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 28-32 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. (C) 2010 Elsevier GmbH. All rights reserved.