Journal of Plant Physiology 168 (2011) 678–686 Contents lists available at ScienceDirect Journal of Plant Physiology journal homepage: www.elsevier.de/jplph Microcystin-LR induces chromatin alterations and modulates neutral single-strand-preferring nuclease activity in Phragmites australis Katalin Jámbrik, Csaba Máthé ∗ , Gábor Vasas, Dániel Beyer, Erika Molnár, George Borbély, Márta M-Hamvas ∗ Department of Botany, University of Debrecen, Faculty of Science and Technology, P.O. Box 14, H-4010 Debrecen, Hungary article info Article history: Received 21 July 2010 Received in revised form 1 October 2010 Accepted 1 October 2010 Keywords: Chromatin condensation Microcystin-LR Phragmites australis Programmed cell death SSP nuclease abstract 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 28–32 kDa in roots and isoenzyme F of 35–38 kDa 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. © 2010 Elsevier GmbH. All rights reserved. Introduction It has long been known that single-strand-preferring nucle- ases (SSP nucleases) cleave primarily single-stranded DNA (ssDNA) and RNA. Based on the reactions they catalyze, SSP nucleases can be classified into endonucleases (their products are oligo- and/or mono-deoxynucleotides), exonucleases (their products are mononucleotides and require free 3 ′ or 5 ′ termini), and endo- exonucleases (Desai and Shankar, 2003). Well-characterized plant SSP nucleases include mung bean nuclease, Avena leaf nuclease, a tobacco culture nuclease (extracellular enzyme) and a senescence- induced bifunctional nuclease of Arabidopsis. Many plant SSP nucleases are type I nucleases (E.C.3.1.30.X) that are extracellu- lar or intracellular heat-stable glycoproteins with endonucleolytic activity on ssDNA and RNA (Wilson, 1975; Farkas, 1982; Pérez- Abbreviations: dsDNase activity, double-stranded DNA cleaving activity; MCY- LR, microcystin-LR; PCD, programmed cell death; ssDNase activity, single-stranded DNA cleaving activity; SSP nucleases, single-strand-preferring nucleases. ∗ Corresponding authors. Tel.: +36 52 512900; fax: +36 52 512943. E-mail addresses: mathe@tigris.unideb.hu (C. Máthé), hamvasm@tigris.unideb.hu (M. M-Hamvas). Amador et al., 2000). Type I nucleases include cereal enzymes co-localized with chromatin (Yupsanis et al., 1996). Their biolog- ical roles involve DNA replication, recombination and repair, or developmental processes such as seed germination and matura- tion (Desai and Shankar, 2003; Le´ sniewicz et al., 2010). Several ssDNase isoenzymes are induced during drought or oxidative stress, have an important functions in resistance to viral, bacte- rial or fungal pathogens as well as in the induction of senescence and programmed cell death (PCD), e.g., during xylogenesis (Wood et al., 1998; Pérez-Amador et al., 2000; Desai and Shankar, 2003; Le´ sniewicz et al., 2010). Microcystin-LR (MCY-LR) is a well-known cyanobacterial toxin, frequently present in eutrophicated freshwaters (Carmichael, 1992). It is a potent inhibitor of type 1 and 2A protein phosphatases in eukaryotic organisms. Due to this property, the cyanotoxin inter- feres with a wide range of cellular processes, involving cell cycle regulation, signal transduction and the regulation of enzyme activ- ities (MacKintosh and MacKintosh, 1994; Luan, 2003). With respect to vascular plants, a variety of toxin effects have been described. These include histological and cell cytoskeletal alterations (Máthé et al., 2007, 2009; Szigeti et al., 2010), interference with photo- synthesis (Abe et al., 1996), the induction of oxidative stress and of corresponding defense enzyme activities (Pflugmacher, 2004). 0176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.10.007