Ioannis-Dimosthenis S. Adamakis

Effects of hexavalent chromium on microtubule organization, ER distribution and callose deposition in root tip cells of Allium cepa 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.

A role for katanin in plant cell division: Microtubule organization in dividing root cells of fra2 and lue1Arabidopsis thaliana mutants

Severing of microtubules by katanin has proven to be crucial for cortical microtubule organization in elongating and differentiating plant cells. On the contrary, katanin is currently not considered essential during cell division in plants as it is in animals. However, defects in cell patterning have been observed in katanin mutants, implying a role for it in dividing plant cells. Therefore, microtubule organization was studied in detail by immunofluorescence in dividing root cells of fra2 and lue1 katanin mutants of Arabidopsis thaliana. In both, early preprophase bands consisted of poorly aligned microtubules, prophase spindles were multipolar, and the microtubules of expanding phragmoplasts were elongated, bended toward and connected to the surface of daughter nuclei. Accordingly, severing by katanin seems to be necessary for the proper organization of these microtubule arrays. In both fra2 and lue1, metaphase/anaphase spindles and initiating phragmoplasts exhibited typical organization. However, they were obliquely oriented more frequently than in the wild type. It is proposed that this oblique orientation may be due to prophase spindle multipolarity and results in a failure of the cell plate to follow the predetermined division plane, during cytokinesis, producing oblique cell walls in the roots of both mutants. It is therefore concluded that, like in animal cells, katanin is important for plant cell division, influencing the organization of several microtubule arrays. Moreover, failure in microtubule severing indirectly affects the orientation of the division plane.

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 100mg/L BPA for 1, 3, 6, 12 and 24h. 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.

The fatal effect of tungsten on Pisum sativum 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.

Tungsten affects the cortical microtubules of Pisum sativum root cells: experiments on tungsten-molybdenum antagonism.

Tungsten (W) is increasingly shown to be toxic to various organisms, including plants. Apart from inactivation of molybdo-enzymes, other potential targets of W toxicity in plants, especially at the cellular level, have not yet been revealed. In the present study, the effect of W on the cortical microtubule array of interphase root tip cells was investigated, in combination with the possible antagonism of W for the pathway of molybdenum (Mo). Pisum sativum seedlings were treated with W, Mo or a combination of the two, and cortical microtubules were examined using tubulin immunofluorescnce and TEM. Treatments with anti-microtubule (oryzalin, colchicine and taxol) or anti-actomyosin (cytochalasin D, BDM or ML-7) drugs and W were also performed. W-affected cortical microtubules were low in number, short, not uniformly arranged and were resistant to anti-microtubule drugs. Cells pre-treated with oryzalin or colchicine and then treated with W displayed W-affected microtubules, while cortical microtubules pre-stabilized with taxol were resistant to W. Treatment with Mo and anti-actomyosin drugs prevented W from affecting cortical microtubules. Cortical microtubule recovery after W treatment was faster in Mo solution than in water. The results indicate that cortical microtubules of plant cells are indirectly affected by W, most probably through a mechanism depending on the in vivo antagonism of W for the Mo-binding site of Cnx1 protein.

Leaf Age-Dependent Photoprotective and Antioxidative Response Mechanisms to Paraquat-Induced Oxidative Stress in Arabidopsis thaliana

Exposure of Arabidopsis thaliana young and mature leaves to the herbicide paraquat (Pq) resulted in a localized increase of hydrogen peroxide (H2O2) in the leaf veins and the neighboring mesophyll cells, but this increase was not similar in the two leaf types. Increased H2O2 production was concomitant with closed reaction centers (qP). Thirty min after Pq exposure despite the induction of the photoprotective mechanism of non-photochemical quenching (NPQ) in mature leaves, H2O2 production was lower in young leaves mainly due to the higher increase activity of ascorbate peroxidase (APX). Later, 60 min after Pq exposure, the total antioxidant capacity of young leaves was not sufficient to scavenge the excess reactive oxygen species (ROS) that were formed, and thus, a higher H2O2 accumulation in young leaves occurred. The energy allocation of absorbed light in photosystem II (PSII) suggests the existence of a differential photoprotective regulatory mechanism in the two leaf types to the time-course Pq exposure accompanied by differential antioxidant protection mechanisms. It is concluded that tolerance to Pq-induced oxidative stress is related to the redox state of quinone A (QA).

Chromium-Induced Ultrastructural Changes and Oxidative Stress in Roots of Arabidopsis thaliana

Chromium (Cr) is an abundant heavy metal in nature, toxic to living organisms. As it is widely used in industry and leather tanning, it may accumulate locally at high concentrations, raising concerns for human health hazards. Though Cr effects have extensively been investigated in animals and mammals, in plants they are poorly understood. The present study was then undertaken to determine the ultrastructural malformations induced by hexavalent chromium [Cr(VI)], the most toxic form provided as 100 μM potassium dichromate (K2Cr2O7), in the root tip cells of the model plant Arabidopsis thaliana. A concentration-dependent decrease of root growth and a time-dependent increase of dead cells, callose deposition, hydrogen peroxide (H2O2) production and peroxidase activity were found in Cr(VI)-treated seedlings, mostly at the transition root zone. In the same zone, nuclei remained ultrastructurally unaffected, but in the meristematic zone some nuclei displayed bulbous outgrowths or contained tubular structures. Endoplasmic reticulum (ER) was less affected under Cr(VI) stress, but Golgi bodies appeared severely disintegrated. Moreover, mitochondria and plastids became spherical and displayed translucent stroma with diminished internal membranes, but noteworthy is that their double-membrane envelopes remained structurally intact. Starch grains and electron dense deposits occurred in the plastids. Amorphous material was also deposited in the cell walls, the middle lamella and the vacuoles. Some vacuoles were collapsed, but the tonoplast appeared integral. The plasma membrane was structurally unaffected and the cytoplasm contained opaque lipid droplets and dense electron deposits. All electron dense deposits presumably consisted of Cr that is sequestered from sensitive sites, thus contributing to metal tolerance. It is concluded that the ultrastructural changes are reactive oxygen species (ROS)-correlated and the malformations observed are organelle specific.

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.

Microtubule integrity and cell viability under metal (Cu, Ni and Cr) stress in the seagrass Cymodocea nodosa

The effects of increasing Cu, Ni and Cr concentrations (0.5, 5, 10, 20 and 40 mg L(-1)) on microtubule organization and the viability of leaf cells of the seagrass Cymodocea nodosa for 13 consecutive days were investigated under laboratory conditions. Increased oblique microtubule orientation, microtubule depolymerization at the 5-40 mg L(-1) Ni treatments after 3 d of exposure, and a complete microtubule depolymerization at all Ni treatments after 5 d were observed. Cu depolymerised microtubules after three to 7 d of exposure, while Cr caused an extensive microtubule bundling after 9 or 11 d of exposure, depending on metal dosage. Fluorescence intensity measurements further consolidated the above phenomena. Cell death, occurring at later time than microtubule disturbance, was also observed at all Cu and Ni treatments and at the 10-40 mg L(-1) Cr treatments and adding to the above quantification of the number of dead cells clearly showed that only a portion of the cell population studied died. The data presented, being the first assessment of microtubule disturbance in seagrasses, indicate that microtubules in seagrass leaf cells could be used as a valuable and early marker of metal-induced stress in biomonitoring programmes.

Tungsten Toxicity in Plants

Tungsten (W) is a rare heavy metal, widely used in a range of industrial, military and household applications due to its unique physical properties. These activities inevitably have accounted for local W accumulation at high concentrations, raising concerns about its effects for living organisms. In plants, W has primarily been used as an inhibitor of the molybdoenzymes, since it antagonizes molybdenum (Mo) for the Mo-cofactor (MoCo) of these enzymes. However, recent advances indicate that, beyond Mo-enzyme inhibition, W has toxic attributes similar with those of other heavy metals. These include hindering of seedling growth, reduction of root and shoot biomass, ultrastructural malformations of cell components, aberration of cell cycle, disruption of the cytoskeleton and deregulation of gene expression related with programmed cell death (PCD). In this article, the recent available information on W toxicity in plants and plant cells is reviewed, and the knowledge gaps and the most pertinent research directions are outlined.