DNA damage in potato plants induced by cadmium,ethyl methanesulphonate and γ-rays

We have calibrated the alkaline protocol of the plant comet (Single Cell Gel Electrophoresis) assay as a method for detecting the extent of induced DNA damage in potato plants (Solanum tuberosum L. cultivar Korela). After 2 and 24 h treatments of the rooted cuttings with the heavy metal cadmium (Cd²⁺), a dose–response increase in DNA damage was noted versus controls in root nuclei. With a 24 h recovery period, the Cd²⁺-induced DNA damage in roots increased significantly. No significant increase in DNA damage was demonstrated in leaf nuclei after 24 h Cd²⁺ treatments, but continuous Cd²⁺ treatments for 2 weeks resulted in an increase in leaf DNA damage. This increase may be however associated with necrotic and apoptotic DNA fragmentation, as the affected plants had inhibited growth and distorted yellowish leaves. For comparison, the monofunctional alkylating agent ethyl methanesulphonate, and γ-rays were assessed for induced DNA damage. Analysis of the accumulation of cadmium by inductively coupled plasma optical emission spectrometry demonstrates that roots accumulate almost 9-fold more cadmium than aboveground parts of the rooted potato cuttings. This may explain the absence of Cd²⁺ genotoxicity in leaves after short-term treatments.     

Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants

Tobacco (Nicotiana tabacum L. var. xanthi) seedlings were treated with aqueous solutions of lead nitrate (Pb²⁺) at concentrations ranging from 0.4 mM to 2.4 mM for 24 h and from 25 μM to 200 μM for 7 days. The DNA damage measured by the comet assay was high in the root nuclei, but in the leaf nuclei a slight but significant increase in DNA damage could be demonstrated only after a 7-day treatment with 200 μM Pb²⁺. In tobacco plants growing for 6 weeks in soil polluted with Pb²⁺ severe toxic effects, expressed by the decrease in leaf area, and a slight but significant increase in DNA damage were observed. The tobacco plants with increased levels of DNA damage were severely injured and showed stunted growth, distorted leaves and brown root tips. The frequency of somatic mutations in tobacco plants growing in the Pb²⁺-polluted soil did not significantly increase. Analytical studies by inductively coupled plasma optical emission spectrometry demonstrate that after a 24-h treatment of tobacco with 2.4 mM Pb²⁺, the accumulation of the heavy metal is 40-fold higher in the roots than in the above-ground biomass. Low Pb²⁺ accumulation in the above-ground parts may explain the lower levels or the absence of Pb²⁺-induced DNA damage in leaves.     

Potato virus X induces DNA damage in leaf nuclei of the host plant Nicotiana tabacum L. var. xanthi

We employed the comet assay (single cell gel electrophoresis) to evaluate induced DNA damage in nuclei isolated from tobacco leaves (Nicotiana tabacum var. xanthi) inoculated with Potato virus X (PVX). The highest DNA damage, expressed by the tail moment value, was observed in the inoculated leaves and decreased in the 1^st to 4^th systemic leaves. DNA damage increased with the time after the inoculation (from day 3 to day 21) and was higher in nuclei isolated from a part of the leaf at the petiole compared to nuclei isolated from the leaf tip. A Pearson moment correlation (r = 0.94) between the induced DNA damage and the PVX titres expressed by ELISA absorbance values was observed. The PVX infection did not induce a significant increase in the rate of somatic mutations evaluated by appearance of dark green, yellow, and double green/yellow sectors on the heterozygous pale green leaves of N. tabacum var. xanthi.     

Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants

Tobacco (Nicotiana tabacum L. var. xanthi) seedlings were treated with aqueous solutions of lead nitrate (Pb2+) at concentrations ranging from 0.4 mM to 2.4 mM for 24 h and from 25 microM to 200 microM for 7 days. The DNA damage measured by the comet assay was high in the root nuclei, but in the leaf nuclei a slight but significant increase in DNA damage could be demonstrated only after a 7-day treatment with 200 microM Pb2+. In tobacco plants growing for 6 weeks in soil polluted with Pb2+ severe toxic effects, expressed by the decrease in leaf area, and a slight but significant increase in DNA damage were observed. The tobacco plants with increased levels of DNA damage were severely injured and showed stunted growth, distorted leaves and brown root tips. The frequency of somatic mutations in tobacco plants growing in the Pb2+-polluted soil did not significantly increase. Analytical studies by inductively coupled plasma optical emission spectrometry demonstrate that after a 24-h treatment of tobacco with 2.4 mM Pb2+, the accumulation of the heavy metal is 40-fold higher in the roots than in the above-ground biomass. Low Pb2+ accumulation in the above-ground parts may explain the lower levels or the absence of Pb2+-induced DNA damage in leaves.     

Identification and characterization of zinc-starvation-induced ZIP transporters from barley roots

Zinc (Zn) is an essential element for plants but limited information is currently available on the molecular basis for Zn²⁺ transport in crop species. To expand the knowledge on Zn²⁺ transport in barley (Hordeum vulgare L.), a cDNA library prepared from barley roots was expressed in the yeast (Saccharomyces cerevisiae) mutant strain Δzrt1/Δzrt2, defective in Zn²⁺ uptake. This strategy resulted in isolation and identification of three new Zn²⁺ transporters from barley. All of the predicted proteins have a high similarity to the ZIP protein family, and are designated HvZIP3, HvZIP5 and HvZIP8, respectively. Complementation studies in Δzrt1/Δzrt2 showed restored growth of the yeast cells transformed with the different HvZIPs, although with different efficiency. Transformation into Fe²⁺ and Mn²⁺ uptake defective yeast mutants showed that the HvZIPs were unable to restore the growth on Fe²⁺ and Mn²⁺ limited media, respectively, indicating a specific role in Zn²⁺ transport. In intact barley roots, HvZIP8 was constitutively expressed whereas HvZIP3 and HvZIP5 were mainly expressed in ?Zn plants. These results suggest that HvZIP3, HvZIP5 and HvZIP8 are Zn²⁺ transporters involved in Zn²⁺ homeostasis in barley roots. The new transporters may facilitate breeding of barley genotypes with improved Zn efficiency and Zn content.     

Deoxymugineic acid increases Zn translocation in Zn-deficient rice plants

Deoxymugineic acid (DMA) is a member of the mugineic acid family phytosiderophores (MAs), which are natural metal chelators produced by graminaceous plants. Rice secretes DMA in response to Fe deficiency to take up Fe in the form of Fe(III)–MAs complex. In contrast with barley, the roots of which secrete MAs in response to Zn deficiency, the amount of DMA secreted by rice roots was slightly decreased under conditions of low Zn supply. There was a concomitant increase in endogenous DMA in rice shoots, suggesting that DMA plays a role in the translocation of Zn within Zn-deficient rice plants. The expression of OsNAS1 and OsNAS2 was not increased in Zn-deficient roots but that of OsNAS3 was increased in Zn-deficient roots and shoots. The expression of OsNAAT1 was also increased in Zn-deficient roots and dramatically increased in shoots; correspondingly, HPLC analysis was unable to detect nicotianamine in Zn-deficient shoots. The expression of OsDMAS1 was increased in Zn-deficient shoots. Analyses using the positron-emitting tracer imaging system (PETIS) showed that Zn-deficient rice roots absorbed less ⁶²Zn-DMA than ⁶²Zn²⁺. Importantly, supply of ⁶²Zn-DMA rather than ⁶²Zn²⁺ increased the translocation of ⁶²Zn into the leaves of Zn-deficient plants. This was especially evident in the discrimination center (DC). These results suggest that DMA in Zn-deficient rice plants has an important role in the distribution of Zn within the plant rather than in the absorption of Zn from the soil. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Motofumi Suzuki and Takashi Tsukamoto equally contributed to this work.     

Application of chelator-buffered nutrient solution technique in studies on zinc nutrition in rice plant (Oryza sativa L.)

It has been difficult to impose different degrees of Zn deficiency on Poaceae species in nutrient solution because most chelators which would control Zn to low activities also bind Fe³⁺ so strongly that Poaceae species cannot obtain adequate Fe. Recently, a method has been developed to provide buffered Fe²⁺ at levels adequate for rice using Ferrozine (FZ), and use of other chelators to buffer the other micronutrient cations. The use of Fe²⁺ buffered with FZ in nutrient solutions in which Zn is buffered with HEDTA or DTPA was evaluated for study of Zn deficiency in rice compared to a conventional nutrient solution technique. The results showed that growth of rice plants in FZ+HEDTA-buffered nutrient solution was similar to that in the conventional nutrient solution. Severe zinc deficiency symptoms were observed in 28-day-old rice seedlings cultured with HEDTA-buffered nutrient solution at Zn²⁺ activities < 10^-10.6 M. With increasing free Zn²⁺ activities, concentrations of Zn, Fe, Cu, and Mn in shoots and roots were quite similar for the FZ+HEDTA-buffered nutrient solution and the conventional nutrient solution techniques. The percentages of water soluble Zn, Fe, Cu and Mn in shoots with HEDTA-buffered nutrient solution were also similar to those with the conventional solution. However, with DTPA-buffered nutrient solution, the rice seedlings suffered severe Fe deficiency; adding more FeFZ₃ corrected the Fe-chlorosis but shifted microelement buffering. Further, much higher total Zn concentrations are required to provide adequate Zn²⁺ in DTPA-buffered solutions, and the contents of Mn and Cu in shoots and roots cultured with DTPA-buffered solutions were much higher than those with the conventional or HEDTA-buffered solutions. In conclusion, DTPA-buffered nutrient solutions are not suitable but the FZ/HEDTA-buffered nutrient solution technique can be used to evaluate genotypic differences in zinc efficiency in rice.     

The Relationship between Salinity and Cadmium Stress in Barley

Distribution of cadmium between roots and shoots of barley was manipulated by the cadmium concentration (0.01 and 0.005 mM Cd²⁺), pH (4.6 and 5.9) as well as treatment duration. The prolongation of treatment increased dry mass and content of cadmium in plants. The cadmium is accumulated mainly in roots. Presence of both, 0.005 mM Cd²⁺ and 100 mM NaCl in medium at pH 5.9 (Cd-NaCl plants) resulted in the most severe growth inhibition of plants, but about one half accumulation of cadmium in roots then in a case of only Cd-treated plants. In the Cd-NaCl plants, the net photosynthetic and transpiration rates were less reduced then in a case of only NaCl-treated plants. The treatments also influenced uptake of Ca, Cd, Cu, K, Mg, Na and Zn predominantly in roots. This revised version was published online in July 2006 with corrections to the Cover Date.     

Does zinc concentration in the substrate influence the onset of flowering in Arabidopsis arenosa (Brassicaceae)?

We investigated the impact of low zinc (Zn) concentrations in the substare on the onset of flowering in Arabidopsis arenosa (Brassicaceae). Experiments were carried out in controlled conditions using plants from four different populations. The research was aimed to verify experimentally the following hypotheses: (1) Zn content in the growth medium promote the onset of flowering in A. arenosa, (2) Changes in the onset of flowering induced by Zn depend on Zn concentration employed; (3) Zn-induced early onset of flowering is an universal plant response present within the species and is not an effect of stress or physiological adaptation to high Zn content in the environment. Investigated plants were subjected to four different Zn concentrations: 0.4 (control), 155, 775 and 1,550???M Zn²⁺. To asses stress level in investigated plants we calculated biomass accumulation and employed fluorometric methods. Zn content was estimated in shoots using atomic absorption spectroscopy. Differences in the onset of flowering were assessed using Kaplan?CMeier curves. Our results showed that Zn was transported form growth medium to roots and shoots of investigated plants and that the content of Zn increased with the increase of Zn concentration in the growth medium. We evidenced that apart from one (1,550???M Zn²⁺) applied Zn concentrations did not caused stress in investigated plants what was confirmed by two independent experimental approaches: measurement of biomass accumulation and chlorophyll a fluorescence. Flowering curves obtained on the basis of calculation of Kaplan?CMeier estimator showed that: (1) control plants originating from four different populations did not differ in terms of the onset of flowering, (2) plants from each population tested tends to enter flowering phase earlier in response to applied Zn concentrations than control plants, (3) plants treated with the lowest tested Zn concentration (155???M Zn²⁺) tend to flower earlier than plants treated with the higher concentration (775???M Zn²⁺), (4) the impact of Zn on the onset of flowering did not depend on the origin on the plant material used (Zn-rich or Zn-poor soils). Our results indicate that Zn ions present in the growth medium promote early flowering in A.arenosa and that this effect may depend on Zn concentration used. Zn-induced early flowering in A. arenosa seems to be an universal plant response present within the species and is not an effect of stress or physiological adaptation to high Zn content in the environment.     

Assessing of tolerance to metallic and saline stresses in the halophyte Suaeda fruticosa: The indicator role of antioxidative enzymes

Many areas are simultaneously affected by high concentrations of salts and trace metal elements (TME), the latter constituting a serious threat to human health. In the present study, we determined the combined effect of high salinity and toxic levels of trace elements on physiological behavior of the halophytic species Suaeda fruticosa. Plants were cultivated for three months with an irrigation solution supplemented separately with different concentrations of Pb²⁺ and Zn²⁺ (0, 200, 400 and 600 μM) with and without 200 mM NaCl. Growth, total chlorophyll, water status and ion nutrition were quantified and antioxidant enzyme activities [ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and catalase (CAT)] were studied. Our results revealed that S. fruticosa has a strong ability to tolerate lead and zinc. This halophyte accumulated higher concentrations of TME in their roots. Growth parameters of S. fruticosa were not significantly affected by TME. An enhancement of Ca²⁺ concentration accompanied by a decrease of Mg²⁺ content was observed under Pb²⁺ or Zn²⁺ treatments whereas K⁺ content was not affected by TME. Of the antioxidant enzymes, the activity of CAT and APX was increased by metal stress. However, the activity of GPX was diminished by increasing TME concentrations. It was concluded that NaCl 200 mM had a positive impact on the response of S. fruticosa to Zn²⁺ toxicity, acting through a decrease in Zn absorption.     

Adding Zn2+ induces DNA fragmentation and cell condensation in cultured human Chang liver cells

Zinc (Zn) is a trace element in human cells and regarded as an essential nutrient with established deficiency states affecting multiple organs in the body. However, it has been reported that Zn uptake is associated with some serious harmful effects, such as inhibition of DNA synthesis and enhanced toxicity from reactive oxygen species. We have previously shown that in vivo administration of Zn²⁺ in C57/6J mice induces weight loss and massive hair loss where the normal course hair becomes replaced by fine vello hair, simulating the side effects from cancer chemotherapy where oxidative free radical damage is implicated in association with DNA fragmentation and programmed cell death (PCD). Here, in vitro flow cytometric studies on human Chang liver showed Zn²⁺ causing cell condensation with DNA fragmentation that occurred in a dose-dependent manner, an effect replicated by micrococcal nuclease digestion. Specific terminal deoxynucleotidyl transferase- (TdT) mediated labeling of 3′-OH ends of DNA nicks corroborated the flow cytometric profiles of propidium iodide-DNA binding where degradation of both 2 and 4N genomic DNA resulted in a solitary 1N peak presentation. DNA degradation concomitant with cell condensation is seen as an estabilished hallmark of PCD. We further showed that Zn²⁺ could enhance the generation of hydroxyl free radicals (OH^?) by the transition metal vanadium. Glutathione, the cell's main reducing agent, underwent corresponding reduction. The results suggested that Zn supplementation could induce features resembling PCD.     

Phytoremediation Potential and Nutrient Status of Barringtonia acutangula Gaerth. Tree Seedlings Grown Under Different Chromium (CrVI) Treatments

To investigate the effect of different chromium (CrVI) treatments on seedlings of semi-aquatic plant Barringtonia acutangula, hydroponic experiments were conducted. Results revealed that B. acutangula could tolerate much higher CrVI concentration accumulated about 751–2,703 mg kg^?1 dry weight in roots and 50–1,101 mg kg^?1 dry weight in shoots, respectively, under 1.0, 2.0, 3.0, 4.0, and 5.0 mM chromium treatments. CrVI exposure at 1.0–4.0 mM does not exhibit toxicity signs; however, up to 4.0 mM CrVI exposure causes significant decline in growth parameters. Content of macronutrients such as Ca and K decreased under different Cr treatments in roots and shoots, while Mg content of roots and shoots did not influence at the range of 1.0–4.0 mM Cr; however, significant decrease at 5.0 mM Cr, besides P content, significantly shows increasing trends, respectively. Interestingly, sulfur content of roots and shoots show increasing trends at 1.0–2.0 mM Cr; however, severe decrease of up to 3.0–5.0 mM is shown in CrVI treatments. Furthermore, micronutrients content were enhanced under CrVI treatments excluding Cu and Fe since they show significant reduction in shoots as well as in roots. Bioaccumulation factor were also calculated on the basis of results obtained which shows the value of >1 without viewing chromium toxicity symptoms. This study demonstrated that B. acutangula could tolerate CrVI concentrations up to 1.0–4.0 mM Cr which may be useful in chromium phytoremediation programs.     

Role of Organic Acids in Sunflower Tolerance to Heavy Metals

Exposure of Helianthus annuus L. seedlings to Al³⁺, Cd²⁺ or Zn²⁺ resulted in a marked decrease of fresh and dry masses of the shoots and the roots. The increase of Al³⁺, Cd²⁺ or Zn²⁺ uptake was accompanied by a significant decrease of nitrate, phosphorus and K⁺ uptake. There was a significant increase of malic and citric acid contents in the shoots and roots of heavy metal-treated seedlings whereas the change in fumaric acid was insignificant. Al³⁺ and Zn²⁺ alone stimulated excretion of malic and citric acids to the rhizosphere. Addition of high concentrations of malic or citric acid alleviate to some extent the inhibitory effect of Al³⁺ and Zn²⁺ on plant growth. This revised version was published online in July 2006 with corrections to the Cover Date.     

Diacylglycerol kinase in plasma membranes from wheat

Diacylglycerol kinase activity was demonstrated in highly purified plasma membranes isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partitioning. The active site of the diacylglycerol kinase was localized to the inner cytoplasmic surface of the plasma membrane using isolated inside-out and right-side-out plasma membrane vesicles from roots. The enzyme activity in plasma membrane vesicles from shoots showed a broad pH optimum around pH 7. The reaction was Mg²⁺ and ATP dependent, and maximal activity was observed around 0.5 mM ATP and 3 mM MgCl₂. The Mg²⁺ requirement could be substituted only partially by Mn²⁺ and not at all by Ca²⁺. The phosphorylation of endogenous diacylglycerol was strongly inhibited by detergents indicating an extreme dependence of the lipid environment. Inositol phospholipids stimulated the activity of diacylglycerol kinase in plasma membranes from shoots and roots, whereas the activity was inhibited by R59022, a putative inhibitor of several diacylglycerol kinase isoenzymes involved in uncoupling diacylglycerol activation of mammalian protein kinase C.     

Determination of ACC-induced cell-programmed death in roots of Vicia faba ssp. minor seedlings by acridine orange and ethidium bromide staining

Fluorescence staining with acridine orange (AO) and ethidium bromide (EB) showed that nuclei of cortex root cells of 1-aminocyclopropane-1-carboxylic acid (ACC)-treated Vicia faba ssp. minor seedlings differed in color. Measurement of resultant fluorescence intensity (RFI) showed that it increased when the color of nuclear chromatin was changed from green to red, indicating that EB moved to the nuclei via the cell membrane which lost its integrity and stained nuclei red. AO/EB staining showed that changes in color of the nuclear chromatin were accompanied by DNA condensation, nuclei fragmentation, and chromatin degradation which were also shown after 4,6-diamidino-2-phenylindol staining. These results indicate that ACC induced programmed cell death. The increasing values of RFI together with the corresponding morphological changes of nuclear chromatin were the basis to prepare the standard curve; cells with green unchanged nuclear chromatin were alive while those with dark orange and bright red nuclei were dead. The cells with nuclei with green–yellow, yellow–orange, and bright orange chromatin with or without their condensation and fragmentation chromatin were dying. The prepared curve has became the basis to draw up the digital method for detection and determination of the number of living, dying, and dead cells in an in planta system and revealed that ACC induced death in about 20% of root cortex cells. This process was accompanied by increase in ion leakage, shortening of cells and whole roots, as well as by increase in weight and width of the apical part of roots and appearance of few aerenchymatic spaces while not by internucleosomal DNA degradation.     

Effect of heavy metal ions on growth and biochemical characteristics of photosynthesis of barley and maize seedlings

The effects of Cu²⁺, Zn²⁺, Cd²⁺ and Pb²⁺ on growth and the biochemical characteristics of photosynthesis were more expressed in barley (Hordeum vulgare L.) than in maize (Zea mays L.) seedlings. The barley and maize seedlings exhibited retardation in shoot and root growth after exposure of Cu²⁺, Cd²⁺ and Pb²⁺. The Zn²⁺ions practically did not influence these characteristics. The total protein content of barley and maize roots declined with an increase in heavy metal ion concentrations. The protein content of barley shoots was only slighly decreased with an increase in heavy metal ion concentrations, but the protein content in maize shoots was increased under the same conditions. The chlorophyll content was decreased in barley shoots and increased in maize. The ribulose-l,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activities were decreased drastically by Cu²⁺, Cd²⁺ and Pb²⁺ in thein vivo experiments. The tested heavy metal ions affect photosynthesis probably mainly by inhibition of these key carboxylating enzymes: this mechanism was studied in thein vitro experiments.     

不同浓度Fe2+与Zn2+对羊草幼苗生长和生理特性的影响

以羊草幼苗为研究对象,通过调整全营养培养基(CK,0.05 mmol/L Fe²⁺、0.015 mmol/L Zn²⁺)中铁或者锌含量设置0、10倍、20倍Fe²⁺(Zn²⁺)浓度处理Fe₀(Zn₀)、Fe₁₀(Zn₁₀)、Fe₂₀(Zn₂₀),以及在高铁培养基中单独添加0.15 mmol/L Zn²⁺或同时添加10 mmol/L Ca²⁺、5 mmol/L Mg²⁺、20 mmol/L K⁺处理,测定培养6 d后幼苗生长指标和矿质元素含量、以及高铁(Fe₂₀)处理下幼苗根中抗氧化指标和相关基因表达量,探究不同浓度Fe²⁺、Zn²⁺对羊草幼苗生长、矿质元素吸收积累及抗氧化指标、基因表达的影响。结果表明：(1)缺锌(Zn₀)显著抑制羊草幼苗鲜重的增加和Zn元素的积累,但促进Fe、Mg元素的积累;高浓度锌(Zn₁₀、Zn₂₀)显著促进幼苗叶片生长和Zn元素的积累;缺铁(Fe₀)显著抑制幼苗的根长、鲜重和Fe元素的积累,促进Mg、Zn元素的积累;高浓度铁(Fe₁₀、Fe₂₀)显著抑制羊草幼苗根叶生长、根毛发育和Ca、Zn、Mg、K元素的积累。(2)增加Zn²⁺和Ca²⁺、Mg²⁺、K⁺浓度无法恢复高铁胁迫对幼苗生长的抑制作用。(3)高浓度铁(Fe₂₀)处理羊草幼苗48 h后,根部过氧化物酶、超氧化物歧化酶、过氧化氢酶、抗坏血酸过氧化物酶、谷胱甘肽还原酶活性和丙二醛、抗坏血酸、还原型谷胱甘肽含量显著升高;烟酰胺合成酶基因、过氧化物酶基因表达量显著下调,植物类萌发素蛋白基因表达量显著上调。研究发现,羊草幼苗生长发育和矿质元素积累对环境中Zn²⁺浓度变化不敏感,却受到环境中高浓度Fe²⁺的显著抑制,并造成严重的氧化胁迫伤害,这种伤害无法在添加Zn²⁺或同时添加Ca²⁺、Mg²⁺、K⁺的条件下恢复。