Extracts of Non-Microcystin-Producing Cyanobacteria Affect the Plant Cytoskeleton and Cell Cycle ^†

Abstract

Studies on the toxicity of cyanobacterial products on plant cytoskeleton have so far focused on the effects of microcystins (MCs), cyanobacterial toxins that inhibit protein phosphatases 1 and 2A, enzymes which are involved in plant cytoskeleton (microtubules and F-actin) organization and cell cycle progression. In this study, we investigated the effects of extracts from two non-microcystin-producing (NMP) cyanobacterial strains, Microcystis viridis TAU-MAC 1810 and Planktothrix agardhii TAU-MAC 0514, on the cytoskeleton and cell cycle of Oryza sativa (rice) root cells. Rice seedling roots were exposed for various time periods (1, 12 and 24 h) to aqueous extracts of the aforementioned strains. Treated root tips underwent either immunostaining for α-tubulin or staining of F-actin with fluorescent phalloidin, and DAPI staining of DNA. Fluorescent specimens were observed by confocal laser scanning microscopy (CLSM). Corrected total cell fluorescence (CTCF) was measured to quantify F-actin disorder. To assess cell cycle alterations, cell cycle stage frequencies were calculated. In addition, Evans Blue staining was applied to determine dead cells. Treatment with the extracts affected microtubules and F-actin, as well as the cell cycle. These findings suggest that bioactive cyanobacterial compounds, apart from MCs, can disrupt the cytoskeleton and cell cycle progression in plant cells.

1. Introduction

Microcystins (MCs) are monocyclic heptapeptides, produced by several species of cyanobacteria [1]. They are potent hepatotoxins [2], but are also known to negatively affect plants [3,4]. In particular, the plant cytoskeleton (F-actin and microtubules) is an established target of MC variants [5,6], the toxicity of which lies in their ability to inhibit protein phosphatases 1 (PP1) and 2A (PP2A) [7], which participate in cytoskeleton organization [8] and cell cycle progression [9].

To date, extracts from cyanobacterial strains that produce MCs have been extensively used to study cytoskeletal and other physiological defects [3,5,10]. In this study, we investigated the effects of extracts from non-microcystin-producing (NMP) cyanobacterial strains on Oryza sativa (rice) meristematic root cells, focusing on the plant cytoskeleton and cell cycle.

2. Experiments

2.1. Culture of Cyanobacterial Strains and Biomass Extraction

Two NMP cyanobacterial strains of the TAU-MAC culture collection, Microcystis viridis TAU-MAC 1810 and Planktothrix aghardii TAU-MAC 0514 [11,12], were cultured using BG-11 medium in a 12:12 h light:dark cycle under white fluorescent lamps. Biomass was collected at the exponential growth phase, frozen and lyophilized. Dry biomass (150 mg for each strain) was dissolved in 75% (v/v) methanol, sonicated and eventually evaporated, according to [13]. The pellet was resuspended in 5 mL of double-distilled water (ddH₂O). Extracts were filtered and stored at −20 °C.

2.2. Plant Material, Cyanobacterial Biomass Extraction and Treatments

Rice (Oryza sativa cv Axios) seeds were kindly provided by the National Cereal Institute (Thessaloniki, Greece) and were germinated on soaked filter paper at room temperature (24 ± 1 °C) in the dark. Four- to five-day-old seedlings were treated with aqueous cyanobacterial extracts for 1, 12 or 24 h, their roots being submerged in tubes containing the extract. For control, seedlings, similarly treated with ddH₂O were used.

2.3. Tubulin Immunolabeling

Control and extract-treated root rips were fixed in 4% (w/v) paraformaldehyde (PFA) solution in PEM buffer (50 mM PIPES, 5 mM EGTA, 5 mM MgSO₄, pH 6.8) +5% (v/v) dimethyl sulfoxide (DMSO) for 1 h. Cell wall digestion was performed with a 3% (w/v) Macerozyme R-10 + 3% (w/v) cellulase Onozuka R-10 solution in PEM buffer for 90 min. Root tips were squashed on coverslips coated with poly-l-lysine, and the squashes were left to dry. The adherent cells were extracted for 1 h with a 5% (v/v) DMSO + 1% (v/v) Triton X-100 solution in phosphate-buffered saline (PBS, pH 7.2). Rat anti-α-tubulin (YOL 1/34) as primary antibody (diluted 1:50 in PBS) and anti-rat IgG AlexaFluor 488 as secondary antibody (diluted 1:300 in PBS) were used. Specimens were incubated with each antibody overnight at room temperature. DNA was counterstained with 0.9 μM 4′, 6-diamidino-2-phenylindole (DAPI) for 5–10 min. Fluorescent specimens were mounted with anti-fade medium (PBS 1:2 glycerol (v/v) + 0.5% (w/v) p-phenylenediamine) and examined under a Zeiss Observer.Z1 microscope, equipped with the LSM780 confocal laser scanning (CLSM) module and the appropriate filters for each fluorophore. Imaging was achieved with ZEN2011 software, according to the manufacturer’s instructions.

2.4. F-Actin Labeling

F-actin of control and extract-treated root tips was pre-stabilized with 300 μM m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) in PEM + 0.1% (v/v) Triton X-100 for 30 min in the dark. Fixation was performed with 4% (w/v) PFA in PEM + 5% (v/v) DMSO + 0.1% (v/v) Triton X-100 + DyLight 554-phalloidin 1:400, and fixed samples were washed with PEM buffer, extracted with 5% (v/v) DMSO + 1% (v/v) Triton X-100 in PBS for 1 h and incubated with DyLight 554-phalloidin (1:40 in PBS) at 37 °C for 2h. DAPI was used for DNA counterstaining. Samples were mounted with anti-fade medium and observed with the Zeiss Observer.Z1 microscope, equipped with the LSM780 confocal laser scanning module.

2.5. Fluorescence Intensity Measurements

Single cortical CLSM sections and maximum intensity projections of serial CLSM sections of control and extract-treated roots were measured for fluorescence intensity using ImageJ. The corrected total cell fluorescence (CTCF) was calculated using the equation:

CTCF = Integrated Density − (Area of Selected Cell × Mean Fluorescence of Background Readings)

Thirty individual cells from three different roots were measured for each treatment and data were statistically analyzed (ANOVA with Dunnett’s test).

2.6. Cell Cycle Analysis

Alterations in the cell cycle progression of extract-treated meristematic root cells were assessed with CLSM observations of whole-mount specimens, labelled for F-actin and stained with DAPI. Cell cycle stages (interphase, preprophase/prophase, metaphase/anaphase and cytokinesis) were recognized according to F-actin arrangement and/or chromatin state. At least 1000 meristematic cells from three different roots per treatment were counted. Statistical analysis of data (chi-squared test, df = 6) was performed, with a significance of P < 0.05.

2.7. Detection of Dead Cells

Control and extract-treated roots were stained with Evans Blue for the detection of dead cells, according to [14]. Roots were incubated in 0.25% (w/v) aqueous Evans Blue extract for 15 min, washed twice and incubated overnight in double-distilled water. Samples were then observed with a stereomicroscope.

3. Results

3.1. Cytoskeletal Alterations

3.1.1. Effects on F-Actin Organization

The extracts of both NMP strains induced time-dependent alterations on F-actin. The fine actin filaments observed in control root tips (Figure 1A) were significantly affected, starting from 1 h of treatment. Roots treated with the 1810 extract exhibited more severe alterations, ranging from bundling after 1 h of treatment (Figure 1B) to the formation of thick F-actin aggregates after 12 h (Figure 1C) and, eventually, the total collapse of F-actin network after 24 h (Figure 1D). Roots treated with the 0514 extract exhibited milder alterations, mainly disorientation, which was detectable at all time points and intensified progressively (Figure 1E–G).

3.1.2. Effects on F-Actin Fluorescence Intensity

CTCF measurements (Figure 2) showed that fluorescence intensity in extract-treated meristematic root cells decreased progressively. Even after short treatments (1 h), the intensity was significantly lower compared to control cells. The lowest levels were recorded after 24 h.

3.2. Effects on Microtubules and Chromatin

Both cyanobacterial extracts induced alterations in the microtubule network (Figure 3). Meristematic cells treated with the 1810 extract exhibited major defects after 1 h of treatment (Figure 3G–J), which eventually led to the disappearance of microtubules after 12 h, in parallel with abnormal chromatin condensation (Figure 3K). The effect of 0514 extract was not as acute, as only minor defects were observed after 1 h of treatment (L-R). No significant alterations were detected after longer treatments.

3.3. Effects on Cell Cycle Progression

In root tips treated with the extracts, cell cycle stage frequencies were significantly altered, starting from 1h of treatment (

Table 1. Percentages (%) of occurrence of cell cycle stages in Oryza sativa root tips, either control or treated for various time periods (1, 2 and 24 h) with extracts from the NMP cyanobacterial strains, Microcystis viridis TAU-MAC 1810 and Planktothrix aghardii TAU-MAC. Kruskal−Wallis analysis of variance showed a significant time-dependent variation in the percentage of cells in different cell cycle phases (chi-squared; χ²: 15.8, n = 35, df = 6, P < 0.05).

Cell Cycle Stage	Control 1	TAU-MAC 1810			TAU-MAC 0514
		1 h	12 h	24 h	1 h	12 h	24 h
Interphase	91.6	90.16	0	0	91.2	91.53	96.7
Preprophase/Prophase	3.73	4.11	0	0	3.4	3.09	2.5
Metaphase/Anaphase	1.02	0.08	0	0	0.58	0.3	0
Cytokinesis	3.65	3.49	0	0	2.66	2.59	0
Abnormal Chromatin	0	2.17	100	100	2.16	2.49	0.8

¹ Data from [5].

). Treatment with the 1810 extract dramatically affected chromatin state after 12h, as no cell cycle stages could be distinguished. On the other hand, treatment with the 0514 extract hindered the cell cycle stage progression after 12 h and, after 24 h, almost all cells observed were halted at interphase.

3.4. Induction of Cell Death

The occurrence of dead cells in extract-treated roots was confirmed by Evans Blue staining (Figure 4). Dead cells were detected at all time points and the effects were particularly harsh after 24 h of treatment with the 1810 extract (Figure 4B).

4. Discussion

In general, extracts from NMP cyanobacterial strains have been shown to negatively affect meristematic root cells of Oryza sativa [15]. Extracts from the aforementioned NMP strains (TAU-MAC 1810, 0514) affected both F-actin and microtubules in a time-dependent manner but in different ways, as the 1810 extract exhibited more adverse effects, leading to the eventual collapse of the plant cytoskeletal system after longer treatments (12 and 24 h). In plant cells, MCs are known to disrupt both microtubules [16] and F-actin [5]. Although Microcystis is a genus notorious for producing MCs, Microcystis viridis TAU-MAC 1810 strain was not found to produce any during LC-MS/MS analysis [12]. However, apart from MCs, Microcystis species have been found to produce a wide range of bioactive compounds, such as microginins, microviridins and cyanopeptolins [1]. Similarly, Plankothrix produces a variety of secondary metabolites, many of which are yet to be characterized [1]. Investigations on the toxicity of such compounds against the plant cytoskeleton could be a focal point of future research.

F-actin appeared to be more sensitive than microtubules. This is especially highlighted by treatment with the 0514 extract. These results imply a possible independent mechanism of toxicity for each cytoskeletal element in plant cells. The cytoskeletal alterations, along with the abnormal condensation of chromatin observed in treated cells (especially with the 1810 extract), justify the observed decrease in dividing cells. Interestingly, the vast majority of root meristematic cells treated with the 0514 extract for 24 h were arrested at interphase. MCs are able to not only cause cytoskeletal abnormalities in mitotic cells, but also to alter the mitotic index of treated plant tissues [16,17]. Our findings suggest that similar effects on plant cell division may be exerted by cyanobacterial bioactive compounds other than MCs.

These cytoskeletal alterations and the disruption of the cell cycle progression could be associated with the cell death effects observed in roots treated with the extracts [18]. Despite the fact that the exact nature of these cell death phenomena has not been determined yet, this is an important indication that the presence of certain NMP cyanobacterial strains in the aquatic environment could pose a threat to plant species exposed to their compounds, which are present in water and are often accessible to crop species (such as rice) through irrigation.

5. Conclusions

• Extracts from NMP cyanobacterial strains are able to negatively affect both F-actin and microtubules in root meristematic cells of Oryza sativa, as well as the cell cycle. Therefore, bioactive cyanobacterial compounds, other than MCs, could be able to disrupt the cytoskeleton and cell cycle progression in plant cells.
• Treatment with the extracts induced cell death in root tips.
• Not only strains that produce MCs, but also NMP strains may be toxic for plants.