Microcystin-LR induces mitotic spindle assembly disorders in Vicia faba by protein phosphatase inhibition and not reactive oxygen species induction

Abstract

We aimed to reveal the mechanisms of mitotic spindle anomalies induced by microcystin-LR (MCY-LR), a cyanobacterial toxin in Vicia faba, a well-known model in plant cell and molecular biology. MCY-LR inhibits type 1 and 2A phosphoserine/threonine specific protein phosphatases (PP1 and PP2A) and induces reactive oxygen species (ROS) formation. The cytoskeleton is one of the main targets of the cyanotoxin during cytopathogenesis. Histochemical-immunohistochemical and biochemical methods were used. A significant number of MCY-LR induced spindle alterations are described for the first time. Disrupted, multipolar spindles and missing kinetochore fibers were detected both in metaphase and anaphase cells. Additional polar microtubule (MT) bundles, hyperbundling of spindle MTs, monopolar spindles, C-S- shaped, additional and asymmetric spindles were detected in metaphase, while midplane kinetochore fibers were detected in anaphase cells only. Several spindle anomalies induced mitotic disorders, i.e. they occurred concomitantly with altered sister chromatid separation. Alterations were dependent on the MCY-LR dose and exposure time. Under long-term (2 and mainly 6 days') exposure they were detected in the concentration range of 0.1-20μgmL(-1) MCY-LR that inhibited PP1 and PP2A significantly without significant ROS induction. Elevated peroxidase/catalase activities indicated that MCY-LR treated V. faba plants showed efficient defense against oxidative stress. Thus, although the elevation of ROS is known to induce cytoskeletal aberrations in general, this study shows that long-term protein phosphatase inhibition is the primary cause of MCY-LR induced spindle disorders.

Production and Characterization of Tissue Cultures of Four Crocus Species from the Carpathian Basin

“…Extracts containing equal amounts (FW) of plant samples were loaded onto native 7.5% polyacrylamide gels. POD activity gels (native polyacrylamide gels) were prepared and assayed using a pyrogallol staining method as described previously (Demeter et al, 2014;Garda et al, 2016). In-gel analysis of CAT activity was performed according to the protocol described by Weidert and Cullen, 2010: native gels were incubated in 0.03% H 2 O 2 , washed and incubated in 0.76 mM K 3 [Fe(CN) 6 ] and 1.5 mM FeCl 3 .…”

The effects of microcystin-LR inOryza sativaroot cells: F-actin as a new target of cyanobacterial toxicity

“…A possible reason for this could be that the duration of treatments here was far shorter than in the previously mentioned studies. In most of the relevant studies, exposure to MC-LR spanned from 4 h to 20 days (Beyer et al, 2012; Garda et al, 2016; Mathé et al, 2009). However, in the present study microtubules were severely affected only after treatment with the crude toxic extract for a very short time (30 min and 1 h), which indicates the possible involvement of other compounds in the effect mechanism.…”

“…All reports about the adverse effects of MCs on plant cytoskeleton have been based on applying purified MCs (e.g. Garda et al, 2016; Máthé et al, 2013), as purified toxins offer high precision in determining the exact concentrations. On the other hand, crude extracts of MC-producing cyanobacterial strains, known to negatively affect plant growth, are helpful in simulating the natural exposure of plants to MCs during lab experiments (Pflugmacher et al, 2007; Prieto et al, 2011).…”

Effects of microcystin-LR and cylindrospermopsin on plant-soil systems: A review of their relevance for agricultural plant quality and public health

“…Along with the specific inhibition of PP1 and PP2A (Dawson, 1998), the increase in antioxidant defenses induced by MC-LR suggests that oxidative stress is a major mechanism contributing to the phytotoxicity of this toxin (Pflugmacher et al, 2006(Pflugmacher et al, , 2007aPichardo and Pflugmacher, 2011). However, although the inhibition of photosynthetic processes due to increased concentrations of ROS has been documented (Noctor and Foyer, 1998), recently Garda et al (2016) have shown that under long-term exposure PP inhibition was the primary cause of MC-LR induced mitotic spindle disorders in Vicia faba and not ROS induction. Nevertheless, in a study performed by Gutiérrez-Praena et al (2014) in which tomato plants were exposed to MC-LR, changes were detected in the function of various proteins related to ATP synthesis, carbon fixation, photosynthesis and carbohydrate metabolism that appear to be linked with the observed decrease in photosynthetic efficiency.…”