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.     

Monitoring toxicity, DNA damage, and somatic mutations in tobacco plants growing in soil heavily polluted with polychlorinated biphenyls

Heterozygous tobacco (Nicotiana tabacum var. xanthi) plants were cultivated in soil from a dump site highly polluted with polychlorinated biphenyls (PCBs) at Lhenice in South Bohemia, Czech Republic. The total amount of PCBs in the polluted soil, measured by gas chromatography varied from 165 to 265mgkg(-1) of soil. In tobacco plants cultivated for 8 weeks in the polluted soil the amount of PCB in the leaves varied from 11 to 28 and in the roots from 104 to 308mgkg(-1) dry mass. The average leaf area of tobacco plants growing in the PCB-polluted soil was significantly reduced and the DNA damage in leaf nuclei, measured by the comet assay, was slightly but significantly increased compared with controls. The tobacco plants with increased DNA damage showed reduced growth and had distorted leaves. No increase in the frequency of somatic mutations was detected in tobacco plants growing in the PCB-polluted soil.     

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.     

Zinc induces DNA damage in tobacco roots

We applied the alkaline version of the single-cell gel electrophoresis (comet assay) to seedlings of heterozygous tobacco (Nicotiana tabacum L. var. xanthi) treated with zinc acetate dihydrate (20 to 80 mM Zn²⁺ for 2 h or 2 to 12 mM Zn²⁺ for 24 h). A dose dependent increase in DNA damage expressed by the tail moment values were observed in nuclei isolated from the roots after 2 and 24 h Zn²⁺ treatments. In contrast, Zn²⁺ did not induce significant DNA damage to leaf nuclei, with the exception of 10 or 12 mM Zn²⁺ for 24 h. Somatic mutations, identified as dark green, yellow, and dark green/yellow double sectors on the pale green tobacco leaves were not detected after any Zn²⁺ treatments. The accumulation of Zn in roots and shoots was determined by inductively coupled plasma optical emission spectrometry and the Zn content in roots was about three times higher than in shoots.     

Evaluation of lead toxicity in Erica andevalensis as an alternative species for revegetation of contaminated soils

Although revegetation using native flora is a low cost way to stabilize soil and restore the landscape contaminated with metals, little is known regarding the Pb-tolerance of many of these species. For this purpose, we evaluated the tolerance of Erica andevalensis to Pb by growing plants in nutrient solutions with increasing concentrations of Pb (up to 100 microM). Plant growth and different physiological parameters were determined to ascertain tolerance to metal stress. Additionally, an electron microscopy study coupled with EDX-analysis was performed to get clues on the Pb uptake and translocation from roots into stem and leaves. The LOEC (the lowest observed effect concentration) of Pb was 40 microM while the IC50 (inhibition concentration) was 80 microM Pb. Chemical analysis revealed a root > stem > leaf accumulation pattern. There was a severe reduction in fresh biomass and chlorophyll concentration at the highest Pb dose. The SEM-EDX study indicated that Pb was mostly located in root epidermal tissues. The blockage of Pb on the root probably avoided its toxic effects by limiting Pb transport to other tissues.     

UV-B component of sunlight causes measurable damage in field-grown maize (Zea mays L.): developmental and cellular heterogeneity of damage and repair

Ultraviolet radiation has diverse morphogenetic and damaging effects on plants. The end point of damage is reduced plant growth, but in the short term UV radiation damages specific cellular components. We measured cyclobutane pyrimidine dimers in maize DNA from plants grown in natural solar radiation. Green maize tissues had detectable DNA damage, roots had less damage, and anthers had much more damage than green leaves. This heterogeneity in damage levels may reflect differences in dose received or in damage repair. The architecture of green tissues had no measurable effects on DNA damage levels, as leaf sheath and leaf blade were equivalent. We observed a slight increase in damage levels in plants sampled at the end of the day, but there was no accumulation of damage over the growing season. We measured photoreactivation, and found substantial levels of this light-dependent repair in both the epidermis and inner cell layers of leaves, and in all organelles that contain DNA – the nucleus, chloroplasts and mitochondria. We conclude that maize has efficient mechanisms for photorepair of daily UV-induced DNA damage that prevent accumulation.     

硅对土壤-烟草系统中铅迁移及形态分布的影响

通过盆栽试验,以烟草为对象,研究了硅对土壤 烟草系统中铅的迁移以及土壤、烟草中铅形态分布的影响.结果表明： 施硅使非根际土壤可交换态铅向铁锰氧化物结合态转化,使根际土中交换态铅向铁锰氧化物结合态和残渣态转化,降低了土壤中铅的植物有效性与迁移性.施硅显著提高了烟草根部和叶部的生物量,显著降低了烟草铅的总吸收量和烟草各器官的铅含量,其中烟草铅的总吸收量降低了6.5%～44.0%,烟叶铅含量降低了3.1%～60.4%.施硅使烟草根、茎和叶中乙醇提取态、去离子水提取态和氯化钠提取态向盐酸提取态和残渣态转化,降低了烟草体内铅的毒性与迁移性.土壤-烟草系统中土壤向烟草根部的移动指数和根部向茎部的移动指数随施硅量的增加而降低,烟草茎部向叶部的移动指数随着施硅量的增加呈先增高后降低的趋势.硅通过降低土壤铅有效性、缓解铅对烟草的毒害、改变烟草体内铅的形态分布,进而抑制土壤中铅向烟草叶部的迁移,降低烟叶中的铅含量.施硅是降低土壤铅的迁移性及烟叶铅含量的有效措施.     

Preliminary study of Lead (Pb) immobilization by meat and bone meal combustion residues (MBMCR) in soil: Assessment of Pb toxicity (phytotoxicity and genotoxicity) using the tobacco model (Nicotiana tabacum var. xanthi Dulieu)

Lead (Pb) is a major chemical pollutant in the environment. The present investigation evaluates the possible use of Meat and Bone Meal Combustion Residues (MBMCR), to sequester Pb from the soil compartment using the heterozygous tobacco model (Nicotiana tabacum var. xanthi Dulieu) characterized by the a1+ /a1 a2+ /a2 system. The toxic potential of Pb-contaminations (50, 100, 1,000, 2,000 and 10,000 mg Pb kg(-1)) as Pb(NO3) in standard soil was investigated in lab conditions according to three endpoints: (i) acute toxicity of plants (mortality, height and surface area parameters), (ii) Pb-accumulation in roots, stems and leaves, and (iii) genetic effects as the expression of reversion in the leaf of plants. Moreover, chemical investigations of Pb interactions with soil were realized to complete the toxicity evaluation. The results demonstrated that: (i) MBMCR were not acutely toxic or genotoxic to tobacco plants, (ii) Pb is acutely toxic to tobacco plants at 10,000 mg Pb kg(-1) of soil, (ii) but is not genotoxic, and (iii) Pb-bioaccumulation is significant in leaves, stems and roots (from 1,000, 2,000, and 50 mg Pb kg(-1) of soil, respectively). In contrast, in the presence of MBMCR, the toxic impacts of Pb were inhibited and Pb-accumulation in tobacco plants was reduced. In complement, chemical analyses highlighted the high capacity of the standard soil to immobilize Pb. The results suggest that even if Pb is bioavailable from soils to plants, complex mechanisms could occur in plants protecting them from the toxic impact of Pb.     

Exogenous salicylate application affects the lead and copper accumulation characteristics of Lemna gibba L

Previous studies have shown that salicylates can change the ion permeability of root cells. Therefore the possible effects of exogenous salicylate application on lead (Pb) and copper (Cu) accumulation and its protective role against DNA damage due to metal exposure in Lemna gibba were studied. L. gibba was exposed to 5, 10, and 25 microM Pb and Cu for six days in the presence and absence of sodium salicylate (SA) (0.1, 0.5, and 1 mM). At all concentrations tested, SA application decreased Pb accumulation. On the other hand, application of 0.5 mM SA increased Cu accumulation. SA did not reduce DNA damage resulting from Pb and Cu toxicity. In summary, SA may be useful for reducing Pb accumulation, and application of SA at 0.5 mM may be useful for the phytoextraction of Cu.     

Expression levels of the Na + /K + transporter OsHKT2;1 and vacuolar Na + /H + exchanger OsNHX1 , Na enrichment,maintaining the photosynthetic abilities and growth performances of indica rice seedlings under salt stress

Salt affected soil inhibits plant growth, development and productivity, especially in case of rice crop. Ion homeostasis is a candidate defense mechanism in the salt tolerant plants or halophyte species, where the salt toxic ions are stored in the vacuoles. The aim of this investigation was to determine the OsNHX1 (a vacuolar Na+/H+ exchanger) and OsHKT2;1 (Na+/K+ transporter) regulation by salt stress (200 mM NaCl) in two rice cultivars, i.e. Pokkali (salt tolerant) and IR29 (salt susceptible), the accumulation of Na+ in the root and leaf tissues using CoroNa Green® staining dye and the associated physiological changes in test plants. Na+ content was largely increased in the root tissues of rice seedlings cv. Pokkali (15 min after salt stress) due to the higher expression of OsHKT2;1 gene (by 2.5 folds) in the root tissues. The expression of OsNHX1 gene in the leaf tissues was evidently increased in salt stressed seedlings of Pokkali, whereas it was unchanged in salt stressed seedlings of IR29. Na+ in the root tissues of both Pokkali and IR29 was enriched, when subjected to 200 mM NaCl for 12 h and easily detected in the leaf tissues of salt stressed plants exposed for 24 h, especially in cv. Pokkali. Moreover, the overexpression of OsNHX1 gene regulated the translocation of Na+ from root to leaf tissues, and compartmentation of Na+ into vacuoles, thereby maintaining the photosynthetic abilities in cv. Pokkali. Overall growth performance, maximum quantum yield (Fv/Fm), photon yield of PSII (ΦPSII) and net photosynthetic rate (Pn) was improved in salt stressed leaves of Pokkali than those in salt stressed IR29.     

Induction and repair of DNA damage as measured by the Comet assay and the yield of somatic mutations in gamma-irradiated tobacco seedlings

The advantage of using the tobacco (Nicotiana tabacum var. xanthi) mutagenicity assay is the ability to analyze and compare on the same plants under identical treatment conditions both the induced acute DNA damage in somatic cells as measured by the Comet assay and the yield of induced leaf somatic mutations. Gamma-irradiation of tobacco seedlings induced a dose-dependent increase in somatic mutations from 0.5 (control) to 240 per leaf (10Gy). The increased yield of somatic mutations was highly correlated (r = 0.996) with the increased DNA damage measured by the Comet assay immediately after irradiation. With increased dose of gamma-irradiation, the averaged median tail moment values ( +/- S.E.) significantly increased from 1.08 +/- 0.10 (control) to 20.26 +/- 1.61 microm (10Gy). Nuclei isolated from leaves 24h after irradiation expressed tail moment values that were not significantly different from the control (2.08 +/- 0.11). Thus a complete repair of DNA damage induced by gamma-irradiation and measurable by the Comet assay was observed, whereas the yield of somatic mutations increased in relation to the radiation dose. Data on the kinetics of DNA repair and of DNA damage induced by gamma-radiation on isolated tobacco nuclei, and on nuclei isolated from irradiated leaves and roots are presented.     

Decreased reactive oxygen species concentration in the elongation zone contributes to the reduction in maize leaf growth under salinity

Reactive oxygen species (ROS) in the apoplast of cells in the growing zone of grass leaves are required for elongation growth. This work evaluates whether salinity-induced reductions in leaf elongation are related to altered ROS production. Studies were performed in actively growing segments (SEZ) obtained from leaf three of 14-d-old maize (Zea mays L.) seedlings gradually salinized to 150 mM NaCl. Salinity reduced elongation rates and the length of the leaf growth zone. When SEZ obtained from the elongation zone of salinized plants (SEZs) were incubated in 100 mM NaCl, the concentration where growth inhibition was approximately 50%, O2*- production, measured as NBT formazan staining, was lower in these than in similar segments obtained from control plants. The NaCl effect was salt-specific, and not osmotic, as incubation in 200 mM sorbitol did not reduce formazan staining intensity. SEZs elongation rates were higher in 200 mM sorbitol than in 100 mM NaCl, but the difference could be cancelled by scavenging or inhibiting O2*- production with 10 mM MgCl2 or 200 microM diphenylene iodonium, respectively. The actual ROS believed to stimulate growth is *OH, a product of O2*- metabolism in the apoplast. SEZ(s) elongation in 100 mM NaCl was stimulated by a *OH-generating medium. Fusicoccin, an ATPase stimulant, and acetate buffer pH 4, could also enhance elongation in these segments, although both failed to increase ROS activity. These results show that decreased ROS production contributes to the salinity-associated reduction in grass leaf elongation, acting through a mechanism not associated with pH changes.     

Above-ground competition does not alter biomass allocated to roots in Abutilon theophrasti

We tested whether plants allocate proportionately less biomass to roots in response to above-ground competition as predicted by optimal partitioning theory. Two population densities of Abutilon theophrasti were achieved by planting one individual per pot and varying spacing among pots so that plants in the two densities experienced the same soil volume but different degrees of canopy overlap. Density did not affect root:shoot ratio, the partitioning of biomass between fine roots and storage roots, fine root length, or root specific length. Plants growing in high density exhibited typical above-ground responses to neighbours, having higher ratios of stem to leaf biomass and greater leaf specific area than those growing in low density. Total root biomass and shoot biomass were highly correlated. However, storage root biomass was more strongly correlated with shoot biomass than was fine-root biomass. Fine-root length was correlated with above-ground biomass only for the small subcanopy plants in crowded populations. Because leaf surface area increased with biomass, the ratio between absorptive root surface area and transpirational leaf surface area declined with plant size, a relationship that could make larger plants more susceptible to drought. We conclude that A. theophrasti does not reallocate biomass from roots to shoots in response to above-ground competition even though much root biomass is apparently involved in storage and not in resource acquisition.     

Flooding-induced changes in photosynthesis and oxidative status in maize plants

Maize plants (Zea mays L.) were subjected to soil flooding for 72, 96, and 120 h. A noticeable decrease in the rate of net photosynthesis (P_N) and the activity of ribulose-1,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) were observed. The values of intercellular CO₂ concentrations (c_i) increased in all flooded plants without significant changes in stomatal conductance (g_s). The activity of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) increased twofold 120 h after soil flooding. Flooding of maize plants led to a decrease in chlorophyll and protein levels and to slight increase of proline content. Flooded plants exhibited a large accumulation of leaf acidity. An increase in the values of some important parameters associated with oxidative stress, namely peroxides production, lipid peroxidation, and electrolyte leakage, confirmed the suggestion that root oxygen deficiency caused photooxidative damage in maize leaves.     

Evaluation of the nuclear DNA Diffusion Assay to detect apoptosis and necrosis

We applied the nuclear DNA Diffusion Assay, described as an accurate tool to estimate apoptotic and necrotic cells [N.P. Singh, A simple method for accurate estimation of apoptotic cells, Exp. Cell Res. 256 (2000) 328-337] to tobacco root and leaf cells. In this assay, isolated nuclei are embedded in an agarose microgel on a microscope slide and low molecular-weight DNA fragments diffuse into the microgel. Exposure of the roots to hydrogen peroxide significantly increased the average nuclear area of isolated nuclei. After 4 and 24 h of recovery, all DNA damage was repaired. The data clearly demonstrate that the manifestation of diffused nuclei upon exposure to hydrogen peroxide is not the result of non-repairable apoptotic or necrotic DNA fragmentation, but represents repairable genotoxin-induced DNA damage. In contrast, treatment with the alkylating agent ethyl methanesulphonate (EMS) followed by 24 h of recovery produced a significant increase in the average nuclear area. The contribution of apoptosis to this increase cannot be excluded. Heat treatment of leaves at 50 degrees C for 1-15 min leading to necrosis, and treatment of isolated nuclei with DNase-I, which digests DNA to nucleosome-sized fragments as during apoptosis, also led to a dose-dependent increase in the nuclear area. The use of different fluorochromes (ethidium bromide, DAPI or YOYO-1) for DNA staining yielded similar results in the DNA Diffusion Assay. As all types and sizes of diffused nuclei were observed after EMS and hydrogen peroxide treatments, we were unable to differentiate, on the basis of the structure of the nuclei, between apoptotic or necrotic DNA fragmentation and other types of genotoxin-induced DNA damage in plants.     

DNA Damage Measured by the Comet Assay in Eight Agronomic Plants

For most crops growing in polluted areas or treated with agricultural chemicals, no genotoxicity assays are available. We have studied the possibility of using the alkaline protocol of the plant-based molecular assay — the Single Cell Gel Electrophoresis (SCGE) assay (also called Comet assay) as a method for detecting induced DNA damage in 8 agronomic important plants (ordered according to the diameter of the nuclei): sugar beet, alfalfa, tobacco, lentil, maize, potato, hard wheat, and bread wheat. The monofunctional alkylating agent ethyl methanesulphonate (EMS) was applied as a model genotoxic agent on young excised leaves of the tested crops for 18 h at 26 °C in the dark. With increasing concentrations of 2 to 10 mM EMS, the DNA damage, expressed by the averaged median tail moment values, significantly increased in nuclei of all crops studied. No correlation between the diameter of nuclei and sensitivity to EMS treatment was observed. The data obtained demonstrate the feasibility of using the Comet assay for detecting induced DNA damage in crops. This revised version was published online in July 2006 with corrections to the Cover Date.     

镉和铅污染对烤烟根区土壤微生物及烟叶品质的影响

通过盆栽试验,研究了Cd和Pb污染对烤烟根区微生物和烟叶品质的影响.结果表明,随着Cd、Pb处理浓度的增加,烤烟根区4种微生物数量均呈减少趋势;低浓度Cd(≤10 mg·kg^-1)和Pb(≤300 mg·kg^-1)下,烤烟根区4种微生物数量降幅最大;烤烟烟叶中品质指标糖/碱比和氮/碱比升高,化学成分组成趋于不协调,不利于烟叶香味的形成.烟叶K含量与Cd和Pb污染浓度呈负相关,与Cd污染下根区钾细菌数量呈显著(或极显著)正相关,与Pb污染根区钾细菌数量相关不显著.     

An investigation of boron toxicity in barley using metabolomics

Boron (B) is an essential micronutrient that affects plant growth at either deficient or toxic concentrations in soil. The aim of this work was to investigate the adaptation of barley (Hordeum vulgare) plants to toxic B levels and to increase our understanding of B toxicity tolerance mechanisms. We used a metabolomics approach to compare metabolite profiles in root and leaf tissues of an intolerant, commercial cultivar (cv Clipper) and a B-tolerant Algerian landrace (cv Sahara). After exposure to elevated B (200 and 1,000 microM), the number and amplitude of metabolite changes in roots was greater in Clipper than in Sahara. In contrast, leaf metabolites of both cultivars only responded following 1,000 microM treatment, at which B toxicity symptoms (necrosis) were visible. In addition, metabolite levels were dramatically altered in the tips of leaves of the sensitive cultivar Clipper after growth in 1,000 microM B compared to those of Sahara. This correlates with a gradual accumulation of B from leaf base to tip in B-intolerant cultivars. Overall, there were always greater differences between tissue types (roots and leaves) than between the two cultivars. This work has provided insights into metabolic differences of two genetically distinct barley cultivars and information about how they respond metabolically to increasing B levels.     

伴生植物盐角草和星花碱蓬不同生育期几种阳离子含量变化分析

为了分析伴生植物盐角草和星花碱蓬在一个生育期内不同器官中Na⁺、K⁺、Ca²⁺及Mg²⁺含量的变化,利用火焰分光光度法和原子吸收法测定了两种植株不同发育时期的根、茎、叶中这4种阳离子含量。结果显示这两种植物根和茎中4种离子的积累趋势和叶器官中不同：Na⁺、K⁺元素含量随着植物的发育呈下降趋势,而在叶器官中则呈上升的趋势;两种植物根和茎及盐角草的叶器官中Ca²⁺、Mg²⁺元素的含量随生育期的发育呈由低到高再降低的趋势,而星花碱蓬的叶器官中这两种离子的含量则表现为由高到低再升高的趋势。两种植物除了Ca²⁺、Mg²⁺的积累趋势在两种植物间存在有差异外,其余指标的变化趋势基本一致。尤其对Na⁺的积累,两种植物Na⁺的含量占根部干重的3%左右,占茎部干重的5%左右,占叶部干重的10%左右。表明伴生植物盐角草和星花碱蓬具有改良盐碱地的良好潜能,可以作为改良盐碱地的优良种植资源。     

断根结合生长素和钾肥施用对烤烟生长及糖碱比、有机钾指数的影响

河南济源烟区存在着烟叶糖碱比高、有机钾指数较低的生产现状,通过田间试验研究了打顶时断根(C)、打顶时断根结合喷施IAA(C+I)、打顶时断根结合追施钾肥(C+K)、打顶时断根结合喷施IAA并追施钾肥(C+K+I)和对照(CK)5种处理对烤烟生物量及糖碱比、有机钾指数的影响。结果表明:不同调控措施在烟株成熟期较对照均能增加根部、叶片的干重,减少烟碱、总糖在中上部烟叶的累积。断根结合喷施IAA能降低各部位烟叶的糖碱比,且以C+K+I处理的降幅最大,在下、中、上叶位中较对照分别降低了19.12%、15.33%和8.15%。与对照相比,各处理均能提高不同部位烟叶K⁺含量,降低Cl^-和SO₄^2-含量。C+K+I处理能极显著提高各部位烟叶的有机钾指数,且在中上部烟叶中增幅最大,较对照分别提高了125.00%和209.43%。总的看来,打顶时断根结合喷施IAA并追施钾肥能促进烟叶中的干物质积累、降低糖碱比、提高有机钾指数,进而提高烟叶的内在品质。