Effects of bisphenol A on the microtubule arrays in root meristematic cells of Pisum sativum L.

Bisphenol A (BPA), a widely used chemical in the plastics industry that displays weak oestrogenic properties, is an emerging environmental pollutant, potentially harmful to living organisms. The presumed cytotoxicity of BPA to plant cells has been poorly studied. To understand how BPA might influence plant cell division and affect the underlying cytoskeleton, the effects of BPA on the microtubule (MT) arrays of meristematic root-tip cells of Pisum sativum L. were investigated. Root tips of young seedlings were exposed to 20, 50 and 100 mg/L BPA for 1, 3, 6, 12 and 24 h. The effects of each treatment were determined by means of confocal laser scanning microscopy after immunolabelling of tubulin and counterstaining of DNA, and by use of light and transmission electron microscopy. It was found that BPA affected normal chromosome segregation, hampered the completion of cytokinesis and deranged interphase and mitotic MT arrays. BPA effects were dependent on the stage of each cell at the time of BPA entrance. Moreover, BPA induced the formation of macrotubules with a mean diameter of 32 ± 0.14 nm, compared with 23 ± 0.70 nm for the MT arrays in untreated cells. Finally, all MT arrays and macrotubules were depolymerised upon longer treatment. Taken together, the data suggest that BPA exerts acute anti-mitotic effects on meristematic root-tip cells of P. sativum, MT arrays constitute a primary sub-cellular target of BPA toxicity, and the manifested chromosomal abnormalities could be attributed to the disruption of the MT cytoskeleton.     

Effects of hexavalent chromium on microtubule organization,ER distribution and callose deposition in root tip cells of <Emphasis Type="Italic">Allium cepa</Emphasis> L.

The subcellular targets of hexavalent chromium [Cr(VI)] were examined in Allium cepa root tips with confocal laser scanning microscopy. Cr(VI) exerted dose- and time-dependent negative effects on root growth rate, the mitotic index and microtubule (MT) organization during cell division cycle. Interphase MTs were more resistant than the mitotic ones, but when affected they were shorter, sparse and disoriented. The preprophase band of MTs became poorly organized, branched or with fragmented MTs, whilst neither a perinuclear array nor a prophase spindle was formed. Metaphase spindles converged to eccentric mini poles or consisted of dissimilar halves and were unable to correctly orient the chromosomes. Anaphase spindles were less disturbed, but chromatids failed to separate; neither did they move to the poles. At telophase, projecting, lagging or bridging chromosomes and micronuclei also occurred. Phragmoplasts were unilaterally developed, split, located at unexpected sites and frequently dissociated from the branched and misaligned cell plates. Chromosomal aberrations were directly correlated with MT disturbance. The morphology and distribution of endoplasmic reticulum was severely perturbed and presumably contributed to MT disassembly. Heavy callose apposition was also induced by Cr(VI), maybe in the context of a cellular defence reaction. Results indicate that MTs are one of the main subcellular targets of Cr(VI), MT impairment underlies chromosomal and mitotic aberrations, and MTs may constitute a reliable biomonitoring system for Cr(VI) toxicity in plants.     

Differential Responsiveness of Cortical Microtubule Orientation to Suppression of Cell Expansion among the Developmental Zones of Arabidopsis thaliana Root Apex

Τhe bidirectional relationship between cortical microtubule orientation and cell wall structure has been extensively studied in elongating cells. Nevertheless, the possible interplay between microtubules and cell wall elements in meristematic cells still remains elusive. Herein, the impact of cellulose synthesis inhibition and suppressed cell elongation on cortical microtubule orientation was assessed throughout the developmental zones of Arabidopsis thaliana root apex by whole-mount tubulin immunolabeling and confocal microscopy. Apart from the wild-type, thanatos and pom2-4 mutants of Cellulose SynthaseA3 and Cellulose Synthase Interacting1, respectively, were studied. Pharmacological and mechanical approaches inhibiting cell expansion were also applied. Cortical microtubules of untreated wild-type roots were predominantly transverse in the meristematic, transition and elongation root zones. Cellulose-deficient mutants, chemical inhibition of cell expansion, or growth in soil resulted in microtubule reorientation in the elongation zone, wherein cell length was significantly decreased. Combinatorial genetic and chemical suppression of cell expansion extended microtubule reorientation to the transition zone. According to the results, transverse cortical microtubule orientation is established in the meristematic root zone, persisting upon inhibition of cell expansion. Microtubule reorientation in the elongation zone could be attributed to conditional suppression of cell elongation. The differential responsiveness of microtubule orientation to genetic and environmental cues is most likely associated with distinct biophysical traits of the cells among each developmental root zone.     

Hexavalent chromium disrupts mitosis by stabilizing microtubules in Lens culinaris root tip cells

Hexavalent chromium [Cr(VI)] is an accumulating environmental pollutant due to anthropogenic activities, toxic for humans, animals and plants. Therefore, the effects of Cr(VI) on dividing root cells of lentil (Lens culinaris) were investigated by tubulin immunofluorescence and DNA staining. In Cr(VI)‐treated roots, cell divisions were perturbed, the chromosomes formed irregular aggregations, multinucleate cells were produced and tubulin clusters were entrapped within the nuclei. All cell cycle‐specific microtubule (MT) arrays were affected, indicating a stabilizing effect of Cr(VI) on the MTs of L. culinaris. Besides, a time‐ and concentration‐dependent gradual increase of acetylated α‐tubulin, an indicator of MT stabilization, was observed in Cr(VI)‐treated roots by both immunofluorescence and western blotting. Evidence is also provided that reactive oxygen species (ROS) caused by Cr(VI), determined with the specific marker dichlorofluorescein, may be responsible for MT stabilization. Combined treatments with Cr(VI) and oryzalin revealed that Cr(VI) overcomes the depolymerizing ability of oryzalin, as it does experimentally introduced hydrogen peroxide, further supporting its stabilizing effect. In conclusion, it is suggested that the mitotic aberrations caused by Cr(VI) in L. culinaris root cells may be the result of MT stabilization rather than depolymerization, which consequently disturbs MT dynamics and their related functions.     

Aberrant microtubule organization in dividing root cells of p60-katanin mutants

Aberrant microtubule organization has been recently recorded in dividing root cells of fra2 and lue1 p60-katanin Arabidopsis thaliana mutants. Here, we report similar defects in the bot1 and ktn1-2 mutants of the same plant, proposing that they constitute a consistent phenotype of p60-katanin mutants. In addition, we show that the Targeting Protein for Xklp2 (TPX2) protein co-localizes with microtubules on the surface of prophase nuclei of the mutants, probably participating in multipolar spindle assembly. As microtubule organization defects are not observed in metaphase/anaphase spindles and initiating phragmoplasts, we also discuss the putative association of the observed aberrations with the nuclear envelope and we emphasize on the mechanism of bipolar metaphase spindle organization in the mutants. It seems that chromosome-mediated spindle assembly, probably minimally dependent on microtubule severing by p60-katanin, dominates after nuclear envelope breakdown, restoring bipolarity.     

The fatal effect of tungsten on <Emphasis Type="Italic">Pisum sativum</Emphasis> L. root cells: indications for endoplasmic reticulum stress-induced programmed cell death

Programmed cell death (PCD) is a widespread response of plants against abiotic stress, such as heavy metal toxicity. Tungsten (W) is increasingly considered toxic for plants since it irreversibly affects their growth. Therefore, we investigated whether W could induce some kind of PCD in plants, like other heavy metals do. The morphology of cell and nucleus, the integrity of the cytoskeleton, Evans Blue absorbance and the expression of PCD-related genes were used as indicators of PCD in W-treated roots of Pisum sativum (pea). TEM and fluorescence microscopy revealed mitotic cycle arrest, protoplast shrinkage, disruption of the cytoskeleton and chromatin condensation and peripheral distribution in the nucleus of W-affected cells. Moreover, Evans Blue absorbance in roots increased in relation to the duration of W treatment. These effects were suppressed by inhibitors of the 26S proteasome, caspases and endoplasmic reticulum stress. In addition, silencing of DAD-1 and induction of HSR203J, BiP-D, bZIP28 and bZIP60 genes were also recorded in W-treated pea roots by semi-quantitative RT-PCR. The above observations show that W induces a kind of PCD in pea roots, further substantiating its toxicity for plants. Data imply that endoplasmic reticulum stress-unfolded protein response may be involved in W-induced PCD.     

The distribution of TPX2 in dividing leaf cells of the fern Asplenium nidus

Plant cell division requires the dynamic organisation of several microtubule arrays. The mechanisms of regulation of the above arrays are under rigorous research. Among several factors that are involved in plant microtubule dynamics, the Targeting Protein for Xklp2 (TPX2) has been found to play a role in spindle organisation, in combination with Aurora kinases, in dividing cells of angiosperms. Microtubule organisation in dividing cells of ferns exhibits certain peculiarities. Accordingly, the presence and distribution of a TPX2 homologue might be helpful in understanding the patterns and regulatory mechanisms of microtubule arrays in this plant group. In this study, a putative TPX2 homologue was identified using Western blotting in the fern Asplenium nidus. It was found, using immunostaining and CLSM, that it is co‐localised with perinuclear preprophase microtubules and the prophase spindle, and follows the microtubule pattern during metaphase/anaphase and telophase. During cytokinesis, while in angiosperms TPX2 is degraded, in A. nidus the TPX2 signal persists, co‐localising with the phragmoplast. In early post‐cytokinetic cells, a TPX2 signal is present on the nuclear surface facing the daughter cell wall and, thereafter it is co‐localised with the fern‐specific microtubule aggregation that lines the new wall, which is possibly involved in cortical microtubule assembly.     

Cortical microtubule patterning in roots of Arabidopsis thaliana primary cell wall mutants reveals the bidirectional interplay with cell expansion

Cell elongation requires directional deposition of cellulose microfibrils regulated by transverse cortical microtubules. Microtubules respond differentially to suppression of cell elongation along the developmental zones of Arabidopsis thaliana root apex. Cortical microtubule orientation is particularly affected in the fast elongation zone but not in the meristematic or transition zones of thanatos and pom2–4 cellulose-deficient mutants of Arabidopsis thaliana. Here, we report that a uniform phenotype is established among the primary cell wall mutants, as cortical microtubules of root epidermal cells of rsw1 and prc1 mutants exhibit the same pattern described in thanatos and pom2–4. Whether cortical microtubules assume transverse orientation or not is determined by the demand for cellulose synthesis, according to each root zone''s expansion rate. It is suggested that cessation of cell expansion may provide a biophysical signal resulting in microtubule reorientation.