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.     

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.     

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.     

Genomic damage induced in tobacco plants by chlorobenzoic acids--metabolic products of polychlorinated biphenyls

Tobacco seedlings (Nicotiana tabacum var. xanthi) were treated for 24 h with mono-(2- and 3-CBA), di-(2,5- and 3,4-CBA), and tri-(2,4,6- and 2,3,5-CBA)-chlorobenzoic acids (CBAs) and with the mixture of polychlorinated biphenyls--Delor 103, or cultivated for 1 or 2 weeks in soil polluted with the CBAs. DNA damage in nuclei of leaves and roots was evaluated by the comet assay. A significant increase in DNA damage was observed only at concentrations of CBAs that caused withering of leaves or had lethal effects within 2-4 weeks after the treatments. As the application of CBAs did not induce somatic mutations, the induced DNA migration is probably caused by necrotic DNA fragmentation and not by DNA damage resulting in genetic alteration. In contrast, the application of the monofunctional alkylating agent ethyl methanesulphonate as a positive control resulted in a dose-response increase of DNA damage and an increase of somatic mutations. Thus, the EMS-produced DNA migration is probably associated with genotoxin-induced DNA fragmentation. The data demonstrate that the comet assay in plants should be conducted together with toxicity studies to distinguish between necrotic and genotoxin-induced DNA fragmentation. The content of 2,5-CBA in tobacco seedlings was measured by reverse-phase high pressure liquid chromatography.     

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.     

Genomic damage induced in tobacco plants by chlorobenzoic acids—Metabolic products of polychlorinated biphenyls

Tobacco seedlings (Nicotiana tabacum var. xanthi) were treated for 24 h with mono-(2- and 3-CBA), di-(2,5- and 3,4-CBA), and tri-(2,4,6- and 2,3,5-CBA)-chlorobenzoic acids (CBAs) and with the mixture of polychlorinated biphenyls – Delor 103, or cultivated for 1 or 2 weeks in soil polluted with the CBAs. DNA damage in nuclei of leaves and roots was evaluated by the comet assay. A significant increase in DNA damage was observed only at concentrations of CBAs that caused withering of leaves or had lethal effects within 2–4 weeks after the treatments. As the application of CBAs did not induce somatic mutations, the induced DNA migration is probably caused by necrotic DNA fragmentation and not by DNA damage resulting in genetic alteration. In contrast, the application of the monofunctional alkylating agent ethyl methanesulphonate as a positive control resulted in a dose–response increase of DNA damage and an increase of somatic mutations. Thus, the EMS-produced DNA migration is probably associated with genotoxin-induced DNA fragmentation. The data demonstrate that the comet assay in plants should be conducted together with toxicity studies to distinguish between necrotic and genotoxin-induced DNA fragmentation. The content of 2,5-CBA in tobacco seedlings was measured by reverse-phase high pressure liquid chromatography.     

Studies on Leaf Surface Lipid of Tobacco I. Changes in Leaf Surface Lipid and Duvatrienediol during Growth,Senescence and Curing of Tobacco Leaves

Duvatrienediol is a diterpene specifically occurred in tobacco plants and thought to be a precursor of tobacco aroma. Green tobacco leaves contained 0.2~1% of duvatrienediol per dry weight and it was corresponded to 30~60% of leaf surface lipid. Leaves on upper stalk position contained more of leaf surface lipid and duvatrienediol. In leaves on each stalk position, leaf surface lipid and duvatrienediol contents increased with leaf growth and decreased by over-maturation. Production of leaf surface lipid and duvatrienediol was affected by soil conditions or applied amount of nitrogen fertilizer. Both leaf surface lipid and duvatrienediol were decreased during curing of tobacco leaves, but the change in the latter was more drastic. Comparing to leaf surface lipid, changes in cytoplasmic lipid were less during growth and senescence of tobacco leaves.     

烟蚜为害的经济损失研究

陕西渭北烟草种植区迁飞到烟田的蚜虫有6种,但只有桃蚜Myzus persicae(Sulzer)能够建立种群,造成为害。从烟草旺长开始,按烟株上蚜量和蚜虫分泌蜜露,引起霉菌滋生,污染叶面积的比例分为0、I、II、III、IV级。以未受蚜害的0级为对照,受蚜害的I、II、III、IV级的中上等烟下降分别为6.93%、13.05%、28.51%和30.94%,经济损失率分别为9.86%、23.81%、36.12%和39.59%。化学成分分析表明：I、II、III和IV级烟叶的烟碱含量分别降低10.61%、19.29%、10.61%和25.40%,还原糖分别降低15.04%、19.23%、40.98%和44.86%,蛋白质含量分别提高16.17%、31.99%、37.02%和38.72%。这就表明桃蚜为害既影响烤烟的外观质量、又影响内在品质。1头蚜虫经济损失率＝0.0695%。     

Fe,Mn and the grey effect in hydroponically-cultured flue-cured tobacco

A study of the influence of Fe or Mn (supplied as 5, 20, 35, 50 or 65 ppm) on the grey disorder in flue-cured tobacco (Nicotiana tabacum L.) was conducted with mater-cultured plants grown under greenhouse conditions and fertilized with Hoagland's nutrient solution. The solution was highly unbuffered as is the podzolic soil in which grey tobacco plants grow. When plants were treated with 5 to 35 ppm Fe, the trend in grey index (a measure of grey tobacco symptoms) in top and middle leaves was similar to the trend in Fe content of tobacco leaves. The highest grey index was found in these leaf positions with the 35 ppm Fe treatment. When Fe in solution was increased from 35 to 65 ppm grey index in top leaves decreased but was unchanged in middle leaves. Leaf Fe content for these leaf positions changed little. No apparent relationship between grey index and Fe in bottom leaves was observed. Initially, pH of the nutrient solution increased from a mean of 4.75 to 6.50 (indicating preferential anionic absorption) followed by an abrupt decrease in pH to 4.2 (suggesting preferential cationic absorption which included the Fe treatment in solution). The grey effect identical to that found in field-grown plants developed in the cured leaf of plants treated with high levels of Fe and was considered to be one of the major causal agents in grey flue-cured tobacco. None of the levels of Mn induced the grey effect.     

降雨量增减对黄河三角洲滨海湿地土壤呼吸和芦苇光合特性的影响

滨海湿地地下水位浅,淡咸水垂直交互作用明显,全球气候变化背景下降水变异改变其土壤表层水盐状况,从而影响植物光合作用与土壤呼吸.为探究降雨量变化对黄河三角洲滨海湿地土壤呼吸和光合特性的影响,采用固定式遮雨顶棚和雨水输送管道相结合的方法设置增减雨处理小区,于2015年生长季测定土壤呼吸和光合作用光响应曲线,同时连续测定土壤温度、土壤含水量、土壤含盐量等土壤环境因子.结果表明: 根据土壤含水量波动情况可将生长季分为3个阶段:干旱期、湿润期、淹水期. 不同土壤水分阶段,土壤呼吸和芦苇光合特性对降雨量增减的响应不同. 在干旱期,增雨处理下土壤呼吸速率显著提高了31.8%,同时芦苇叶片气孔导度和光合能力显著增强;减雨处理下土壤呼吸速率降低41.1%,芦苇叶片气孔阻塞,光合能力降低. 在湿润期,增雨和减雨处理使土壤呼吸速率及其温度敏感性指数(Q₁₀)均出现下降,但二者未对芦苇各光合参数和净光合速率产生显著影响. 在淹水期,增减雨处理未对土壤呼吸产生显著影响,但芦苇对淹水胁迫较为敏感,增减雨分别加重和降低了淹水对芦苇植株的伤害,光合速率由高到低为减雨>对照>增雨.     

Changes in chloroplast DNA during development in tobacco, Medicago truncatula, pea, and maize

We examined the DNA from chloroplasts obtained from young and fully expanded leaves of tobacco (Nicotiana tabacum L.), Medicago truncatula, pea (Pisum sativum L.), and maize (Zea mays L.). The changes in plastid DNA content and structure were monitored by four independent methods: 4′,6-diamidino-2-phenylindole (DAPI) staining with intact chloroplasts, in situ DAPI staining of cytological sections, ethidium bromide staining at the single-molecule level after exhaustive deproteinization of lysed chloroplasts, and pulsed-field gel electrophoresis. During leaf development, we found a decline of chloroplast DNA (cpDNA) in all four plants. For tobacco, for which plants can readily be regenerated from somatic cells, cpDNA persisted longer than in the other three plants. We also found a striking progression from complex multigenomic DNA molecules to simple subgenomic molecules during plastid development. Although the decrease in molecular size and complexity paralleled the decrease in DNA content per plastid, 6% of the chloroplasts in a fully expanded tobacco leaf still contained DNA in complex branched structure, whereas no such complex structures were found in mature leaves for the hard-to-regenerate maize.     

Soil-dependent variability of leaf iron accumulation in transgenic tobacco overexpressing ferritin

Ferritin overexpression in transgenic plants has been recently reported to increase leaf and seed iron content. We investigated the influence of various soil conditions on this increase in leaf iron content. One control transgenic tobacco and two transgenic tobaccos overexpressing ferritin in the plastids or in the cytoplasm, respectively, were grown on five different soils, two of them being amended with sewage sludge. Although a significant increase in leaf iron concentration was measured in transgenics overexpressing ferritin grown on three out of five soils, this increase was not a general rule. On some soils, leaf iron concentration of control plants was as high as in transgenics grown on other soils. In addition, an increased phosphorus concentration in the two sewage sludge amended soils correlated with a high leaf iron concentration in control plants, similar to the one measured in ferritin transformed plants. Indeed, growing plants in vitro with various increasing phosphate concentrations revealed a direct P involvement in iron loading of control plants, at a similar level as overexpressing ferritin plants. Also, with one of the soil tested, not only iron but also manganese, zinc and cadmium, and to a much lesser extent copper, nickel and lead were found more abundantly in ferritin transformed plants than in control plants. These data indicate that the iron fortification of leaves, based on ferritin overexpression, could be limited in its biotechnological application because of its high soil dependence.     

Reduction of Cadmium Availability to Tobacco (Nicotiana tabacum) Plants using Soil Amendments in Low Cadmium-contaminated Agricultural Soils: A Pot Experiment

Cadmium (Cd) concentration in field-grown tobacco leaves usually ranges from < 0.5 to 5 mg Cd kg^–1 dry matter (DM). Reducing bioavailability of soil Cd by adding amendments to the soil could be suitable to mitigate Cd uptake by tobacco plants. However, little is known on the effect of inorganic amendments on agricultural soils with low Cd concentrations. Therefore, we performed a pot experiment with tobacco plants that were grown during 56 days in two neutral to alkaline agricultural soils with low total Cd concentrations (soil 1 = 0.4, soil 2 = 0.7 mg kg^–1). Both soils were amended or not with 1 or 5% of sepiolite, zeolite, hydroxyapatite and apatite II™. Major and trace elements were measured in mid-stalk position leaves. Soil metals were measured in a DTPA soil extraction to assess the effect of the amendments on metal bioavailability. Some amendments significantly reduced Cd concentration in tobacco leaves, but the effect differed between the two soils tested. In soil 1, the use of zeolite at the 1% dose was the most efficient, reducing the average Cd concentration from 0.6 to 0.4 mg kg^–1. In soil 2, the 5% hydroxyapatite treatment led to the maximal reduction in Cd concentration (50%), with an average final Cd concentration in leaves of 0.7 mg kg^–1 (control: 1.5 mg kg^–1). There was a dose effect for some amendments in soil 2 (containing more Cd), suggesting a reduced efficiency of the amendment at the lowest addition rate. DTPA extractable Cd and Zn measured at the end of the pot experiment were correlated to the metal concentrations in tobacco leaves suggesting that (1) the reduction in leaf Cd concentration was due to a reduction in metal availability to tobacco and (2) DTPA may be a suitable extractant to estimate Cd availability to tobacco plants in these two soils. In addition, a batch experiment was performed with the same soils to test a larger number of amendments, including the four tested in the pot experiment. Results were compared to those of the pot experiment to assess whether a batch experiment may predict the efficiency of an amendment on a given soil. It gave results compatible with those from the pot experiment except for the sepiolite and highlighted the broad range of potential amendments available for heavy metal remediation in crop plants.     

Effects of water stress on leaf growth in tall fescue, Italian ryegrass and their hybrid: rheological properties of expansion zones of leaves, measured on growing and killed tissue

In Expt 1, plants of tall fescue (Festuca arundinacea Schreb.), Italian ryegrass (Lolium multiflorum Lam.) and their F1 hybrid were grown in soil-based compost in a controlled environment, and subjected to full or partial irrigation for 20 d. In Expt 2, plants of the parent species were grown in nutrient solution in the same environment and subjected to osmotic stress (0.76 MPa) for 2 d. In both experiments, distribution of growth in the leaf growing zone (at the base of the growing leaf) was determined, and elastic and plastic compliances were measured on methanol-killed samples of growing zone and of mature lamina using an extensiometer. In Expt 2 plastic compliance coefficient of extension, extensibility, and hydraulic conductance were calculated from changes in leaf extension rate occasioned by imposing linear stress. 'Plastic and elastic compliances of growing zones were 10-20 times greater than those of mature laminae. In both species, drought reduced (a) leaf extension rate, (b) the length of the growing zone, the height of maximum growth, (d) the plastic compliance of whole bases (Expt 1), and (e) hydraulic conductance. The elastic compliance of whole leaf bases was unaffected by drought, but when expressed per unit length of growing zone was increased by drought. Killing with methanol reduced the plastic compliance of leaf bases in control plants, but not in droughted plants.F. arundinacea differed from L. multiflorum in having (a) a lower leaf extension rate (although drought reduced extension by the same proportion in both species), (b) a longer growing zone in droughted plants in Expt 2, a lower elastic and plastic compliance of whole killed leaf bases and laminae, (d) slightly higher plastic compliance in attached growing leaves, and (e) lower plastic compliance per unit length of growing zone in attached leaves. The hybrid was generally intermediate between the parents. the results are discussed in relation to methodology and to crop improvement.Key words: Extensibility, extension coefficient, hydraulic conductance, elastic compliance, plastic compliance, leaf growth, leaf extension rate.      

Adaptation of tobacco plants to elevated CO2: influence of leaf age on changes in physiology, redox states and NADP-malate dehydrogenase activity

Transgenic tobacco plants (Nicotiana tabacum L. cv. Xanthi) with altered chloroplast NADP-malate dehydrogenase (NADP-MDH) content were grown under ambient or under doubled atmospheric CO2 in order to analyse the effect of elevated CO2 on the redox state of the chloroplasts. Since large differences exist between the individual leaves of tobacco plants, gas exchange characteristics, enzyme capacities and metabolite contents were measured separately for each leaf of the plants. Large variations between leaves of different age were found in nearly every parameter analysed, and the differences between younger and older leaves were, in most cases, larger than the differences between comparable leaves at ambient or elevated CO2. For all parameters (chlorophyll fluorescence, P700 reduction, NADP-MDH activation) that are indicative for the redox situation in the electron transport chains and in the chloroplast stroma, more oxidized values were determined under elevated CO2. The increased redox state of ferredoxin, observed at ambient conditions in the NADP-MDH-under-expressing plants, disappeared under elevated CO2. It was concluded that the reduced rate of photorespiration under elevated CO2 decreases the amount of excess electrons. Interestingly, this lowered not only the activation state of NADP-MDH, but also the expression of the enzyme in the wild-type plants. The results are discussed with respect to a possible interaction between stromal reduction state and gene expression.     

Ultrastructural and Morphological Characteristics of Cultivated Wheat Growing on Copper-Polluted Fields

A cultivated Greek variety of wheat (Triticum aestivum L. cv. Vergina) growing in fields naturally polluted by outcrops of copper ores was investigated. Wheat plants show a negative response to increasing quantities of soil copper, including reduced growth and chlorosis. Copper toxicity was demonstrated in the laboratory by a rooting test; the frequency of mitoses declines sharply with increasing copper concentration in the nutrient solution. The mesophyll cells of polluted plants display a circular shape (in transverse sections) with a few chloroplasts parietally distributed, in contrast with the elongate or pleomorphic shape of control leaves that contain numerous chloroplasts crowded at the cell periphery. Ultrastructurally, the chloroplasts of polluted plants contain a poorly developed internal membrane system consisting of thylakoids arranged parallel to each other with only a few, rudimentary grana. In addition, a number of statistically significant differences were found, including the number of starch grains and plastoglobuli, chloroplast surface area, volume fraction of starch grains and, most important, the volume fraction of the internal membrane system. All ultrastructural changes are attributed to the toxic effect of high concentrations of soil copper.     

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.     

桑-蚕系统中镉的吸收、累积与迁移

对桑－蚕系统中镉的吸收,累积与迁移行为研究结果表明：（１）桑树对土壤镉污染有一定的耐性,桑树镉累积量和相对累积率与土壤镉浓度的关系可分别用回归方程Ｔａ＝ａ＋ｂｌｏｇ（Ｓｃ）和ｌｏｇ（Ｒａ）＝ａ＋ｂｌｏｇ（Ｓｃ）描述。（２）随着土壤镉浓度的增加,镉在桑树根部的分布率明显增加,地上部分的分布率有所降低,运转到叶片的比率明显降低。（３）蚕体,蚕砂和蚕茧的镉含量,镉累积量,随着桑叶镉含量的增加而增加。     

Effects of simulated winter browsing on mountain birch foliar chemistry and on the performance of insect herbivores

Winter browsing by mammalian herbivores is known to induce a variety of morphological and physiological changes in plants. Browsing has been suggested to decrease the carbohydrate reserves in woody plants, which might lead to reduced tannin production in leaves during the following summer, and consequently, to increased herbivore damage on leaves. We conducted a clipping experiment with mature mountain birch trees and measured the effects of clipping on birch growth, leaf chemistry and toughness, as well as on the performance of insect herbivores. Leaves grew larger and heavier per unit area in the clipped ramets and had a higher content of proteins than leaves in the control trees. Clipping treatment did not affect the total content of sugars in the leaves (mg g^?1), suggesting that a moderate level of clipping did not significantly reduce the carbohydrate pools of fully‐grown mountain birch trees. Furthermore, the contents of proanthocyanidins (condensed tannins) and gallotannins were slightly higher in the leaves of clipped ramets, contrary to the hypothesis of reduced tannin production. The effects of clipping treatment on leaf and shoot growth and on foliar chemistry were mainly restricted to the clipped ramets, without spreading to untreated ramets within the same tree individual. The effects of clipping on leaf characters varied during the growing season; for instance, leaf toughness in clipped ramets was higher than toughness in control trees and ramets only when leaves were mature. Accordingly, clipping had inconsistent effects on insect herbivores feeding at different times of the growing season. The generally small impact of clipping on herbivore performance suggests that the low intensity of natural browsing at the study area, simulated by our clipping treatment, does not have strong consequences for the population dynamics of insect herbivores on mountain birch via enhanced population growth caused by browsing‐induced changes in food quality.