Aquatic Toxicology 92 (2009) 122–130
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/aquatox
Microcystin-LR induces abnormal root development by altering microtubule
organization in tissue-cultured common reed (Phragmites australis) plantlets
Csaba Máthéa,∗, Dániel Beyera, Ferenc Erd˝ odib, Zoltán Serf˝ oz˝ oe, Lóránt Székvölgyic, Gábor Vasasa,
Márta M-Hamvasa, Katalin Jámbrika, Sándor Gondaa, Andrea Kissb, Zsuzsa M. Szigetid, Gyula Surányia
aUniversity of Debrecen, Department of Botany, Faculty of Science and Technology, PO Box 14, H-4010 Debrecen, Hungary
bUniversity of Debrecen, Department of Medical Chemistry and Cell Biology and Signaling Research Group of the Hungarian Academy of Sciences,
Research Center for Molecular Medicine, Medical and Health Science Center, H-4012 Debrecen, Hungary
cUniversity of Debrecen, Department of Biophysics and Cell Biology, Medical and Health Science Center, H-4032 Debrecen, Hungary
dUniversity of Debrecen, Institute of Food Processing, Quality Control and Microbiology, Center of Agricultural Sciences, Böszörményi út 138, H-4032 Debrecen, Hungary
eUniversity of Debrecen, Department of Experimental Zoology, Balaton Limnological Research Institute, Hungarian Academy of Sciences,
Klebelsberg Kuno u. 3, H-8237 Tihany, Hungary
a r t i c l ei n f o
Received 12 January 2009
Received in revised form 3 February 2009
Accepted 8 February 2009
a b s t r a c t
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
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?gml−1(0.001–40.2?M) MC-LR for 2–20 days. At
0.5?gml−1MC-LR and at higher cyanotoxin concentrations, the inhibition of protein phosphatase activ-
ity by MC-LR induced alterations in reed root growth and morphology, including abnormal lateral root
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
and cytokinesis of late mitosis. High cyanotoxin concentrations (10–40?gml−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?gml−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.
© 2009 Elsevier B.V. All rights reserved.
Microcystin-LR (MC-LR) is a widely occurring heptapeptide
toxin produced by several freshwater cyanobacterial genera
indole; MAP, microtubule-associated protein; MC-LR, microcystin-LR; MT, micro-
tubule; PP1, type 1 serine–threonine protein phosphatase; PP2A, type 2A
serine–threonine protein phosphatase; PPB, preprophase band.
∗Corresponding author. Tel.: +36 52 512 900; fax: +36 52 512 943.
E-mail address: email@example.com (C. Máthé).
CMT, cortical microtubule; DAPI, 4?,6?-diamidino-2-phenyl-
(Carmichael, 1992). Many studies have reported that mass devel-
opment of toxic cyanobacteria called water blooming has impacts
on aquatic ecosystems and adverse effects on animal and human
health as well (see for example Codd et al., 2005; Falconer, 1993;
Falconer et al., 1999). MC-LR is a member of the microcystin
family that inhibits type 1 and 2A serine–threonine protein phos-
phatases (PP1 and PP2A) of eukaryotic cells with similar potency
(MacKintosh and Diplexcito, 2003; MacKintosh and MacKintosh,
1994). PP1 and PP2A are involved in the regulation of key enzymes
of carbon and nitrogen metabolism in plants and play a crucial
role in the regulation of cell cycle and plant development (Luan,
2003; Smith and Walker, 1996). Due to its biochemical effects,
0166-445X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
C. Máthé et al. / Aquatic Toxicology 92 (2009) 122–130
MC-LR is currently used for the study of the influence of protein
dephosphorylation on animal and plant cell structure, function
and metabolism (MacKintosh and MacKintosh, 1994; Pflugmacher,
2002; Smith and Walker, 1996). Several plant systems, including
rice, white mustard (Sinapis alba), runner bean (Phaseolus vulgaris)
and common reed (Phragmites australis) are available for the study
of the uptake and effects of MC-LR on plant physiological events
(Abe et al., 1996; Chen et al., 2004; Kurki-Helasmo and Meriluoto,
1998; M-Hamvas et al., 2003; Máthé et al., 2007). It is known that
the cyanotoxin induces stress responses at the biochemical level
in higher plants. Such responses include the increase of single-
stranded DNase and peroxidase activities (M-Hamvas et al., 2003;
chromatin structure by MC-LR as well as the apoptogenic effects of
the cyanotoxin are well documented (see for example, Fladmark
et al., 1999; McDermott et al., 1998; Toivola and Eriksson, 1999). In
organization in plant cells has not been analyzed in detail.
The organization of microtubular cytoskeleton in the cells of
higher plants bears several particularities among eukaryotic cells
(Barlow and Baluˇ ska, 2000). The microtubule system consists of
cortical arrays in interphase meristematic cells and differentiated
cells. Mitosis starts with the formation of preprophase band (PPB)
peculiar to plant cells. PPB probably influences the plane of cell
division. At late prophase, formation of mitotic spindle begins and
directs chromosome alignment and chromatid separation during
assembly of spindle structure, phragmoplast structures develop.
Such MT organization is particular to plant cells, and it is impor-
tant for the formation of cell plate and subsequent new cell wall
development during cytokinesis (Barlow and Baluˇ ska, 2000). MT
dynamics in the cell cycle and in non-dividing cells is subject to
fine regulation directed by many factors including microtubule-
be related to specific changes in chromatin organization (Ayaydin
et al., 2000; Barlow and Baluˇ ska, 2000; Baskin and Wilson, 1997;
Common reed (P. australis) is a widespread emergent aquatic
macrophyte. It is important for wetland ecology due, among oth-
ers, to its effect of diminishing freshwater pollution by its tolerance
to heavy metals (Pflugmacher, 2002; Ye et al., 1998). Little is known
about its cytology and in particular, the changes at cellular levels
induced by adverse environmental factors in this plant. Due to the
major ecological importance of P. australis, the decline of its pop-
ulations in Europe has attracted the attention of many researchers
uptake (Pflugmacher et al., 2001). In a previous study (Máthé et al.,
2007), we added purified MC-LR to the culture media for embryo-
genic callus-derived reed plantlets. We excluded any additional
experiments were carried out under axenic conditions. We have
proved that tissue cultures of P. australis are a good model experi-
mental system for the study of growth inhibitory and histological
alterations induced by the protein phosphatase inhibitor, MC-LR
(Máthé et al., 2000, 2007).
Our goal was to understand MC-LR toxicity at the cellular level
in higher plants and among them, aquatic macrophytes that are
subject to cyanotoxin exposure in the environment. In the present
induced changes of microtubule and mitotic chromatin organi-
zation in P. australis roots. For the morphological and cytological
studies, we have chosen the roots of reed plantlets originating from
tissue culture, because out of embryogenic calli, shoots and roots,
the latter system proved to be the most sensitive to microcystin as
judged from growth inhibition and histological alterations (Máthé
et al., 2007). Many aspects of the cytological effects of MC-LR in
plants are reported here for the first time and the possible cellular
mechanisms related to those alterations are discussed.
2. Materials and methods
2.1. The purification of MC-LR
MC-LR was purified from Microcystis aeruginosa BGSD243 iso-
et al. (1995) with slight modifications. Briefly, after cell extraction,
we performed ion-exchange chromatography on DEAE cellulose
(DE-52, Whatman) followed by desalting with Waters Sep-Pak®
cartridges according to Kós et al. (1995). The purity of MC-LR
used in this study was of NMR grade and checked by HPLC and
capillary electrophoresis methods as described by Vasas et al.
2.2. Plant material and cyanotoxin treatments
Common reed (P. australis/Cav./Trin. Ex Steud.) plantlets were
produced from stem node derived embryogenic calli as previously
described (Máthé et al., 2000). MC-LR treatments of reed plantlets
were performed as described for histological investigations (Máthé
et al., 2007). Briefly, cyanotoxin was added to 2ml liquid MS
basal medium (Murashige and Skoog, 1962) supplemented with
2% (w/v) sucrose (Reanal, Budapest, Hungary), Gamborg’s vitamins
(Gamborg et al., 1968) and 2.7?M ?-naphtaleneacetic acid, a syn-
of root growth. For the study of growth, morphology and cytology,
cyanotoxin treatments were applied in a concentration range of
0.1–40?gml−1(0.1–40.2?M) and for a time interval of 2–20 days.
that developed after the start of cyanotoxin treatment. At least four
independent measurements were performed for each exposure
results plotted with the aid of Sigma Plot®8.0 software. All stud-
ies, including those for mitotic index, chromatin and microtubule
labelling were performed in the absence of ?-naphtaleneacetic
acid as well (data not shown) in order to assure that auxin
does not influence MC-LR induced root growth or cytological
2.3. The assay of protein phosphatase activity
Reed plantlets were exposed for 10 days to 0, 0.001, 0.01, 0.1,
1 and 10?gml−1(0.001–10.05?M) MC-LR. Roots were detached
from plantlets, thoroughly washed in MS medium, then frozen in
liquid nitrogen followed by grounding to a fine powder in a pre-
frozen mortar and pestle. Samples were homogenized on ice in a
buffer containing 50mM Tris–HCl (pH 7.5), 0.1mM EDTA, 0.2mM
EGTA, 0.1% (w/v) DTT and 0.2mM PMSF (all chemicals were from
Sigma–Aldrich, Budapest, Hungary). Homogenates were rapidly
frozen in liquid nitrogen. After thawing, they were centrifuged at
10,000×g for 10min at 4◦C. Supernatants were assayed for protein
content by the method of Bradford (1976). The protein phosphatase
assay mixture consisted of the supernatant containing 10?g pro-
tein of cell lysates and 2?M [32P]-myosin-light chain substrate.
The phosphorylated 20kDa myosin-light chain of smooth muscle
was used as substrate because it is known to be dephosphory-
lated primarily by both PP1 and PP2A in cell extracts (Erd˝ odi et
al., 1995). The release of [32Pi] was measured according to Erd˝ odi
et al. (1995). Protein phosphatase activity (pmol [32Pi] released
mgprotein−1) of the cell extract was calculated and plotted against
C. Máthé et al. / Aquatic Toxicology 92 (2009) 122–130
ware. The phosphatase activity of control root extracts was taken
2.4. Cytological methods: the study of microtubule and mitotic
chromatin organization in P. australis root cells
The direct immunolabelling of MTs by a Cy3-conjugated anti-?
tubulin antibody (Sigma, Budapest, Hungary) and the subsequent
staining of chromatin with 4?,6?-diamidino-2-phenylindole (DAPI,
Fluka, Buchs, Switzerland) were performed according to the man-
ufacturers’ instructions adapted to plant cells (Zhang et al., 1992),
and modified accordingly in our laboratory. The procedure was as
follows. For cytological and root morphology studies, reed roots
(PBS). In order to assure microtubule integrity in the vacuolized
cells from the elongation and differentiated root developmental
zones, fixed roots were treated with 40% (w/v) sucrose (Reanal,
Budapest, Hungary) dissolved in PBS, followed by cryosection-
ing with a Leica Jung Histoslide 2000 microtome. Ten to 15?m
thick sections were subjected to direct microtubule labelling, fol-
lowed by chromatin staining. Sections were washed with PBS
and then cells were permeabilized by 10min treatment with PBS
containing 0.5% (v/v) Triton X-100 (Reanal, Budapest, Hungary).
After three washes for 5min, MTs were labelled for 16h with
the antibody diluted with PBS containing 1% (w/v) bovine serum
albumin (Sigma–Aldrich, Budapest, Hungary). Labelling was fol-
lowed by washing with PBS and staining with 3?gml−1DAPI
for 40min. Sections washed with PBS were mounted on micro-
scopic slides in a drop of antifading buffer containing 0.1% (w/v)
p-phenylenediamine (Sigma–Aldrich, Budapest, Hungary) in 90%
(v/v) glycerol (Reanal, Budapest, Hungary), with pH adjusted to
8.0. Microscopic examinations were carried out with an Olym-
pus Provis AX-70/A fluorescence microscope equipped with an
Olympus Camedia 4040 digital camera. Microtubules were visu-
alized with the aid of a 540–580nm excitation filter, while nuclear
DNA was observed with the aid of a 320–360nm excitation fil-
ter. To obtain a better resolution of microtubule and chromatin
structures, we used a Zeiss LSM 510 confocal laser scanning
microscope. Excitation wavelengths were 543 and 351/364nm,
respectively. Fluorescence emission was detected through 560–615
and 385–470nm band-pass filters. Images were taken in multi-
track mode to prevent cross-talk between the channels. 512×512
pixel image stacks of 1.0–1.5?m thick optical sections were
obtained with a 63× Plan-Apochromat oil immersion objective (NA
For the calculation of total mitotic indices and the percentage of
cells in certain mitotic phases, we used samples labelled with anti-
? tubulin and subsequently stained with DAPI. At least 500 cells
were examined per root section and at least six roots were exam-
root tip meristems were excluded when the percentage of dividing
cells was investigated. Due to the small size of reed chromosomes,
the microtubule organization as well (the presence of PPBs and late
prophase spindles, metaphase and anaphase spindles and phrag-
moplasts). The same principles were applied when we counted the
percentage of mitotic anomalies out of total mitotic cells.
For the study of MT alterations in interphase meristematic or
differentiated cells, we took into consideration the percentage of
Fig. 1. The effect of microcystin-LR (MC-LR) on root growth, morphology and protein phosphatase activity of Phragmites australis roots regenerated from embryogenic calli.
(a) The time- and concentration-dependent effects of MC-LR on primary root growth: control (black circles), 0.5?gml−1MC-LR (open circles), 2.5?gml−1MC-LR (open
triangles), 5?gml−1MC-LR (black squares), 10?gml−1MC-LR (open squares). (b) The effect of MC-LR on the protein phosphatase activity of root extracts. (c) Control primary
root. (d) Early and fused lateral roots (arrowheads) from plant treated for 5 days with 0.5?gml−1MC-LR. (e) Formation of a callus-like tissue from rhizodermis and outer
cortical cells of thin primary roots from plants exposed to 5?gml−1MC-LR for 20 days. (f) Radial swelling of primary root exposed to 20?gml−1MC-LR for 20 days. Bars for
c–f: 400?m. (g) Early lateral root (arrowhead) with radial swelling of cells in the elongation and root hair zone from plant exposed to 20?gml−1MC-LR for 20 days. Bar:
C. Máthé et al. / Aquatic Toxicology 92 (2009) 122–130
roots that contained those anomalies. Sections of at least 10 roots
were investigated for each treatment.
All cytological experiments were repeated at least four times,
standard errors were calculated and results were plotted with the
aid of Sigma Plot®8.0 software.
3.1. The effect of microcystin-LR on P. australis root growth,
morphology and protein phosphatase activity
MC-LR inhibited reed root growth in a time- and dose-
dependent manner (Fig. 1a). At short-term (5 days) of exposure,
10?gml−1MC-LR significantly reduced root length, and as expo-
sure time increased, lower cyanotoxin concentrations were also
able to induce growth inhibition. Finally at 20 days exposure, con-
inhibition of root growth (Fig. 1a). Starting from 0.001?gml−1
(1nM) MC-LR, 10 days of cyanotoxin treatment inhibited signif-
icantly the total PP1 and PP2A activity of reed roots (Fig. 1b).
Inhibition of protein phosphatase activity by 50% (IC50) occurred
at 0.1?gml−1MC-LR. At 0.5?gml−1MC-LR the enzyme activ-
ity was 38% of controls (inhibition of PP1 and PP2A by 62%).
10?gml−1MC-LR inhibited total PP1 and PP2A activity by 80%
We observed characteristic alterations of root morphology of
plantlets treated with the cyanotoxin. Starting from 0.5?gml−1
primordia appeared at 1.5–2.0mm distance from root tip, a posi-
tion where usually the elongation zone of roots develop. Control
primary roots did not develop lateral roots at such a distance from
root tip (Fig. 1c and d). We observed this phenomenon at MC-LR
treatments in the presence as well as the absence of ?-naphtalene
bility that exogenous auxin could induce the fusion of lateral roots.
At long-term (20 days) of 5?gml−1MC-LR treatment, the radial
swelling of rhizodermis and adjacent cortical cells were clearly vis-
ible in primary roots (Fig. 1e). Starting from short-term (5 days) of
phenomenon extended to all developing rhizodermis and cortical
generalization of altered anisotropic cell growth led to the devel-
opment of deformed, swollen primary and lateral roots (Fig. 1f and
3.2. The effect of MC-LR on microtubule organization in
interphase cells of primary and lateral P. australis root meristems
and in differentiating root tissues
Control interphase meristematic cells showed normal cortical
microtubule (CMT) structure (Fig. 2c). MC-LR treatment caused
the disruption of CMTs in a time- and concentration-dependent
manner. Such alteration occurred in a significant percentage of
primary reed root tips (Fig. 2a). Those changes were detected
as diffuse labelling of cells with anti-? tubulin and the lack
of MTs (Fig. 2d). At short-term (2–5 days) of cyanotoxin expo-
sure, those alterations were observed at high concentrations
(10–40?gml−1), while starting from 10 days of treatment, they
could be detected at low concentrations, too (1?gml−1MC-LR,
Fig. 2a and d).
The MT organization of lateral root meristems and differenti-
ating cells was normal in controls (Fig. 2e). MC-LR induced MT
disorganization in laterals, as shown by the disappearance of MTs
and high background tubulin labelling in the tips as well as dif-
ferentiating lateral root cells (Fig. 2f). This phenomenon could be
observed after as early as 2 days of treatment with the cyanotoxin,
at concentrations of 5?gml−1and above, and at higher expo-
sure times it was concomitant with the appearance of radial cell
swelling and irregularly shaped laterals (Figs. 1g, and 2b and f).
Mitotic anomalies could be detected. The mitotic alterations
were similar to those observed in the tips of primary roots
and will be presented at the next section of this study (Section
In the elongation and differentiated zones of primary roots
treated with 1?gml−1or higher MC-LR concentrations, we
detected the alteration of CMT organization in cortex cells at all
time intervals studied (Fig. 2b). In contrast to normal CMT in con-
of cell elongation and radial swelling (Fig. 2i).
3.3. The effect of microcystin-LR on mitosis and mitotic
alterations in reed root cells
MC-LR induced characteristic changes in the mitotic activity of
reed primary root meristematic cells as shown by the calculation
of mitotic indices. At 2–10 days of treatment, only concentrations
of 10?gml−1cyanotoxin and above induced significant alterations
in cell division activity, that is, the inhibition of mitosis (Fig. 3a).
In contrast, at long-term (20 days) exposure, MC-LR induced a
significant increase of mitotic index at a concentration range of
0.1–5?gml−1, while at 20–40?gml−1it decreased mitotic activ-
ity of meristematic cells (Fig. 3a). In an attempt to understand
more precisely the mitotic changes induced, we analyzed the dis-
tinct mitotic phases separately. At short-term (5 days) of MC-LR
exposure, 1?gml−1MC-LR induced the increase of the percent-
age of cells in early mitosis (data not shown). Twenty days of
0.5–10?gml−1MC-LR treatment induced the increase of the fre-
quency of late mitotic (telophase) and cytokinetic cells, as shown
by the increase in the number of cells with telophase/cytokinetic
and cytokinetic cells was at 1?gml−1cyanotoxin (Fig. 3b, black
circles). At the same time interval the occurrence of high per-
centage of early mitotic cells was characteristic for 1–5?gml−1
MC-LR concentration, with the highest occurrence of such cells at
cells in early mitosis, when they contained PPBs, prophase spin-
dles or microtubular and chromatin organization characteristic for
With the aid of microscopic analysis of chromatin and MT orga-
nization, we detected characteristic mitotic anomalies in MC-LR
treated primary root meristems. At short-term (2 days) expo-
sure, such anomalies were detected starting from 1?gml−1MC-LR
and culminated at 40?gml−1cyanotoxin (Fig. 3c, black circles).
At 5–20 days of treatment, we could detect abnormal mitosis
starting from 0.5?gml−1MC-LR with the highest percentage of
abnormal cell divisions occurring at 10?gml−1cyanotoxin (Fig. 3c,
open circles). We detected anomalies both at the transition from
metaphase to anaphase and in late mitosis. Control meristem-
atic cells were characterized by normal metaphase and anaphase
spindles as well as intact phragmoplasts. Chromatin organiza-
tion was normal throughout mitosis (Fig. 4a, f and j). The altered
metaphase–anaphase transition in MC-LR treated plants meant
the formation of disrupted or even split metaphase–anaphase
mitotic spindles that could be detected starting from short-term
(2 days) exposure and 1?gml−1MC-LR (Fig. 4b–e). The highest
occurrence of such anomalies was at 20?gml−1MC-LR after 20
days of treatment (Fig. 3d, black circles). At later stages of mito-
sis, both anaphase and telophase/cytokinesis anomalies occurred.
First, we detected asymmetric anaphase spindles with abnormal
sister chromatid separation (Fig. 4g–i). Second, we observed lag-
C. Máthé et al. / Aquatic Toxicology 92 (2009) 122–130
Fig. 2. The effects of MC-LR on microtubule (MT) organization in interphase and non-dividing reed root cells. (a) The time- and dose-dependent effect of the cyanotoxin
on MT degradation in primary root tips. Black circles: 2 days; open circles: 5 days; black triangles: 10 days of MC-LR exposure. (b) The effect of MC-LR on MT degradation
in the elongation zone of primary roots (black circles), and in the tips of lateral roots (open circles), in reed plantlets exposed for 2 days to the cyanotoxin. (c) The cortical
microtubule (CMT) system of a control primary root tip shown by anti-? tubulin labelling of interphase and dividing (arrow) meristematic cells. (d) Anti-? tubulin labelling
of primary root tip meristem from reed plants exposed to 2.5?gml−1MC-LR for 20 days, showing the lack of normal MTs and the high background fluorescence. (e) Anti-?
tubulin labelling of a control lateral root at the transition between the division and elongation zone. (f) Anti-? tubulin labelling of a lateral root at the transition between
the division and elongation zone, exposed for 20 days to 20?gml−1MC-LR showing the lack of normal MT structure, and high background fluorescence. Radially swollen
C. Máthé et al. / Aquatic Toxicology 92 (2009) 122–130
Fig. 3. The effect of MC-LR on mitosis and mitotic anomalies in P. australis primary root tip meristem. (a) The time and dose-dependent effects on the number (percentage)
of mitotic cells. Black bars: 2 days; grey bars: 10 days; open bars: 20 days of MC-LR exposure. (b) Telophase and cytokinesis (black circles), and early mitosis (open circles)
indices in reed root tips exposed to MC-LR for 20 days. Cells in telophase and cytokinesis were considered by the presence of daughter chromatin structure and nuclei
as well as the presence of phragmoplasts. Early mitotic cells showed chromatin and microtubule (MT) organization characteristic for prophase, including the presence of
preprophase bands (PPBs) as well as prophase and prometaphase spindles. (c) The time- and dose-dependent effects of MC-LR on the occurrence of total mitotic anomalies
in the respect of MT and chromatin disorders. Black circles: 2 days; open circles: 20 days of MC-LR exposure. (d) Cyanotoxin concentration dependent frequency of the
anomalies of metaphase–anaphase transition (black circles) and late mitosis (anaphase, telophase and cytokinesis) microtubular/chromosome organization (open circles) in
the percentage of all mitotic cells in reed root tips exposed to MC-LR for 20 days.
ging chromosomes that remained in the equatorial plane of cells
after the start of chromatid separation (Fig. 4k). This phenomenon
occurred concomitantly with the formation of phragmoplast and
the maintenance of remnants of mitotic spindle characteristic
for early telophase (Fig. 4l and m). The alteration of telophase
led to anomalous cytokinesis. At short-term (2 days) of expo-
sure, 1?gml−1MC-LR induced the formation of anomalous late
mitotic (anaphase, telophase and cytokinetic) cells. At longer expo-
sures (5–20 days), alterations of late mitosis were observed at
0.5?gml−1MC-LR, too (data not shown). The highest occurrence
of such anomalies was at 10?gml−1MC-LR (Fig. 3d, open cir-
In relation to the effects of MC-LR on mitotic events in reed
roots, we can state that (1) the high percentage of anomalous
metaphase–anaphase transition occurs at higher cyanotoxin con-
centrations than the aberrant late mitosis events (Fig. 3d); (2) the
highest percentage of mitotic anomalies is characteristic for higher
cyanotoxin concentrations, compared to the MC-LR concentrations
that induce the maximal increase of the percentage of cells in cer-
tain mitotic phases (Fig. 3b and d).
We have shown that MC-LR concentrations of 0.5?gml−1and
above are able to induce morphological and cytological alterations
in parallel with more than 50% inhibition of protein phosphatase
activity in reed roots. Starting at low MC-LR concentrations
(0.5?gml−1), many lateral root primordia develop and eventually
fuse together at low distance from root tip, but those primordia fail
to form fully developed laterals (Fig. 1d). The early development
of laterals can be related to the cyanotoxin induced inhibition of
cell elongation in the differentiation zone of primary roots (Fig. 1d
and f). The relationship between protein phosphorylation and lat-
eral root development has been demonstrated in Arabidopsis. For
example, the hyperphosphorylation of the KRP2 protein leads to
morphological effects through the induction of protein hyperphos-
Smith et al. (1994) reported that the protein phosphatase
inhibitors calyculin-A and okadaic acid induced abnormal expan-
cells are shown by arrows. Bars for (c–f): 20?m. (g) CMTs of control, differentiated reed primary root cortex cells shown by anti-? tubulin labelling. (h) The start of CMT
disorganization (arrows) in differentiated primary root cortex cells exposed for 2.5?gml−1MC-LR for 20 days. (i) The lack of MT cytoskeleton and autofluorescence of plastids
in differentiated primary root cortex cells exposed to 20?gml−1MC-LR for 10 days. Bars for (g–i): 50?m.
C. Máthé et al. / Aquatic Toxicology 92 (2009) 122–130
Fig. 4. The effect of MC-LR on the development of mitotic anomalies in tips of P. australis primary roots, as shown by anti-? tubulin labelling (red, in color images) and staining
of chromatin with DAPI (blue, in color images). (a) Control metaphase. (b) Disrupted spindle at the transition from metaphase to anaphase in a cell from roots exposed to
1?gml−1MC-LR for 5 days. (c–e) Triple spindle in root treated with 20?gml−1MC-LR for 2 days, chromatin staining with DAPI (c), anti-? tubulin labelling (d) and merged
image of concomitant labelling for chromatin and MTs (e). (f) Control anaphase. (g–i) Abnormal chromatid separation and asymmetric spindle as shown by DAPI staining
(g), anti-? tubulin labelling (h), and merged image (i) respectively in roots exposed to 1?gml−1MC-LR for 5 days. Bars for (a–i): 5?m. (j) Control telophase. (k–m) Root tip
meristem cell exposed to 2.5?gml−1MC-LR for 5 days, abnormal telophase as shown by DAPI staining of incompletely separated chromatin (k), anti-?-tubulin labelling of
telophase microtubule (MT) structure (l), and merged image (m). Bars for (j–m): 10?m.
sion of Arabidopsis cells of peripheral root tissues. We observed a
similar phenomenon in MC-LR treated reed roots, when a callus-
like tissue developed from rhizodermis and outer cortical cell
abnormal radial swelling of reed primary and lateral roots, just
above meristematic tissue, which coincided with more than 50%
inhibition of root growth (Fig. 1a and f–g). Similar phenotype was
observed in the Arabidopsis rcn pp2aa2-1 and rcn pp2aa3-1 double
mutants impaired in PP2A activity (Zhou et al., 2004).
It is worth mentioning that the MC-LR concentrations of
0.5?gml−1and higher proved to be effective in altering reed
root growth and development in this study, may occur under
natural conditions in reed stands, as suggested in previous studies
(Falconer et al., 1999; Máthé et al., 2007). Blooms of M. aeruginosa
concentrated at the littoral zone of freshwaters might influence
growth and development of reed plants in natural populations
(Máthé et al., 2007).
Concomitantly with the altered development and morphology,
we have shown that MC-LR induces MT depolymerization in pri-
mary and lateral reed roots (Fig. 2). We suggest that this alteration
is directly related to the PP1 and PP2A inhibition by the cyanotoxin,
because it can be induced by exposure to low MC-LR concentra-
tion (1?gml−1) in the elongation zone and differentiated reed root
tissues and higher cyanotoxin concentrations increase the percent-
age of roots with disrupted MTs (Fig. 2b and g–i). Several studies
indicate that actin and MT cytoskeleton are both involved in the
Wilson, 1997; Mathur, 2004; Smith et al., 1994; Zhou et al., 2004).
The phosphorylation state of several proteins including MAPs is
involved in the organization of MTs (Baskin and Wilson, 1997). The
TONNEAU mutation, known to affect the “B” regulatory subunit of
PP2A, induces the alteration of MT organization and consequently
influences root cell shape, leading to the development of dwarf,
thick Arabidopsis seedlings (Camilleri et al., 2002). Our present
data prove that MC-LR, a protein phosphatase inhibitor, induces
anomalies in reed primary and lateral root growth, development
and morphology. The inhibition of longitudinal root growth and
the promotion of radial cell swelling in primary and lateral roots
can be directly related to the alterations of MT organization in the
elongation and root hair zone (see Figs. 1 and 2).
The disruption of microtubular cytoskeleton observed in reed
tantly with the changes of mitotic activity and the development
of abnormal cell divisions induced by MC-LR. At 20-day exposure,
low MC-LR concentrations increase, whereas high MC-LR concen-
C. Máthé et al. / Aquatic Toxicology 92 (2009) 122–130
trations decrease mitotic index in reed roots (Fig. 3a). In animal
cells (CHO-K1 cells) MC-LR induces a transient increase of mitotic
model of the effects of protein phosphatase inhibitors, originally
reported for animal cells (Gehringer, 2004) is valid for plant cells,
too. According to this hypothesis, cell proliferation is stimulated by
low concentrations of MC-LR and okadaic acid (probably by the
activation of MAP kinases), whereas high concentrations inhibit
mitosis. The inhibition of cell division is related to the near 100%
inhibition of protein phosphatases (Gehringer, 2004).
We observed the accumulation of cells in prophase and
telophase in reed root tips (Fig. 3b). It is worth mentioning that
the phenomenon of MC-LR induced increase of the number of cells
in prophase and cytokinesis could be observed in white mustard
(S. alba) seedlings, too (M. Hamvas et al., unpublished results).
The Arabidopsis TONNEAU2 mutation totally inhibits the activity
of a PP2A required for the formation of PPB at the onset of mito-
sis (Camilleri et al., 2002). However, if the activity of PP2A is only
partially inhibited by low concentrations of okadaic acid, tobacco
cells are arrested in early prophase as shown by the abundance
of PPBs. This phenomenon can be related to the hyperphosphoryla-
1992). Treatment with low concentration of endothall (1?M) leads
to the premature activation of a plant-specific cyclin dependent
dles during prometaphase. High endothall concentrations (10?M)
induce the arrest of alfalfa cells in prophase (Ayaydin et al., 2000).
In contrast, MC-LR does not induce PPB or early mitotic spindle
anomalies. We measured relatively high mitotic indices at MC-
LR concentrations that induced the increase of early mitosis and
telophase indices (Fig. 3a and b). Therefore, we suggest that the
cyanotoxin increases the duration of early mitosis and those cells
can undergo later stages of cell division (Fig. 4b), rather than arrest-
ing cells in prophase. This difference between the effects of distinct
of PP1 and PP2A inhibition by MC-LR, and the more potent inhi-
bition of PP2A by okadaic acid and endothall (MacKintosh and
MacKintosh, 1994, and this study). Low concentration of endothall
(1?M) leads to the increase of phragmoplast frequency of synchro-
nized alfalfa cells (Ayaydin et al., 2000). Based on our studies, we
suggest that MC-LR does not arrest cells in telophase and cytoki-
nesis, but it increases the duration of these mitotic stages. The
completion of cytokinesis in plants that is, phragmoplast and cell
plate expansion, depends on the phosphorylation state of several
proteins including MAPs (Soyano et al., 2003; Sasabe et al., 2006).
Significant increase of the percentage of mitotic anomalies
appears at low MC-LR concentrations (0.5–1?gml−1) and short
exposure times (2–5 days). The highest ratio of altered mito-
sis to all mitotic cells occurs at higher MC-LR concentrations
(10–40?gml−1). Higher cyanotoxin concentrations induce the
inhibition of mitosis, meaning that even if some cells still divide
at those treatments, a significant percentage of divisions is
anomalous. We could detect mitotic spindle anomalies at the
metaphase–anaphase transition and anaphase, including the
formation of disrupted or asymmetric spindles (Figs. 3d and 4a–i).
Many studies have proven that PP1 and PP2A activity is required
for the correct onset of metaphase and anaphase in eukaryotic
cells (for a review, see Wolniak and Larsen, 1992). In Drosophila,
the loss of a PP1 activity by the mutation in bimG gene disturbs the
completion of anaphase (Doonan and Morris, 1989). The involve-
ment of a PP2A in the transition from metaphase to anaphase was
described in the pig kidney cell line LLC-PK (Vandré and Wills,
1992). In animal cells, protein phosphatase inhibitors induce the
formation of multipolar and disrupted spindles that alter the entry
into anaphase and chromatid separation (Vandré and Wills, 1992;
Cheng et al., 1998; Lankoff et al., 2003; Bonness et al., 2006). Nat-
urally occurring protein phosphatase inhibitors, like okadaic acid
and microcystin variants, increase the metaphase transit time (i.e.
the duration of nuclear envelope breakdown to anaphase onset),
and induce incomplete chromatid separation in Tradescantia
stamen hair cells: the movement of some chromatids towards
cell poles is temporarily delayed (Wolniak and Larsen, 1992). The
authors did not analyze in detail the MT anomalies related to such
phenomena. Our study is the first one reporting the MC-LR induced
multipolar metaphase–anaphase spindles, asymmetric anaphase
spindles and consequent incomplete chromatid separation during
anaphase and telophase in MC-LR treated reed and generally, plant
cells (Fig. 4f–m).
The disturbance of normal mitotic events induced by MC-LR
is similar to cytological observations that link protein hyperphos-
phorylation to the alterations of cyclin dependent kinase (CDK)
activities or functioning of MAPs in animal and plant cells. CDKs,
known to be regulated by PP1 and PP2A, co-localize with micro-
their crucial role in MT dynamics at those stages (Weingartner et
al., 2001). The inhibition of protein phosphatase activity disturbs
the p34cdc2kinase mediated exit from mitosis (Vandré and Borisy,
1989; Vandré and Wills, 1992). It is noteworthy that the mainte-
nance of the hyperphosphorylated state of certain MAPs increases
sition (Vásquez et al., 1999).
Our present data prove that the induction of mitotic anoma-
lies in reed root cells coincides with MC-LR concentrations where
the inhibition of protein phosphatase activity is higher than 50%
(Figs. 1b, 3c and d, and 4a–m). The increase of the percent-
age of meristematic cells in early and late mitosis, can also
be related to the inhibition of protein dephosphorylation on
phosphoserine–threonine residues by the cyanotoxin. We proved
that MC-LR as an inhibitor of PP1 and PP2A is capable of altering
the normal segregation of chromosomes during mitosis and the
correct chromatin/MT organization during metaphase–anaphase
transition and cytokinesis.
The model experimental system used in this study involved P.
australis, an emergent freshwater aquatic macrophyte. Therefore
our results could contribute to the understanding of the cellular
effects of MC-LR in aquatic ecosystems. We have shown that MC-
PP1 and PP2A activity by more than 50% in P. australis roots, in good
agreement with the cyanotoxin concentrations that induce the
accumulation of root meristematic cells in certain mitotic phases,
the induction of mitotic anomalies and alteration of CMT orga-
nization. We suggest for the first time, that the MC-LR induced
disruption of metaphase–anaphase spindles leads to abnormal sis-
ter chromatid segregation at telophase. The relationship between
the disruption of MT structures and the abnormal development of
roots is proved in this study. Our results show that the inhibition of
protein dephosphorylation by MC-LR is involved in every stage of
mitotic and CMT organization in plant cells.
This work was supported by Hungarian national research
Grants OTKA F046493, K68416 and GVOP-3.2.1.-2004-04-0110/3.0.
C. Máthé, G. Vasas, M. M-Hamvas and G. Surányi were supported by
the Bolyai J. Research Scholarship. Z. Serf˝ oz˝ o was supported by the
OTKA Postdoctoral Grant no. PD75276. The authors wish to thank
Mrs. Ágnes Németh for excellent technical assistance.
Abe, T., Lawson, T., Weyers, D.B., Codd, G.A., 1996. Microcystin-LR inhibits photo-
synthesis of Phaseolus vulgaris primary leaves: implications for current spray
irrigation practice. New Phytol. 133, 651–658.
130 Download full-text
C. Máthé et al. / Aquatic Toxicology 92 (2009) 122–130
Armstrong, J., Armstrong, W., 2001. An overview of the effects of phytotoxins on
Phragmites australis in relation to die-back. Aquat. Bot. 69, 251–268.
Ayaydin, F., Vissi, E., Mészáros, T., Miskolczi, P., Kovács, I., Fehér, A., Dombrádi, V.,
Erd˝ odi, F., Gergely, P., Dudits, D., 2000. Inhibition of serine/threonine-specific
protein phosphatases causes premature activation of cdc2MsF kinase at G2/M
transition and early microtubule organisation in alfalfa. Plant J. 23, 85–96.
Barlow, P.W., Baluˇ ska, F., 2000. Cytoskeletal perspectives on root growth and mor-
phogenesis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, 289–322.
Baskin, T.I., Wilson, I.E., 1997. Inhibitors of protein kinases and phosphatases alter
root morphology and disorganize cortical microtubules. Plant Physiol. 113,
Bonness, K., Aragon, I.V., Rutland, B., Ofori-Acquah, S., Dean, N.M., Honkanen, R.E.,
2006. Cantharidin-induced mitotic arrest is associated with the formation of
aberrant mitotic spindles and lagging chromosomes resulting, in part, from the
suppression of PP2A?. Mol. Cancer Ther. 5, 2727–2736.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein-dye binding. Anal.
Biochem. 72, 248–254.
The Arabidopsis TONNEAU2 gene encodes a putative novel protein phosphatase
2A regulatory subunit essential for the control of the cortical cytoskeleton. Plant
Cell 14, 833–845.
Carmichael, W.W., 1992. Cyanobacterial secondary metabolites—the cyanotoxins. J.
Appl. Bacteriol. 72, 445–459.
Casimiro, I., Beeckman, T., Graham, N., Bhalerao, R., Zhang, H., Casero, P., Sandberg,
G., Bennett, M.J., 2003. Dissecting Arabidopsis lateral root development. Trends
Plant Sci. 8, 165–171.
Chen, J., Song, L., Dai, J., Gan, N., Liu, Z., 2004. Effects of microcystins on the growth
and the activity of superoxide dismutase and peroxidase of rape (Brassica napus
L.) and rice (Oryza sativa L.). Toxicon 43, 393–400.
Cheng, A., Balczon, R., Zuo, Z., Koons, J.S., Walsh, A.H., Honkanen, R.E., 1998.
Fostriecin-mediated G2-Mphase growth arrest correlate with abnormal centro-
some replication, the formation of aberrant mitotic spindles, and the inhibition
of serine/threonine protein phosphatase activity. Cancer Res. 58, 3611–3619.
for health protection. Toxicol. Appl. Pharmacol. 203, 264–272.
Doonan, J.H., Morris, N.R., 1989. The bimG gene of Aspergillus nidulans, required for
completion of anaphase, encodes a homolog of mammalian phosphoprotein
phosphatase 1. Cell 57, 987–996.
thioanhydride inhibits protein phosphatases-1 and-2A in vivo. Am. J. Physiol.
Falconer, I.R., 1993. Mechanism of toxicity of cyclic peptide toxins from blue-green
algae. In: Falconer, I.R. (Ed.), Algal Toxins in Seafood and Drinking Water. Aca-
demic Press, Harcourt Brace and Company, pp. 177–209.
Falconer, I., Bartram, J., Chorus, I., Kuiper-Goodman, T., Utkilen, H., Burch, M., Codd,
G.A., 1999. Safe levels and safe practices. In: Chorus, I., Bartram, J. (Eds.), Toxic
Cyanobacteria in Water. E & FN Spon, London/NY, pp. 155–178.
Fladmark, K.E., Serres, M.H., Larsen, N.L., Yasumoto, T., Aune, T., Døskeland, S.O.,
1999. Sensitive detection of apoptogenic toxins in suspension cultures of rat
and salmon hepatocytes. Toxicon 36, 1101–1114.
Gamborg, O.L., Miller, R.A., Ojima, K., 1968. Nutrient requirements of suspension
cultures of soybean root cells. Exp. Cell Res. 50, 151–158.
Gehringer, M.M., 2004. Microcystin-LR and okadaic acid-induced cellular effects: a
dualistic response. FEBS Lett. 557, 1–8.
Kós, P., Gorzó, G., Surányi, G., Borbely, G., 1995. Simple and efficient method for
alba L.). Anal. Biochem. 225, 49–53.
Kurki-Helasmo, K., Meriluoto, J., 1998. Microcystin uptake inhibits growth and pro-
tein phosphatase activity in mustard (Sinapis alba L.) seedlings. Toxicon 36,
Lankoff, A., Banasik, A., Obe, G., Deperas, M., Kuzminski, K., Tarczynska, M., Jurczak,
T., Wojcik, A., 2003. Effect of microcystin-LR and cyanobacterial extract from
Polish reservoir of drinking water on cell cycle progression, mitotic spindle, and
apoptosis in CHO-K1 cells. Toxicol. Appl. Pharmacol. 189, 204–213.
Luan, S., 2003. Protein phosphatases in plants. Annu. Rev. Plant Biol. 54, 63–92.
M-Hamvas, M., Máthé, Cs., Molnár, E., Vasas, G., Grigorszky, I., Borbely, G., 2003.
Microcystin-LR alters the growth, anthocyanin content and single-stranded
DNase enzyme activities in Sinapis alba L. seedlings. Aquat. Toxicol. 62, 1–9.
MacKintosh, C., MacKintosh, R.W., 1994. Inhibitors of protein kinases and phos-
phatases. Trends Biochem. Sci. 19, 444–448.
MacKintosh, C., Diplexcito, J., 2003. Naturally occurring inhibitors of ser-
ine/threonine phosphatases. In: Wells, J., Hunter, T., Karin, M., Farquhar, M.,
Thompson, B. (Eds.), Handbook of Cell Signalling, vol. 1. Elsevier Science Pub-
lication, USA, pp. 607–611.
Máthé, Cs., M.Hamvas, M., Grigorszky, I., Vasas, G., Molnár, E., Power, J.B., Davey,
M.R., Borbély, G., 2000. Plant regeneration from embryogenic cultures of Phrag-
mites australis (Cav.) Trin. Ex Steud. (common reed). Plant Cell Tiss. Org. 63,
Borbély, G., 2007. Microcystin-LR, a cyanobacterial toxin, induces growth inhi-
bition and histological alterations in common reed (Phragmites australis) plants
regenerated from embryogenic calli. New Phytol. 176, 824–835.
Mathur, J., 2004. Cell shape development in plants. Trends Plant Sci. 9, 583–590.
McDermott, C.M., Nho, C.W., Howard, W., Holton, B., 1998. The cyanobacterial toxin,
microcystin-LR, can induce apoptosis in a variety of cell types. Toxicon 36,
of Lake Ferto/Neusiedlersee. Hydrobiologia 506, 681–686.
tobacco tissue cultures. Physiol. Plantarum 15, 473–497.
Nakai, T., Kato, K., Shinmyo, A., Sekine, M., 2006. Arabidopsis KRPs have distinct
inhibitory activity toward cyclin D2-associated kinases, including plant-specific
B-type cyclin-dependent kinase. FEBS Lett. 580, 336–340.
Ostendorp, W., 1989. Die-back of reeds in Europe—a critical review of literature.
Aquat. Bot. 35, 5–26.
Pflugmacher, S., Wiegand, C., Beattie, K.A., Krause, E., Steinberg, C.E.W., Codd, G.A.,
2001. Uptake, effects and metabolism of cyanobacterial toxins in the emergent
reed plant Phragmites australis (Cav.) Trin. Ex Steud. Environ. Toxicol. Chem. 20,
Pflugmacher, S., 2002. Possible allelopathic effects of cyanotoxins, with reference to
microcystin-LR, in aquatic ecosystems. Environ. Toxicol. 17, 407–413.
Pflugmacher, S., 2004. Promotion of oxidative stress in the aquatic macrophyte
Ceratophyllum demersum during biotransformation of the cyanobacterial toxin
microcystin-LR. Aquat. Toxicol. 70, 169–178.
Sasabe, M., Soyano, T., Takahashi, Y., Sonobe, S., Igarashi, H., Itoh, T.J., Hidaka, M.,
Machida, Y., 2006. Phosphorylation of NtMAP65-1 by a MAP kinase down-
regulates its activity of microtubule bundling and stimulates progression of
cytokinesis of tobacco cells. Genes Dev. 20, 1004–1014.
Shibaoka, H., 1994. Plant hormone-induced changes in the orientation of cortical
microtubules: alterations in the cross-linking between microtubules and the
plasma membrane. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 527–544.
block root hair growth and alter cortical cell shape of Arabidopsis roots. Planta
Smith, R.D., Walker, J.C., 1996. Plant protein phosphatases. Annu. Rev. Plant Physiol.
Plant Mol. Biol. 45, 527–544.
is a MAPKK that acts in the NPK1 MAPKKK-mediated MAPK cascade and is
required for plant cytokinesis. Genes Dev. 17, 1055–1067.
Toivola, D.M., Eriksson, J.E., 1999. Toxins affecting cell signalling and alteration of
cytoskeletal structure. Toxicol. In Vitro 13, 521–530.
Vandré, D.D., Borisy, G.G., 1989. Anaphase onset and dephosphorylation of mitotic
phosphoproteins occur concomitantly. J. Cell Sci. 94, 245–258.
J. Cell Sci. 101, 79–91.
Analysis of cyanobacterial toxins (anatoxin-a, cylindrospermopsin, microcystin-
LR) by capillary electrophoresis. Electrophoresis 25, 108–115.
Vásquez, R.J., Gard, D.L., Cassimeris, L., 1999. Phosphorylation by CDK1 regulates
XMAP215 function in vitro. Cell Motil. Cytoskel. 43, 310–321.
Weingartner, M., Binarova, P., Drykova, D., Schweighofer, A., David, J.P., Heberle-
Bors, E., Doonan, J., Bögre, L., 2001. Dynamic recruitment of Cdc2 to specific
microtubule structures during mitosis. Plant Cell 13, 1929–1943.
Wolniak, S.M., Larsen, P.M., 1992. Changes in the metaphase transit times and the
pattern of sister chromatid separation in stamen hair cells of Tradescantia after
treatment with protein phosphatase inhibitors. J. Cell Sci. 102, 691–705.
Ye, Z.H., Wong, M.H., Baker, A.J.M., Willis, A.J., 1998. Comparison of biomass and
metal uptake between two populations of Phragmites australis grown in flooded
and dry conditions. Ann. Bot.-London 82, 83–87.
Zhang, K., Tsukitani, Y., John, P.C.L., 1992. Mitotic arrest in tobacco caused by
the phosphoprotein phosphatase inhibitor okadaic acid. Plant Cell Physiol. 33,
Zhou, H.W., Nussbaumer, C., Chao, Y., DeLong, A., 2004. Disparate roles for the regu-
latory A subunit isoforms in Arabidopsis protein phosphatase 2A. Plant Cell 16,