Hydrogen Peroxide Production by the Spot-Like Mode Action of Bisphenol A.

Bisphenol A (BPA), an intermediate chemical used for synthesizing polycarbonate plastics, has now become a wide spread organic pollutant. It percolates from a variety of sources, and plants are among the first organisms to encounter, absorb, and metabolize it, while its toxic effects are not yet fully known. Therefore, we experimentally studied the effects of aqueous BPA solutions (50 and 100 mg L-1, for 6, 12, and 24 h) on photosystem II (PSII) functionality and evaluated the role of reactive oxygen species (ROS) on detached leaves of the model plant Arabidopsis thaliana. Chlorophyll fluorescence imaging analysis revealed a spatiotemporal heterogeneity in the quantum yields of light energy partitioning at PSII in Arabidopsis leaves exposed to BPA. Under low light PSII function was negatively influenced only at the spot-affected BPA zone in a dose- and time-dependent manner, while at the whole leaf only the maximum photochemical efficiency (Fv/Fm) was negatively affected. However, under high light all PSII photosynthetic parameters measured were negatively affected by BPA application, in a time-dependent manner. The affected leaf areas by the spot-like mode of BPA action showed reduced chlorophyll autofluorescence and increased accumulation of hydrogen peroxide (H2O2). When H2O2 was scavenged via N-acetylcysteine under BPA exposure, PSII functionality was suspended, while H2O2 scavenging under non-stress had more detrimental effects on PSII function than BPA alone. It can be concluded that the necrotic death-like spots under BPA exposure could be due to ROS accumulation, but also H2O2 generation seems to play a role in the leaf response against BPA-related stress conditions.

Arbuscular Mycorrhizal Symbiosis Enhances Photosynthesis in the Medicinal Herb Salvia fruticosa by Improving Photosystem II Photochemistry.

We investigated the influence of Salvia fruticosa colonization by the arbuscular mycorrhizal fungi (AMF) Rhizophagus irregularis on photosynthetic function by using chlorophyll fluorescence imaging analysis to evaluate the light energy use in photosystem II (PSII) of inoculated and non-inoculated plants. We observed that inoculated plants used significantly higher absorbed energy in photochemistry (ΦPSII) than non-inoculated and exhibited significant lower excess excitation energy (EXC). However, the increased ΦPSII in inoculated plants did not result in a reduced non-regulated energy loss in PSII (ΦNO), suggesting the same singlet oxygen (1O2) formation between inoculated and non-inoculated plants. The increased ΦPSII in inoculated plants was due to an increased efficiency of open PSII centers to utilize the absorbed light (Fv'/Fm') due to a decreased non-photochemical quenching (NPQ) since there was no difference in the fraction of open reaction centers (qp). The decreased NPQ in inoculated plants resulted in an increased electron-transport rate (ETR) compared to non-inoculated. Yet, inoculated plants exhibited a higher efficiency of the water-splitting complex on the donor side of PSII as revealed by the increased Fv/Fo ratio. A spatial heterogeneity between the leaf tip and the leaf base for the parameters ΦPSII and ΦNPQ was observed in both inoculated and non-inoculated plants, reflecting different developmental zones. Overall, our findings suggest that the increased ETR of inoculated S. fruticosa contributes to increased photosynthetic performance, providing growth advantages to inoculated plants by increasing their aboveground biomass, mainly by increasing leaf biomass.

Tubulin Acetylation Mediates Bisphenol A Effects on the Microtubule Arrays of Allium cepa and Triticum turgidum.

The effects of bisphenol A (BPA), a prevalent endocrine disruptor, on both interphase and mitotic microtubule array organization was examined by immunofluorescence microscopy in meristematic root cells of Triticum turgidum (durum wheat) and Allium cepa (onion). In interphase cells of A. cepa, BPA treatment resulted in substitution of cortical microtubules by annular/spiral tubulin structures, while in T. turgidum BPA induced cortical microtubule fragmentation. Immunolocalization of acetylated α-tubulin revealed that cortical microtubules of T. turgidum were highly acetylated, unlike those of A. cepa. In addition, elevation of tubulin acetylation by trichostatin A in A. cepa resulted in microtubule disruption similar to that observed in T. turgidum. BPA also disrupted all mitotic microtubule arrays in both species. It is also worth noting that mitotic microtubule arrays were acetylated in both plants. As assessed by BPA removal, its effects are reversible. Furthermore, taxol-stabilized microtubules were resistant to BPA, while recovery from oryzalin treatment in BPA solution resulted in the formation of ring-like tubulin conformations. Overall, these findings indicate the following: (1) BPA affects plant mitosis/cytokinesis by disrupting microtubule organization. (2) Microtubule disassembly probably results from impairment of free tubulin subunit polymerization. (3) The differences in cortical microtubule responses to BPA among the species studied are correlated to the degree of tubulin acetylation.

Structural Evidence of Programmed Cell Death Induction by Tungsten in Root Tip Cells of Pisum sativum.

Previous studies have shown that excess tungsten (W), a rare heavy metal, is toxic to plant cells and may induce a kind of programmed cell death (PCD). In the present study we used transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) to investigate the subcellular malformations caused by W, supplied as 200 mg/L sodium tungstate (Na2WO4) for 12 or 24 h, in root tip cells of Pisum sativum (pea), The objective was to provide additional evidence in support of the notion of PCD induction and the presumed involvement of reactive oxygen species (ROS). It is shown ultrastructurally that W inhibited seedling growth, deranged root tip morphology, induced the collapse and deformation of vacuoles, degraded Golgi bodies, increased the incidence of multivesicular and multilamellar bodies, and caused the detachment of the plasma membrane from the cell walls. Plastids and mitochondria were also affected. By TEM, the endoplasmic reticulum appeared in aggregations of straight, curved or concentric cisternae, frequently enclosing cytoplasmic organelles, while by CLSM it appeared in bright ring-like aggregations and was severely disrupted in mitotic cells. However, no evidence of ROS increase was obtained. Overall, these findings support the view of a W-induced vacuolar destructive PCD without ROS enhancement.

Disruption of actin filaments in Zea mays by bisphenol A depends on their crosstalk with microtubules.

Bisphenol A (BPA) is a widespread environmental pollutant, reportedly harmful to living organisms. In plant cells, BPA was shown to disrupt microtubule (MT) arrays and perturb mitosis, but its effects on filamentous actin (F-actin) have not been explored. Here we studied the effects of BPA on actin filaments (AFs) in meristematic root tip and leaf cells of Zea mays, by fluorescent labeling and confocal microscopy. Considering the typical dynamic interaction between MTs and AFs, the effects on these two essential components of the plant cytoskeleton were correlated. It was found that BPA disorganized rapidly AFs in a concentration- and time-dependent manner. The fine filaments were first to be affected, followed by the subcortical bundles, resulting in rod- and ring-like conformations. The observed differences in sensitivity between protodermal and cortex cells were attributed to the deeper location of the latter. Depolymerization or stabilization of MTs by relevant drugs (oryzalin, taxol) revealed that AF susceptibility to BPA depends on MT integrity. Developing leaves required harder and longer treatment to be affected by BPA. Ontogenesis of stomatal complexes was highly disturbed, arrangement of AFs and MT arrays was disordered and accuracy of cell division sequence was deranged or completely arrested. The effect of BPA confirmed that subsidiary cell mother cell polarization is not mediated by F-actin patch neither of preprophase band organization. On the overall, it is concluded that AFs in plant cells constitute a subcellular target of BPA and their disruption depends on their crosstalk with MTs.

Bisphenol A disrupts microtubules and induces multipolar spindles in dividing root tip cells of the gymnosperm Abies cephalonica.

The effects of bisphenol A (BPA), an endocrine chemical disruptor extensively used in the plastic and epoxy resin industry, on dividing root tip cells of the gymnosperm Abies cephalonica Loudon were investigated by confocal laser scanning microscopy after tubulin and endoplasmic reticulum immunolocalization and DNA staining. Microtubule arrays of all mitotic stages were disrupted within a few hours of treatment: preprophase bands exhibited asymmetric width; prometaphase, metaphase and anaphase spindles appeared sharply pointed, sigmoid or multipolar; phragmoplast microtubules were elongated and occasionally bended toward the daughter nuclei. Depending on the mitotic stage, the chromosomes appeared condensed at prophase, as a compact mass at metaphase and anaphase, unsegregated or bridged at telophase. Endoplasmic reticulum patterns were also affected, reflecting those of the respective microtubule arrays. Recovery of the microtubules after oryzalin treatment was more effective in a BPA solution than in water. It is concluded that the plant mitotic apparatus microtubules are very sensitive to BPA, the effect of which depends on the specific cell cycle stage. The formation of multipolar spindles is reminiscent of animal cells and is ascribed to the induction of multiple microtubule nucleation sites, deriving from the centrosomal properties of gymnosperms.

Hexavalent chromium-induced differential disruption of cortical microtubules in some Fabaceae species is correlated with acetylation of α-tubulin.

The effects of hexavalent chromium [Cr(VI)] on the cortical microtubules (MTs) of five species of the Fabaceae family (Vicia faba, Pisum sativum, Vigna sinensis, Vigna angularis, and Medicago sativa) were investigated by confocal laser scanning microscopy after immunolocalization of total tubulin with conventional immunofluorescence techniques and of acetylated α-tubulin with the specific 6-11B-1 monoclonal antibody. Moreover, total α-tubulin and acetylated α-tubulin were quantified by Western immunoblotting and scanning densitometry. Results showed the universality of Cr(VI) detrimental effects to cortical MTs, which proved to be a sensitive and reliable subcellular marker for monitoring Cr(VI) toxicity in plant cells. However, a species-specific response was recorded, and a correlation of MT disturbance with the acetylation status of α-tubulin was demonstrated. In V. faba, MTs were depolymerized at the gain of cytoplasmic tubulin background and displayed low α-tubulin acetylation, while in P. sativum, V. sinensis, V. angularis, and M. sativa, MTs became bundled and changed orientation from perpendicular to oblique or longitudinal. Bundled MTs were highly acetylated as determined by both immunofluorescence and Western immunoblotting. Tubulin acetylation in P. sativum and M. sativa preceded MT bundling; in V. sinensis it followed MT derangement, while in V. angularis the two phenomena coincided. Total α-tubulin remained constant in all treatments. Should acetylation be an indicator of MT stabilization, it is deduced that bundled MTs became stabilized, lost their dynamic properties, and were rendered inactive. Results of this report allow the conclusion that Cr(VI) toxicity disrupts MTs and deranges the MT-mediated functions either by depolymerizing or stabilizing them.

"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.

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.

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).

Aberration of mitosis by hexavalent chromium in some Fabaceae members is mediated by species-specific microtubule disruption.

Because the detrimental effects of chromium (Cr) to higher plants have been poorly investigated, the present study was undertaken to verify the toxic attributes of hexavalent chromium [Cr(VI)] to plant mitotic microtubules (MTs), to determine any differential disruption of MTs during mitosis of taxonomically related species and to clarify the relationship between the visualized chromosomal aberrations and the Cr(VI)-induced MT disturbance. For this purpose, 5-day-old uniform seedlings of Vicia faba, Pisum sativum, Vigna sinensis and Vigna angularis, all belonging to the Fabaceae family, were exposed to 250 µM Cr(VI) supplied as potassium dichromate (K2Cr2O7) for 24, 72 and 120 h and others in distilled water serving as controls. Root tip samples were processed for tubulin immunolabelling (for MT visualization) and DNA fluorescent staining (for chromosomal visualization). Microscopic preparations of cell squashes were then examined and photographed by confocal laser scanning microscopy (CLSM). Cr(VI) halted seedling growth turning roots brown and necrotic. Severe chromosomal abnormalities and differential disturbance of the corresponding MT arrays were found in all mitotic phases. In particular, in V. faba MTs were primarily depolymerized and replaced by atypical tubulin conformations, whereas in P. sativum, V. sinensis and V. angularis they became bundled in a time-dependent manner. In P. sativum, the effects were milder compared to those of the other species, but in all cases MT disturbance adversely affected the proper aggregation of chromosomes on the metaphase plate, their segregation at anaphase and organization of the new nuclei at telophase. Cr(VI) is very toxic to seedling growth. The particular effect depends on the exact stage the cell is found at the time of Cr(VI) entrance and is species-specific. Mitotic MT arrays are differentially deranged by Cr(VI) in the different species examined, even if they are taxonomically related, while their disturbance underlies chromosomal abnormalities. Results furthermore support the view that MTs may constitute a reliable, sensitive and universal subcellular marker for monitoring heavy metal toxicity.

Tungsten disrupts root growth in Arabidopsis thaliana by PIN targeting.

Tungsten is a heavy metal with increasing concern over its environmental impact. In plants it is extensively used to deplete nitric oxide by inhibiting nitrate reductase, but its presumed toxicity as a heavy metal has been less explored. Accordingly, its effects on Arabidopsis thaliana primary root were assessed. The effects on root growth, mitotic cell percentage, nitric oxide and hydrogen peroxide levels, the cytoskeleton, cell ultrastructure, auxin and cytokinin activity, and auxin carrier distribution were investigated. It was found that tungsten reduced root growth, particularly by inhibiting cell expansion in the elongation zone, so that root hairs emerged closer to the root tip than in the control. Although extensive vacuolation was observed, even in meristematic cells, cell organelles were almost unaffected and microtubules were not depolymerized but reoriented. Tungsten affected auxin and cytokinin activity, as visualized by the DR5-GFP and TCS-GFP expressing lines, respectively. Cytokinin fluctuations were similar to those of the mitotic cell percentage. DR5-GFP signal appeared ectopically expressed, while the signals of PIN2-GFP and PIN3-GFP were diminished even after relatively short exposures. The observed effects were not reminiscent of those of any nitric oxide scavengers. Taken together, inhibition of root growth by tungsten might rather be related to a presumed interference with the basipetal flow of auxin, specifically affecting cell expansion in the elongation zone.

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.

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.

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.

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.

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.

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.

Aberrant cell plate formation in the Arabidopsis thaliana microtubule organization 1 mutant.

MICROTUBULE ORGANIZATION 1 encodes a microtubule-associated protein in Arabidopsis thaliana but different alleles have contradictory phenotypes. The original mutant mor1 alleles were reported to have disrupted cortical microtubules, swollen organs and normal cytokinesis, whereas other alleles, embryo-lethal gemini pollen 1 (gem1), have defective pollen cytokinesis. To determine whether MOR1 functions generally in cytokinesis, we examined the ultrastructure of cell division in roots of the original mor1-1 allele. Cell plates are misaligned, branched and meandering; the forming cell plates remain partly vesicular, with electron-dense or lamellar content. Phragmoplast microtubules are abundant but organized aberrantly. Thus, MOR1 functions in both phragmoplast and cortical arrays.