Organospecific responses of lupin seedlings to lead Localization of hydrogen peroxide and peroxidase activity

In an earlier work using tissue printing method, we found that the PR-10 stress protein was observed in leaf petiole of lupin seedling where lead was not detected (Przymusiński et al. 2001). These results suggested the presence of substance(s) mediating a signal transduction from directly affected cells to distant organs. As the hydrogen peroxide was found to be involved in signal transduction pathway, in the present paper, we analysed the level of H₂O₂ in the organ of lupin seedlings exposed to Pb²⁺ with spectrophotometric method and tissue printing technique. It was unequivocally demonstrated that the level of H₂O₂ and the activity of peroxidase increased in every tested organ of lead-treated lupin seedling. Both the level of H₂O₂ and the activity of POX were correlated with amount of Pb²⁺ ions in the cells (Przymusiński et al. 2001) and decreased in tissues more and more distant from the site of metal application. On the other hand, there was no correlation between the histological localization of H₂O₂ and peroxidase. Our results seem to confirm the hypothesis that H₂O₂ may act as a signalling substance involved in the induction of PR protein synthesis. It was indicated that there is high degree of correlation between the localization of H₂O₂ and the histological localization of PR-10 proteins (Przymusiński et al. 2001) in every tested organ of lupin seedling. The presented hypothesis is also supported by the fact that H₂O₂ and PR-10 proteins are detected in organs and tissues where Pb²⁺ was not found at all.     

The effect of lead on cyclin expression in lupine roots

The expression of cell cycle gene, cyclin, was analyzed in lupine roots exposed to lead. The level of cyclin mRNA and coding protein were examined by Northern and Western blot techniques and by using lupine cDNA (CycB1;1) and animal-derived antibody, respectively. The cyclin mRNA level was either unchanged or slightly increased after lead treatment and it was concomitant with decrease of RNase activity. The lead ions caused the decrease of cyclin protein accumulation and this effect may be responsible for previously described reduction of mitotic activity caused by this heavy metal (Przymusiński and Woźny 1985).     

Plant cell responses to heavy metals: molecular and physiological aspects

The effect of lead, cadmium and cooper on protein pattern, free radicals and antioxidant enzymes in root of Lupinus luteus L. were investigated. Heavy metals inhibited growth of lupin roots, which was accompanied by increased synthesis and accumulation of a 16 kDa polypeptide (Przymusiński et al. 1991 Biochem. Physiol. Pflanzen., 187:51–57). This component has been earlier identified as immunologically related to Cu,Zn-superoxide dismutase (Przymusiński et al. 1995 Env.Exp.Bot., 35:485–495). However, more detailed study revealed that this stress-stimulated protein is composed of four to six polypeptides of different electrophoretic mobility. The most abundant polypeptides of the 16kDa region were found to be closely homologous to pathogen related proteins. The number and intensity of these polypeptides was highly variable in roots of individual seedlings, which suggests that they might represent separate allelic forms. Electron paramagnetic spectra revealed that at low lead concentrations the amplitude of the first derivative was similar to the control and distinctly increased at higher metal concentrations. On the other hand, at the lower lead concentrations the activity of antioxidant enzymes increased, whereas at higher metal doses the enzyme activities did not raise further (SOD) or even dropped (CAT, APOX). This implies that the responses of antioxidant system to lead is dose-dependent stimulated by low metal concentrations, whereas at the higher metal level the free radical emission is beyond the quenching capacity of antioxidant enzymes, which in turn might contribute to the reduced root growth. The effect of various heavy metals: Pb²⁺, Cd²⁺ and Cu²⁺ on phytochelatins and antioxidant enzymes depends on the kind of metal ion. Pb²⁺ and Cd²⁺ stimulated the PCs formation whereas Cu²⁺ was not effective. On the other hand, in root exposed to Cu the activity of catalase (CAT) was the highest as was the production of H₂O₂. The strong oxidative effect of Cu²⁺ ions which were not complexed by PCs suggests that these peptides might by involved in the cellular defense system by binding excessive heavy metal ions. On the basis of our results it can be concluded that in lupin roots exposed to heavy metals there is a complex defense system against metal phytotoxicity, which comprises of specific proteins, antioxidant enzymes and phytochelatins.     

Ultrastructure analysis of cadmium-tolerant and -sensitive cell lines of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Previously, a stable cell suspension culture of cucumber tolerant to cadmium (Cd) was established (Gzyl and Gwóźdź, Plant Cell Tissue Organ Cult 80:59–67, 2005). In this study, ultrastructures of Cd-tolerant and -sensitive cells were analyzed by transmission electron microscopy (TEM). Ultrastructural differences between cell lines exposed to 100 μM CdCl₂ were observed both at cellular and organelle levels. Tolerant cells exposed to Cd exhibited well-preserved cellular structures in comparison with sensitive cells. Increased numbers of osmiophilic globules in the cytoplasm and nucleolus-associated bodies as well as electron dense material in vacuoles were observed in cadmium tolerant cells. In contrast, ultrastructure of sensitive cells following exposure to Cd exhibited distinct disturbances including vacuolation, disintegration of cytoplasm, and structural changes in both mitochondria and endoplasmic reticulum. TEM observations confirmed the adaptation of tolerant cells to Cd.     

Actin filaments connected with the microtubules of lipotubuloids, cytoplasmic domains rich in lipid bodies and microtubules

Summary. Lipotubuloids, i.e., cytoplasmic domains containing an agglomeration of lipid bodies surrounded by half-unit membrane, entwined and held together by a system of microtubules, have been found in the ovary epidermis of Ornithogalum umbellatum. Ultrastructural studies demonstrated thin filaments in lipotubuloids that are probably actin filaments arranged parallel to microtubules. It is suggested that interaction of actin filaments with the microtubules determines the driving force for the rotary motion characteristic of lipotubuloids, as this movement is sensitive to cytochalasin B. Correspondence: Department of Cytophysiology, University of Łódź, Pilarskiego 14, 90-231 Łódź, Poland.     

1-(N-chloroacetylamino)-alkylphosphonic acids – synthetic precursors of phosphonopeptides

Summary. General procedures of N-chloroacetylation of the representative 1-aminoalkylphosphonic acids (Gly^P, Ala^P, Val^P, Pgly^P and Phe^P) are described. These 1-(N-chloroacetylamino)-alkylphosphonic acids were converted into the corresponding glycylphosphonodipeptides (Gly-AA^P) and/or related N-alkylglycylphosphonodipeptides (Me_nGly-AA^P) in the course of ammonolysis/aminolysis. Physico-chemical properties of synthesized 1-(N-chloroacetylamino)-alkylphosphonic acids and phosphonodipeptides are characterized. Authors’ address: Z. H. Kudzin, Institute of Chemistry, University of Łódź, Narutowicza 68, Łódź 91-136, Poland     

Metabolic responses of Lemna minor to lead ions II. Induction of antioxidant enzymes in roots

Duckweed Lemna minor L. was grown on Wang culture medium supplemented with lead ions for 24 hours. Metal was tested at 1.5, 3 and 6 mg·dm⁻³ concentrations. The response of antioxidant enzymes, such as superoxide dismutase, catalase and peroxidase in lead-treated roots of duckweed was investigated. Lead ions had no effect on the spectrum of catalase and peroxidase isoenzymes while a new isoform of superoxide dismutase appeared on the Pb treated roots. A lead-depended increase in activities of superoxide dismutase and peroxidase was observed, whereas catalase activity was maintained at relatively constant values at lower lead concentrations and then decreased markedly below control level.     

Effects of Enhanced UV-B Radiation and Tropospheric Ozone on Physiological and Biochemical Characteristics of Field Grown Wheat

Non-vernalized scions were grafted onto vernalized stocks in winter rape (Brassica napus L. var. oleifera, cv. Górczański). The grafted plants were subjected to electric current (30 V for 30 s or 6 V for 24 h) and the percentage of flowering scions was recorded. The negative polarity with cathode (−) attached to the scion and anode (+) left close to the roots inhibited greatly the percentage of flowering. The reverse polarity enhanced flowering markedly under short days and only slightly promoted flowering under long days. Attachment of electrodes without passing a current had no effect on flowering. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phosphorylation of H2AX histones in response to double-strand breaks and induction of premature chromatin condensation in hydroxyurea-treated root meristem cells of Raphanus sativus, Vicia faba, and Allium porrum

Summary. Histone H2A variant H2AX is rapidly phosphorylated on the induction of DNA double-strand breaks by ionizing radiation and hydroxyurea-mediated replication arrest, resulting in the formation of γ-H2AX foci along megabase chromatin domains nearby the sites of incurred DNA damage. In an attempt to establish a relationship between species-specific nuclear architecture and H2AX phosphorylation in S/G₂ phase-arrested root meristem cells, immunocytochemical comparisons using an antibody raised against human γ-H2AX were made among three plants differing with respect to DNA contents: Allium porrum, representing a reticulate type of DNA package, Vicia faba, having semireticulate cell nuclei, and Raphanus sativus, characterised by a chromocentric type of chromatin. Another approach was aimed at determining possible correlations between the extent of hydroxyurea-induced phosphorylation of H2AX histones and the quantities of root meristem cells induced by caffeine to enter aberrant mitotic division (premature chromosome condensation). It was concluded that the higher-order structure of chromatin may contribute to the accessibility of molecular factors engaged in the recognition and repair of genetic lesions. Consequently, in contrast to A. porrum and V. faba, a diffuse chromatin in chromocentric cell nuclei of R. sativus may become more vulnerable both to generate DNA double-strand breaks and to recruit molecular elements needed to arrange the cell cycle checkpoint functions, and thus, more resistant to factors which allow the cells to enter premature chromosome condensation spontaneously. On the other hand, however, caffeine-mediated overriding of the S-M checkpoint control system resulted in the typical appearance of premature chromosome condensation, irrespective of the genomic content of DNA. Correspondence and reprints: Department of Cytophysiology, University of Łódź, Pilarskiego 14, 90-231 Łódź, Poland.     

Response of seeds ofLupinus termis andVicia faba to the interactive effect of salinity and ascorbic acid or pyridoxine

The interactive effect of salinity and presoaking in ascorbic acid or phyridoxine on germination, seedling growth, and some relevant metabolic changes ofLupinus termis andVicia faba seeds were studied. Germination studies indicated that broad bean tolerated NaCl salinity up to 240mM NaCl and lupin to 200mM NaCl. The lengths of roots and shoots and their water content, as well as dry matter yield, remained more or less unchanged up to the level of 80mM NaCl. Salinity induced marked progressive increases of carbohydrates and proline in broad bean and soluble protein in lupin seedlings, irrespective of the salinity level used. The other organic solutes (soluble protein in broad bean and carbohydrates in lupin seedlings) remained more or less unchanged at low and moderate levels of NaCl. However, under the higher salinity levels, in lupin the losses in carbohydrates were accompanied by increases in soluble protein, whereas in broad bean an opposite effect was obtained. The level of 40mM NaCl had a pronounced stimulatory effect on the all the variables studied. Presoaking seeds in either ascorbic acid or pyridoxine counteracted the adverse effects of salinity on germination and seedling growth as well as on some metabolic mechanisms of lupin and broad bean plants. The importance of these processes to the salinity tolerance of broad bean and lupin have been discussed.     

Seasonal changes in antioxidant level of scots pine (Pinus sylvestris L.) needles exposed to industrial pollution. I. ascorbate and thiol content

Current and previous year needles from three 16 years-old populations of Scots pine (Pinus sylvestris L.) trees were seasonally collected at the three experimental areas: Luboń- close to the phosphate fertiliser factory, Głogów — close to the copper foundry and Kórnik — control site. Głogów is the most polluted site, where at 1998 monthly mean daily concentrations of different pollutants were: SO₂ - 17 μg·m⁻³, NO_x - 12 μg·m⁻³ and dust containing heavy metals as Cu, Pb, Cd - 29 μg·m⁻³. Trees growing in Luboń were influenced for many years by high concentration of SO₂ and fluor compounds. A few years ago emissions were markedly reduced, but low pH of soil and high concentration of aluminium ions still influence the growth of trees. Seasonal changes of ascorbate and thiol content were observed in each needle class and population, with the maximum in the winter and minimum in the summer. In needles from trees growing on polluted sites higher level of ascorbic acid and thiols comparing to control site was observed. Significant differences appeared in each population of Scots pine growing under higher pollution stress in the Głogów site. In needles from trees growing in Luboń significant differences in ascorbic acid and thiols content were evidently less numerous. Needles from polluted sites in some seasons contained significantly more malondialdehyde (MDA) and those was more frequent in Głogów than in Luboń. The results indicated that in the Głogów site trees are more influenced by pollution stress than in Luboń and the defense reaction measured as an increase of the antioxidant level is more evident.     

Distribution of lead in human milk fractions

The purpose of this study was to determine the concentration and distribution of lead, calcium, iron, zinc, and copper in major fractions (fat, casein, whey) of mature milk from 38 nursing adult women with low environmental lead exposure. The potential associations between milk lead and maternal blood lead and between milk and blood lead and essential mineral data (nutritional status, dietary intake, and milk concentration) were investigated. Maternal blood lead (geometric mean, 60 μg/L) was negatively associated, although modestly, with dietary calcium intake (r=−0.32, p=0.02). Lead in whole milk (geometric mean, 1.2 μg/L) was positively associated with calcium in whole milk (r=0.56, p=0.005). Distribution of lead in milk fractions was 63%, 28%, and 9%, in whey, fat, and casein, respectively. Milk distribution of essential minerals was 67–76%, 17–18%, and 7–17% in whey, fat, and casein, respectively. Lead in milk whey was positively associated with lead in maternal blood (r=0.49, p=0.02). However, milk lead was not affected by nutritional status, dietary intake, and milk composition of the essential minerals. The high percentage of lead in the milk whey fraction, as seen for the essential minerals, suggests that most lead in human milk is bioavailable to the infant.     

Di-Mercapto Succinic Acid (DMSA) and vitamin C chelating potency in lead intoxication,regarding oxidative stress and apoptotic related proteins in rabbits

Free radicals interact with other molecules within cells. This can cause oxidative damage to proteins, membranes and genes. Oxidative damage has been implicated in the cause of many diseases such as cancer and Alzheimer’s and has an impact on the body’s aging process. External factors such as pollution, sunlight and smoking also trigger the production of free radicals and the process of programmed cell death (PCD) that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. The study was conducted to 42 male Orycytolagus Cuniculus rabbits. These animals were subdivided into seven groups approaching the comparison between treatment with vitamin C and Di-Mercapto Succinic Acid (DMSA) including positive and negative control groups. Lead ions concentration in blood, liver, kidney and brain were measured before and after treatments application. Apoptotic related proteins P53 and Bcl2 were estimated beside histological studies of tissues. Results revealed that it is more saving to use vitamin C as a lead chelator than DMSA. It is also can be stated that severe lead toxicity can be carcinogenic by increasing the oxidative stress in the biological systems as well as altering the balanced concentration of the apoptotic proteins.     

Determination of lead in white lupin by anodic stripping voltammetry

An anodic stripping voltammetry method for the simultaneous determination of trace heavy metals in nutrient solutions, soils and plants has been developed at a hydrodynamic electrochemical sensor. Several parameters were optimized in order to enhance sensitivity. Calibration curves in different media are presented. The study of the uptake of lead by white lupin (Lupinus albus L.) was carried out. Toxicity symptoms were observed and compared with levels of lead measured in roots and leaves.     

The induction of stress-related proteins by lead

Differential inductive effects of lead on protein synthesis in rat fibroblasts and kidney epithelial cells were examined. The lead was administered as lead glutamate, a complex known to introduce lead into cells. Lead exposure induced the synthesis of three proteins which constitute two separate stress protein subgroups. Two of these proteins have been previously identified as the glucose-regulated proteins because their synthesis is induced by reagents which perturb glucose utilization. The third protein is inducible by several sulfhydryl-binding reagents including lead. This third protein has been compared with a protein, p32/6.3, of very similar size and isoelectric point, which has been associated with lead-induced intranuclear inclusion bodies. However, several features, including one-dimensional peptide maps, indicated that the third protein and p32/6.3 are not identical. The three lead-induced proteins are distinguished from the major group of stress proteins by their relative insensitivity to heat stress. Lead glutamate, on the other hand, induces neither the heat shock protein 70 nor metallothionein, both of which are strongly induced by several metals including cadmium, zinc, and mercury.     

On the reliability of the lead salt precipitation method of acid phosphatase localization in plant cells

Summary Fixation of cells with glutaraldehyde (5.0%, pH 6.7) was found to facilitate both the penetration of substrate (p-nitrophenyl phosphate) into cells and the leaking out of intracellular phosphate ions. 64% of the original activity survived the fixation for at least 24 hours. Lead ions added to the incubation medium at 6 mM neither accelerated nonenzymatic hydrolysis of the substrate, nor completely inactivated the enzyme activity. Lead ions at concentrations above 6 mM formed an insoluble compound with p-nitrophenyl phosphate, resulting in a decrease in the concentration of free substrate and lead ions. Phosphate ions liberated from substrate could not be completely trapped by lead ions even at above 6 mM, suggesting the possibility of intracellular migration of phosphate ions.In the presence of 4 mM p-nitrophenyl phosphate, 6 mM lead nitrate, and 0.2 M sucrose at pH 6.5, lead salt precipitates were deposited on the outer surface of cell walls, within cell walls, at tonoplast membranes, in nuclei, and occasionally in proplastids. No deposition of lead salt was formed in the control test from which the substrate was omitted. When cells were treated at first with lead nitrate and then with potassium phosphate, lead salt deposits were formed in the same sites as those of cells incubated in a complete reaction medium.It is concluded that although the result of the lead salt precipitation procedure reflects the presence of enzyme activity, it cannot directly show the site of the enzyme.     

Desiccation tolerance changes in winter rape leaves grown under different environmental conditions

The results of studies performed on winter rape leaves (Brassica napus L. var.oleifera L., cv. Górczański) indicated that independently of the plant cultivation conditions and of the leaf growth rate, all leaves showed significant increase in desiccation tolerance at the beginning of leaf expansion. The increase in tolerance was correlated with the formation of a central vacuole in the mesophyll cells. Therefore, the high desiccation tolerance of vacuolised cells in comparison to the non-vacuolised ones is supposed to be due to the higher ability of the former to avoid cytoplasm dehydration. Development of frost tolerance in the leaves during autumn does not seem to be causally related to desiccation tolerance changes.     

Seasonal changes in antioxidant level of Scots pine (Pinus sylvestris L.) needles exposed to industrial pollution. II. Enzymatic scavengers activities

The involvement of enzymic antioxidant system, superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase in defense reaction to environmental stress evoked by air and soil pollution, was seasonally studied on three populations of Scots pine (Pinus sylvestris L.) growing on experimental areas close two industrial objects in Poland. The first of them (Luboón) is localised near a phosphate fertiliser factory, the second (Głogów) near a copper foundry, and control stand is placed in Kórnik. Głogów is the most polluted site, where in 1998 monthly mean daily concentrations was: SO₂ - 17 μg·m⁻³, NO_x- 12 μg·m⁻³ and dust containing heavy metals (Cu, Pb, Cd) - 29 μg·m⁻³. Trees in Luboń were influenced for many years by high concentration of SO₂ and fluor compounds. Few years ago emissions were markedly reduced, but changes in the soil (low pH and high concentration of aluminium ions) still influence the growth of trees. In needles of two populations: 3 (Russia) and 8 (Poland), from the polluted sites Głogów and Luboń, activities of superoxide dismutase (SOD) and guaiacol peroxidase (PO) were significantly higher compared to Kórnik. However, in one population (16 - Slovakia), such dependance was not evident. Activity of ascorbate peroxidase (AP) measured in winter was also higher in needles from polluted sites. The results indicated that the sensitivity of free radical scavenging system in Scots pine needles differs among populations.     

Mutagenesis and comutagenesis by lead compounds.

We have previously reported that lead(II) is weakly mutagenic to Chinese hamster V79 cells. A transgenic cell line G12 containing a single copy of the E. coli gpt gene was developed in this laboratory from Chinese hamster V79 cells. The gpt locus in the G12 cells is more mutable by radiation and oxidative agents compared with the endogenous hprt locus of wild-type V79 cells. We have investigated the mutagenicity of two lead compounds at the gpt locus in G12 cells. Only at a toxic dose is lead acetate significantly mutagenic to G12 cells. Lead nitrate is not significantly mutagenic at any dose. Although both compounds are water-soluble, lead acetate, but not lead nitrate, forms a fine white insoluble precipitate upon addition to growth medium. A nick translation assay on cells treated with lead compounds and then permeabilized indicated that lead nitrate and, to a greater extent, lead acetate causes the appearance of nicks in chromosomal DNA. Lead ions in the presence of hydrogen peroxide, but not alone, introduced nicks into supercoiled plasmid DNA in vitro, suggesting that lead ions can partake in a Fenton reaction and thereby damage DNA. At lower nonmutagenic concentrations, lead acetate enhances the mutagenicity of MNNG and ultraviolet light. DNA damage by ultraviolet light is not enhanced by lead ions in vitro. Our data support the concept that non-toxic concentrations of lead(II) can inhibit DNA repair. Thus, at biologically relevant doses, lead(II) could act as a comutagen and possibly a cocarcinogen, but is not likely to act as an initiating genotoxic carcinogen.