The effect of water deficit and excess copper on proline metabolism in Nicotiana benthamiana

Fluctuation in proline content is a widespread phenomenon among plants in response to heavy metal stress. To distinguish between the participation of water deficit and copper on changes in proline metabolism, potted plants and floating leaf discs of tobacco were subjected to CuSO₄ treatments. The application of copper increased the proline content in the leaves concomitantly with decreased leaf relative water content and increased abscisic acid (ABA) content in the potted plant. Excess copper increased the expression of two proline synthesis genes, pyrroline-5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT) and suppressed proline catabolism gene, proline dehydrogenase (PDH). However, in the experiment with tobacco leaf discs floating on CuSO₄ solutions, the excess copper decreased proline content and suppressed the expression of the P5CS, OAT and PDH genes. Therefore, proline accumulation in the potted tobacco plants treated with excess Cu treatment might not be the consequence of the increased copper content in tobacco leaves but rather by the accompanied decrease in water content and/or increased ABA content.     

Effect of cadmium and copper on growth of Bacopa monniera regenerants

The effects of cadmium and copper on in vitro growth of Bacopa monniera (L.) Wettst. was monitored. Cadmium (25 and 50 μM CdCl₂) inhibited plantlet growth and addition of 50 or 100 μm CuSO₄ partially alleviated this negative effect. Cadmium increased both protein and proline contents, but to a lesser extent with the additional supply of CuSO₄. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phytoprotective Effect of Ammonium Humate at High Copper Concentrations in the Environment

The influence of ammonium humate obtained from peat on the tolerance of wheat plants to high CuSO₄ concentrations (100, 250, 500, and 1000 μM/L) has been studied. Seeds were germinated on a copper sulfate solution with and without the humate. Then the plants were grown on Hoagland’s solution in an artificial climate chamber. The coefficient of protective action by humate was estimated in the following two ways: by dry weight changes and by the copper ion content in comparison with the plants grown without humate. The protective role of the humate at 100 and 250 μM has been established, which is due to reduction of copper accumulation in the plants. It has been found that the humate at higher concentrations enhances the toxic effect of copper.     

Antioxidant activity in the leaves of <Emphasis Type="Italic">Melilotus albus</Emphasis> and <Emphasis Type="Italic">Trifolium medium</Emphasis> from man-made disturbed habitats in the Middle Urals under the influence of copper

Physiological mechanisms of adaptation to copper-induced stress in two widespread legume plants, white sweet clover (Melilotus albus Merik.) and zigzag clover (Trifolium medium L.), growing in habitats differing in the man-made pollution. An antioxidant plant defense system was activated in response to 10 mM CuSO₄, which is a stress factor. Specific biochemical features related to adaptation to soil contamination with copper were observed in tested plant species. Superoxide dismutase was activated in response to stress in both species from various habitats. M. albus from the impact zone manifested the better capacity of proline accumulation as compared with plants from less polluted habitats. T. medium plants from the impact zone contained more active peroxidase. It was suggested that plants growing for a long time under stressful conditions manifest the greater tolerance to copper ions than plants, which did not experience stress or were subjected to the milder stress.     

Exogenous melatonin improves growth and photosynthetic capacity of cucumber under salinity-induced stress

Melatonin mediates many physiological processes in animals and plants. To examine the potential roles of melatonin in salinity tolerance, we investigated the effects of exogenous melatonin on growth and antioxidant system in cucumber under 200 mM NaCl stress conditions. The results showed that the melatonin-treated plants significantly increased growth mass and antioxidant protection. Under salinity stress, the addition of melatonin effectively alleviated the decrease in the net photosynthetic rate, the maximum quantum efficiency of PSII, and the total chlorophyll content. Our data also suggested that melatonin and the resistance of plants exhibited a concentration effect. The application of 50–150 μM melatonin significantly improved the photosynthetic capacity. Additionally, the pretreatment with melatonin reduced the oxidative damage under salinity stress by scavenging directly H₂O₂ or enhancing activity of antioxidant enzymes (including superoxide dismutase, peroxidase, catalase, ascorbate peroxidase) and concentrations of antioxidants (ascorbic acid and glutathione). Therefore, the melatonin-treated plants could effectively enhance their salinity tolerance.     

<Emphasis Type="Italic">In vitro</Emphasis> evaluation of excess copper affecting seedlings and their biochemical characteristics in <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> L. (variety PBNS-12)

The present study was focused to recognize the changes in the Safflower (Carthamus tinctorius L. variety PBNS-12), when exposed to different concentration of copper (25, 50 and 100 μM) along with control (0.5 μM) for 10 and 20 days. This experiment used Hoagland’s nutrient solution to meet the external nutrient conditions, which includes micro and macronutrients equivalent to soil solution with copper sulphate (CuSO₄. 5H₂O) as a metal stress. The plant samples were harvested after 10 and 20 days. The effect of increased concentrations of copper was indicated by the reduction in overall growth with reduced fresh and dry weight. Copper stress caused significant increase in the non- enzymatic antioxidants (polyphenols and flavonoids) in leaves of treated safflower seedlings as compared to the control. Also, enhanced accumulation of proline was observed in the safflower leaves. In response to excess copper concentration, the level of MDA content was found to be increased. The results showed that the copper has time and dose-dependent effects on safflower seedlings.     

Relationship between ammonium accumulation and senescence of detached rice leaves caused by excess copper

The possibility that ammonium (NH ₄ ⁺ ) accumulation is linked to the senescence of detached rice (Oryza sativa) leaves induced by copper (Cu) was investigated. CuSO₄ was effective in promoting senescence of detached rice leaves. Both CuSO₄ and CuCl₂ induced NH ₄ ⁺ accumulation in detached rice leaves, indicating that NH ₄ ⁺ accumulation is induced by copper. Sulfate salts of Mg, Mn, Zn, and Fe were ineffective in inducing NH ₄ ⁺ accumulation in detached rice leaves. The senescence of detached rice leaves induced by Cu was found to be prior to NH ₄ ⁺ accumulation. Free radical scavengers, such as glutathione and thiourea, inhibited senescence caused by Cu and at the same time inhibited Cu-induced NH ₄ ⁺ accumulation. The current results suggest that NH ₄ ⁺ accumulation is not associated with senescence induced by Cu, but is part of the overall expression of oxidative damage caused by an excess of Cu. Evidence was presented to show that copper-induced ammonium accumulation in detached rice leaves is attributed to a decrease in glutamine synthetase activity and an increase in reduction of nitrate.     

Tomato QM-Like Protein Protects Saccharomyces cerevisiae Cells against Oxidative Stress by Regulating Intracellular Proline Levels

Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H₂O₂, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants.     

Adaptation of the Common Ice Plant to High Copper and Zinc Concentrations and Their Potential Using for Phytoremediation

A facultative halophite Mesembryanthemum crystallinum L. (the common ice plant) was shown to grow successively at the high concentrations of Cu and Zn. Although 25 μM CuSO₄ or 800 μM ZnSO₄ retarded markedly plant growth, they did not interfere with the completion of plant development and the formation of viable seeds. In such plants, leaves accumulated more than 200 μg of Cu and 1700 μg of Zn per 1 g of dry weight. A damaging effect of heavy metals (HMs) was manifested in a reduced content of water in leaves and proline accumulation in them. As copper is a metal with transient valence, copper salts are more toxic than zinc salts, which was manifested in a stronger inhibition of the chlorophyll synthesis. Both HMs induced oxidative stress, as evident from increased activities of guaiacol peroxidase and lipoxygenase. Moderate Cu and Zn concentrations did not damage cell membranes in leaves, as evident from the absence of their action on electrolyte leakage either under optimum conditions or after heat treatment. A capability of a substantial HM accumulation by the common ice plant and their considerable transport to shoots (up to 50 μg of Cu and 560 μg of Zn per plant) make it possible to consider the common ice plant as a promising phytoremediator. __________ Translated from Fiziologiya Rastenii, Vol. 52, No. 6, 2005, pp. 848–858. Original Russian Text Copyright ? 2005 by Kholodova, Volkov, Kuznetsov.     

Antitumor activity of resveratrol is independent of Cu(II) complex formation in MCF-7 cell line

Resveratrol (Rsv) is widely reported to possess anticarcinogenic properties in a plethora of cellular and animal models having limited toxicity toward normal cells. In the molecular level, Rsv can act as a suppressive agent for several impaired signaling pathways on cancer cells. However, Fukuhara and Miyata have shown a non-proteic reaction of Rsv, which can act as a prooxidant agent in the presence of copper (Cu), causing cellular oxidative stress accompanied of DNA damage. After this discovery, the complex Rsv-Cu was broadly explored as an antitumor mechanism in multiples tumor cell lines. The aim of the study is to explore the anticarcinogenic behavior of resveratrol–Cu(II) complex in MCF-7 cell line.Selectivity of Rsv binding to Cu ions was analyzed by HPLC and UV–VIS. The cells were enriched with concentrations of 10 and 50 µM CuSO₄ solution and treated with 25 µM of Rsv. Copper uptake after enrichment of cells, as its intracellular distribution in MCF-7 line, was scanned by ICP-MS and TEM-EDS. Cell death and intracellular ROS production were determined by flow cytometry.Different from the extracellular model, no relationship of synergy between Rsv–Cu(II) and reactive oxidative species (ROS) production was detected in vitro. ICP-MS revealed intracellular copper accumulation to both chosen concentrations (0.33 ± 0.09 and 1.18 ± 0.13 ppb) but there is no promotion of cell death by Rsv–Cu(II) complex. In addition, significant attenuation of ROS production was detected when cells were exposed to CuSO₄ after Rsv treatment, falling from 7.54% of ROS production when treated only with Rsv to 3.07 and 2.72% with CuSO₄.Based on these findings antitumor activity of resveratrol when in copper ions presence, is not mediated by Rsv-Cu complex formation in MCF-7 human cell line, suggesting that the antitumoral reaction is dependent of a cancer cellular model.     

Effects of the algicides CuSO4 and NaOCl on various physiological parameters in the harmful dinoflagellate Cochlodinium polykrikoides

The marine dinoflagellate Cochlodinium polykrikoides has spread worldwide and is responsible for harmful algal blooms. The chemical biocides, copper sulfate (CuSO₄) and sodium hypochlorite (NaOCl), are known to be effective in removing bloom-forming or biofouling organisms. Here, we assessed the biocidal efficiency and toxicological properties of NaOCl and CuSO₄ on the physiological and catalase responses of C. polykrikoides. The endpoints used were cell counts, pigment content, chlorophyll autofluorescence (CAF), and antioxidant catalase (CAT) activity. The test organism showed a dose-dependent decrease in growth rate against the algicides; 72-h median effective concentrations (EC₅₀) were 0.584 and 0.633 mg L^–1 for NaOCl and CuSO₄, respectively. The decrease in pigment levels and CAF intensity showed that NaOCl and CuSO₄ might affect the photosynthetic processes of the exposed cells. Furthermore, a considerable increase in CAT activity in the cells was detected, indicating that the algicides might generate reactive oxygen species, thereby markedly damaging the cells. These results suggest that the test algicides are very effective in removing C. polykrikoides by inducing cellular stress and inhibiting cell recovery at higher concentrations.     

Effect of copper excess in environment on soybean root viability and morphology

The effects of increase copper concentrations in medium (10–150 μM CuSO₄) on growth and viability of the roots of two-week-old soybean seedlings (Glycine max L., cv. Dorintsa) were studied. Copper excess suppressed biomass accumulation and linear plant growth; copper affected root growth much stronger than shoot growth. The presence of 10 μM CuSO₄ in medium suppressed accumulation of plant biomass by 40% and the root length by 70%; in the presence of 25 μM CuSO₄, these indices were equal to 80 and 90%, respectively. In the presence of 50 μM CuSO₄, roots ceased to grow but biomass and shoot length still increased slightly. 150 μM CuSO₄ was lethal for plants. The earliest sign of excessive copper toxicity was the accumulation of MDA, indicating activation of membrane lipid peroxidation. A significant increase in MDA content was observed at plant incubation in medium with 10 μM CuSO₄ for 1 h; in this case, the content of copper in the roots increased from 36 ±1.8 (in control) to 48 ± 2.4 μg/g dry wt. The number of dead cells (permeable for the dye Evans Blue) was doubled in the presence of 200 μg/g dry wt within the root; this occurred in 72 h of growth in medium with 10 μM CuSO₄, in 6 h at 25 μM CuSO₄, in 3 h at 50 μM CuSO₄, and 1 h at 150 μM CuSO₄. Toxicity of copper excess was manifested stronger in dividing and elongation cells of the root apex (root meristem and the zone of elongation) than in more basal root regions. Copper excess resulted in the formation of breaks in the surface cell layers of the root tips and affect root morphology. When plant grew in medium with 10 μM CuSO₄, a distance of lateral root formation zone from the root tip decreased markedly, and spherical swellings were formed on the tips of lateral roots. The higher copper concentrations (50 and 150 μM) suppressed completely the development of lateral roots.     

Cadmium,copper and zinc toxicity effects on growth,proline content and genetic stability of Solanum nigrum L., a crop wild relative for tomato; comparative study

Plants like other organisms are affected by environmental factors. Cadmium, copper and zinc are considered the most important types of pollutants in the environment. In this study, a comparison of growth and biochemical parameters between the crop wild relative (CWR) Solanum nigrum versus its cultivated relative Solanum lycopersicum to different levels of Cu, Zn and Cd stress were investigated. The presence of ZnSO₄ and CuSO₄ in Murashige and Skoog medium affected severely many growth parameters (shoot length, number of roots and leaves, and fresh weight) of both S. nigrum and S. lycopersicum at high levels. On the other hand, CdCl₂ significantly reduced most of the studied growth parameters for both species. S. nigrum exhibited higher tolerance than S. lycopersicum for all types of stress. In addition, results show that as stress level increased in the growing medium, proline content of both S. nigrum and S. lycopersicum increased. A significant difference was observed between the two species in proline accumulation as a result of stress. In addition, a higher accumulation rate was observed in the crop wild relative (S. nigrum) than in cultivated S. lycopersicum. Changes in Inter-simple sequence repeat (ISSR) pattern of CuSO₄ treated S. nigrum and S. lycopersicum plants were also observed. In conclusion, based on growth and biochemical analysis, S. nigrum showed higher level of metals tolerance than S. lycopersicum which indicates the possibility of using it as a crop wild relative for S. lycopersicum.     

Production and partial characterization of Duddingtonia flagrans (AC001) crude extract and its in vitro larvicidal action against trichostrongylid infective larvae

The production and partial characterization of Duddingtonia flagrans (AC001) crude extract and its in vitro larvicidal action against trichostrongylid infective larvae from sheep were studied. D. flagrans was grown in liquid medium with glucose, casein, bibasic potassium phosphate (K₂HPO₄), magnesium sulfate (MgSO₄), zinc sulfate (ZnSO₄), ferrous sulfate (FeSO₄), and copper sulfate (CuSO₄). The proteolytic activity was measured within varied pHs and temperatures. To determine the thermostability, the crude extract was incubated at 28°C for 72 h. To study the effect of different chemical compounds on the activity of the crude extract, the samples were incubated in solutions containing (10 mM): calcium chloride (CaCl₂), copper II sulfate (CuSO₄), zinc sulfate (ZnSO₄), magnesium sulfate (MgSO₄), inhibitor phenylmethylsulfonyl fluoride (PMSF), and 0.5% SDS. Results showed that the highest activity obtained (79.23 U/mL) was at pH 9.0, while the optimum temperature was 60°C (119.6 U/mL). The thermostability analysis demonstrated that after 72 h the activity was maintained or increased. It was found that the CuSO₄, ZnSO₄, and PMSF strongly inhibited the proteolytic activity. Moreover, the MgSO4 and SDS, caused a weak inhibition of the proteolytic activity. There was a significant (P<0.01) reduction in number of treated L₃ when compared to control (94.2%). The results suggest that the crude extract produced by D. flagrans (AC001) in liquid medium exerted larvicidal activity on trichostrongilid L₃ and therefore may contribute to a large-scale industrial production.     

RETRACTED ARTICLE: Influence of arbuscular mycorrhizal fungi and copper on growth, accumulation of osmolyte, mineral nutrition and antioxidant enzyme activity of pepper (Capsicum annuum L.)

The effect of arbuscular mycorrhizal (AM) fungi inoculation on pepper (Capsicum annuum L. cv. Zhongjiao 105) plant growth and on some physiological parameters in response to increasing soil Cu concentrations was studied. Treatments consisted of inoculation or not with Glomus mosseae and the addition of Cu to soil at the concentrations of 0 (control), 2 (low), 4 (medium), and 8 (high) mM CuSO₄. AM fungal inoculation decreased Cu concentrations in plant organs and promoted biomass yields as well as the contents of chlorophyll, soluble sugar, total protein, and the concentrations of P, K, Ca, and Mg. Plants grown in high Cu concentration exhibited a Cu-induced proline accumulation and also an increase in total free amino acid contents; however, both were lower in mycorrhizal pepper. Cu-induced oxidative stress by increasing lipid peroxidation rates and the activity of superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase, and AM symbiosis enhanced these antioxidant enzyme activities and decreased oxidative damage to lipids. In conclusion G. mosseae was able to maintain an efficient symbiosis with pepper plants in contaminated Cu soils, improving plant growth under these conditions, which is likely to be due to reduced Cu accumulation in plant tissues, reduced oxidative stress and damage to lipids, or enhanced antioxidant capacity.     

Salinity-induced changes in two cultivars of Vigna radiata: responses of antioxidative and proline metabolism

The influence of increasing salinity stress on plant growth, antioxidant enzymes and proline metabolism in two cultivars of Vigna radiata L. (cv. Pusa Bold and cv. CO 4) was investigated. Salt stress was imposed on 30-days-old cultivars with four different concentrations of NaCl (0, 100, 200 and 300 mM). The roots and shoots of CO 4 showed greater reduction in fresh weight, dry weight and water content when compared to Pusa Bold with increasing salt stress. Under salinity stress, the roots and shoots of CO 4 exhibited higher Na⁺: K⁺ ratio than Pusa Bold. The activities of reactive oxygen species (ROS) scavenging enzymes and reduced glutathione (GSH) concentration were found to be higher in the leaves of Pusa Bold than in CO 4, whereas oxidized glutathione (GSSG) concentration was found to be higher in the leaves of CO 4 compared to those in Pusa Bold. Our studies on oxidative damage in two Vigna cultivars showed lower levels of lipid peroxidation and H₂O₂ concentration in Pusa Bold than in CO 4 under salt stress conditions. High accumulation of proline and glycine betaine under salt stress was also observed in Pusa Bold when compared to CO 4. The activities of proline biosynthetic enzymes were significantly high in Pusa Bold. However, under salinity stress, Pusa Bold showed a greater decline in proline dehydrogenase (ProDH) activity compared to CO 4. Our data in this investigation demonstrate that oxidative stress plays a major role in salt-stressed Vigna cultivars and Pusa Bold has efficient antioxidative characteristics which could provide better protection against oxidative damage in leaves under salt-stressed conditions.     

Copper tolerance in the cuprophyte Haumaniastrum katangense (S. Moore) P.A. Duvign. & Plancke

Cu tolerance and accumulation have been studied in Haumaniastrum katangense, a cuprophyte from Katanga (DR Congo), previously described as a copper hyperaccumulator. Nicotiana plumbaginifolia, a well-known non-tolerant and non-accumulator species, was used as a control. The germination rate of H. katangense was enhanced by copper and fungicide addition, suggesting that fungal pathogens, which restrain germination in normal conditions, are limiting. In hydroponic culture in the Hoagland medium, H. katangense did not grow well, in contrast to N. plumbaginifolia. Better growth was achieved by adding fungicide or higher copper concentrations. The maximal non-effective concentration (NEC) was 12 µM CuSO₄ for H. katangense grown in hydroponics, i.e. 24 times greater than Cu concentration in the Hoagland medium. By comparison, copper concentrations greater than 0.5 µM had a negative effect on the growth of N. plumbaginifolia. EC₅₀ (50% effective concentration) in hydroponics was 40 µM CuSO₄ for H. katangense and 6 µM CuSO₄ for N. plumbaginifolia. EC₁₀₀ (100% effective concentration) was 100 µM CuSO₄ for H. katangense and 15 µM CuSO₄ for N. plumbaginifolia. In soil, growth was also stimulated by Cu addition up to 300 mg kg^-1 CuSO₄. Surplus copper was also required for cultivating H. katangense in sterile conditions, suggesting that Cu excess may be necessary for needs other than pathogen defence. Cu accumulation in the shoot has been measured for N. plumbaginifolia and H. katangense at their respective NEC. Cu allocation in the two species showed a similar response to increasing Cu concentrations, i.e. root/shoot concentration ratio well above 1. In conclusion, H. katangense is highly tolerant to copper and has elevated copper requirement even in the absence of biotic interactions. Its accumulation pattern is typical of an excluder species.