Effects of sodium tungstate on the ultrastructure and growth of pea (Pisum sativum) and cotton (Gossypium hirsutum) seedlings

Pea (Pisum sativum L. cv. Onmard) and cotton (Gossypium hirsutum L. cv. Campo) seedlings were treated with two concentrations (200 and 500 mg/l) of sodium tungstate (Na₂WO₄) and the developmental effects were investigated. Tungstate retarded seedling growth rate and stopped root elongation in both species. Seedling growth recovered when tungstate was removed, but primary roots continued to be stunted, while lateral root initiation and growth were stimulated. Tungstate induced premature vacuolation in cells of the root apical meristem, with vacuoles having an unusual semi-circular or cap-like shape around the nucleus. In control roots, the nuclei were spherical with prominent nucleoli bearing several randomly distributed fibrillar centres. In the tungstate-treated cells nuclei contained spherical nucleoli with a big nucleolar vacuole. Occasionally, cytoplasmic components, such as mitochondria, were entrapped in the nucleoplasm of interphasic cells of the treated roots. In these roots, most cell plates were fused to only one lateral parental wall suggesting a non-uniform centrifugal extension. The vesicles in these cell plates were dark and fused to each other at a much lower rate than in the dividing cells of the untreated seedlings. Phragmoplast and cortical microtubules were abundant in the untreated cells, but scarcely detected in the treated ones. All these observations are consistent with the view that tungstate causes considerable toxic effects to pea and cotton seedlings.     

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.     

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.     

“CLASPing” tungsten's effects on microtubules with “PINs”

Tungsten, supplied as sodium tungstate, inhibits root elongation in Arabidopsis thaliana, which has been attributed to a diminishing of PIN2 and PIN3 auxin efflux carriers. In this work, we sought to analyze the effect of tungsten on cortical microtubules and CLASP (Cytoplasmic Linker Associated Protein), which are also involved in the anisotropic cell expansion of root cells. Seedlings grown in a tungsten-free substrate for 4 d and then transplanted into a tungsten-containing substrate exhibited randomly oriented microtubules in a time-dependent manner. While tungsten had no effect on roots treated for 3 h, microtubule alignment was obviously affected in the transition and elongation zones after a 6, 12, 24, 48 h tungsten treatment, at prolonged tungsten administrations and in seedlings grown directly in the presence of tungsten. This change in microtubule orientation may be associated with the reduction of CLASP protein expression induced by tungsten, as evidenced in experiments with plants expressing the CLASP-GFP protein. A possible mechanism, by which the coordinated functions of CLASP, PIN2 and microtubules are affected, as revealed by inhibited root growth, is discussed.     

The nitrate reductase inhibitor,tungsten, disrupts actin microfilaments in Zea mays L.

Tungsten is a widely used inhibitor of nitrate reductase, applied to diminish the nitric oxide levels in plants. It was recently shown that tungsten also has heavy metal attributes. Since information about the toxic effects of tungsten on actin is limited, and considering that actin microfilaments are involved in the entry of tungsten inside plant cells, the effects of tungsten on them were studied in Zea mays seedlings. Treatments with sodium tungstate for 3, 6, 12 or 24 h were performed on intact seedlings and seedlings with truncated roots. Afterwards, actin microfilaments in meristematic root and leaf tissues were stained with fluorescent phalloidin, and the specimens were examined by confocal laser scanning microscopy. While the actin microfilament network was well organized in untreated seedlings, in tungstate-treated ones it was disrupted in a time-dependent manner. In protodermal root cells, the effects of tungsten were stronger as cortical microfilaments were almost completely depolymerized and the intracellular ones appeared highly bundled. Fluorescence intensity measurements confirmed the above results. In the meristematic leaf tissue of intact seedlings, no depolymerization of actin microfilaments was noticed. However, when root tips were severed prior to tungstate application, both cortical and endoplasmic actin networks of leaf cells were disrupted and bundled after 24 h of treatment. The differential response of root and leaf tissues to tungsten toxicity may be due to differential penetration and absorption, while the effects on actin microfilaments could not be attributed to the nitric oxide depletion by tungsten.