MeaB or heterotrimeric G protein mediated signaling pathways caused less significant changes in the formation of extracellular proteinases. Taken together, these results indicate that while the accumulation of PrtA and PepJ is tightly coupled to the initiation of autolysis, they are not essential for autolytic cell wall degradation in A. nidulans. Thus, as Aspergillus genomes contain a large group of genes encoding proteinases with versatile physiological functions, selective control of proteinase production in fungal cells is needed for the improved industrial use of fungi. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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.

Histological, cytological and biochemical alterations induced by microcystin-LR and cylindrospermopsin in white mustard (Sinapis alba L.) seedlings

This study compares the histological, cytological and biochemical effects of the cyanobacterial toxins microcystin-LR (MCY-LR) and cylindrospermopsin (CYN) in white mustard (Sinapis alba L.) seedlings, with special regard to the developing root system. Cyanotoxins induced different alterations, indicating their different specific biochemical activities. MCY-LR stimulated mitosis of root tip meristematic cells at lower concentrations (1 μg ml-1) and inhibited it at higher concentrations, while CYN had only inhibitory effects. Low CYN concentrations (0.01 μg ml-1) stimulated lateral root formation, whereas low MCY-LR concentrations increased only the number of lateral root primordia. Both inhibited lateral root development at higher concentrations. They induced lignifications, abnormal cell swelling and inhibited xylem differentiation in roots and shoots. MCY-LR and CYN induced the disruption of metaphase and anaphase spindles, causing altered cell divisions. Similar alterations could be related to decreased protein phosphatase (PP1 and PP2A) activities in shoots and roots. However, in vitro phosphatase assay with purified PP1 catalytic subunit proved that CYN in contrast to MCY-LR, decreased phosphatase activities of mustard in a non-specific way. This study intends to contribute to the understanding of the mechanisms of toxic effects of a protein phosphatase (MCY-LR) and a protein synthesis (CYN) inhibitory cyanotoxin in vascular plants.

Microcystin-LR, a protein phosphatase inhibitor, induces alterations in mitotic chromatin and microtubule organization leading to the formation of micronuclei in Vicia faba

BACKGROUND AND AIMS Microcystin-LR (MCY-LR) is a cyanobacterial toxin, a specific inhibitor of type 1 and 2A protein phosphatases (PP1 and PP2A) with significant impact on aquatic ecosystems. It has the potential to alter regulation of the plant cell cycle. The aim of this study was improved understanding of the mitotic alterations induced by cyanotoxin in Vicia faba, a model organism for plant cell biology studies. METHODS Vicia faba seedlings were treated over the long and short term with MCY-LR purified in our laboratory. Short-term treatments were performed on root meristems synchronized with hydroxylurea. Sections of lateral root tips were labelled for chromatin, phosphorylated histone H3 and β-tubulin via histochemical and immunohistochemical methods. Mitotic activity and the occurrence of mitotic alterations were detected and analysed by fluorescence microscopy. The phosphorylation state of histone H3 was studied by Western blotting. KEY RESULTS Long-term MCY-LR exposure of lateral root tip meristems increased the percentage of either early or late mitosis in a concentration-dependent manner. We observed hypercondensed chromosomes and altered sister chromatid segregation (lagging chromosomes) leading to the formation of micronuclei, accompanied by the formation of disrupted, multipolar and monopolar spindles, disrupted phragmoplasts and the hyperphosphorylation of histone H3 at Ser10. Short-term MCY-LR treatment of synchronized cells showed that PP1 and PP2A inhibition delayed the onset of anaphase at 1 µg mL(-1) MCY-LR, accelerated cell cycle at 10 µg mL(-1) MCY-LR and induced the formation of lagging chromosomes. In this case mitotic microtubule alterations were not detected, but histone H3 was hyperphosphorylated. CONCLUSIONS MCY-LR delayed metaphase-anaphase transition. Consequently, it induced aberrant chromatid segregation and micronucleus formation that could be associated with both H3 hyperphosphorylation and altered microtubule organization. However, these two phenomena seemed to be independent. The toxin may be a useful tool in the study of plant cell cycle regulation.

Osmotic stress responses of individual white oak (Quercus section, Quercus subgenus) genotypes cultured in vitro

White oaks (Quercus section, Quercus subgenus) are widely distributed in Europe. Quercus petraea (sessile oak), an economically important species is predicted to be affected by climate change. Q. pubescens (pubescent oak) and Q. virgiliana (Italian pubescent oak) are economically less important, drought tolerant species. Frequent hybridization of white oaks was observed and currently the introgression of Q. pubescens and Q. virgiliana in non-mediterranean regions of Europe has been reported. Our goal was to use tissue cultures established from individual trees of the above taxa and their putative hybrids, all present in the forest stand of Síkfőkút LTER Research Area (NE Hungary) as simple experimental model systems for studying drought/osmotic stress tolerance. Tissue cultures are more suitable models for such studies, than seedlings, because they are genetically identical to the parent plants. Polyethylene glycol (PEG6000) treatments were used for this purpose. The identification of taxa was based on leaf morphological traits and microsatellite analysis and showed that Q. petraea is genetically distinct to all other taxa examined. We established six callus lines of Quercus. As expected, in Q. petraea cultures PEG6000 induced severe loss of fresh weight and the ability to recover after removal of the osmoticum, which was not characteristic for Q. pubescens and Q. virgiliana. Putative hybrids exhibited an intermediate response to osmotic stress. Activity gels showed the increase of single-strand preferring (SSP) nuclease and no significant change of guaiacol-peroxidase activities in drought-sensitive genotypes/cultures and no significant increase of SSP nuclease activities accompanied with increases of guaiacol-peroxidase activities in drought-tolerant ones. This indicates that drought/osmotic stress tolerance is associated to increased capacity of scavenging reactive oxygen species and hence less susceptibility to DNA damage. Our results confirm that tissue cultures of oak are suitable model systems for studying drought/osmotic stress responses.