sse of photosynthetic parameters for the diagnosis of copper deficiency in Pinus radiata seedlings

Six-month-old water cultures of Pinus radiataI D. Don seedlings showed optimal growth, and the highest CO₂ assimilation and photosystem I-dependent ascorbate/dichlorophenolindophenol → NADP⁺ electron flow, at 3.0 uM Cu²⁺ (excess) in the hydroponic media. In the nine-month-old water cultures, when the early Cu deprivation has been overcome, the optimum for plant growth and CO₂ fixation shifts to 0.3 u M Cu²⁺ (normal); at that time, the 3.0 uM Cu²⁺ water cultures showed toxic symptoms of foliar chlorosis. Under Cu²⁺ deficient levels (0.03 uM) a clear decrease in the photosystem I-linked electron transport and CO₂ assimilation rates, as well as in the whole plant development, could be observed. Both six- and nine-month-old water cultures showed a close relationship between the Cu²⁺ concentration of the media and the foliar Cu content. However, leaf chlorophyll and the Cu content of thylakoid lamellae showed such a correlation only in the Cu²⁺ deficient and Cu²⁺ normal water cultures. The conclusion from these results is that the electron transport rate ascorbate/dicblorophenolindophenol → NADP⁺, and the Cu content of the photosynthetic membranes, can be used to diagnose a Cu deficiency in Pinus radiata plants.     

Decreased cupric ion uptake as the mechanism for cupric ion resistance in Escherichia coli

Abstract Plasmid-encoded copper (Cu²⁺) resistance in Escherichia coli was due to decreased uptake of Cu²⁺. The Cu²⁺-resistant E. coli Rtsl strain contained a 60 MDa plasmid which is known to encode for both Cu²⁺ and kanamycin resistance. A plasmid-free derivative of the same organism exhibited a greater uptake of Cu²⁺, and sensitivity to Cu²⁺ in both respiration and growth studies than the E. coli Rtsl strain.     

Photosynthetic and respiratory electron transport in Cu2+-deficient Dunaliella

Growth inhibition of the green alga Dunalietla parva Lerche has been observed during cultivation in low Cu²⁺ media. A minimum endogenous Cu concentration for unrestricted growth of 100 to 200 nmol ml⁻¹ packed cell volume was estimated. At lower concentrations, Cu deficiency causes a decrease in photosynthesis and respiration. Assay of photosynthetic electron transport rates as well as the determination of several redox components showed that the target of Cu deprivation in the photosynthetic apparatus is the synthesis of Cu-containing plastocyanin. Consequently, inhibited formation of plastocyanin resulted in low activities of photosynthetic electron transport. A secondary, indirect effect of Cu deficiency is the reduction of thylakoid formation resulting in an additional decrease of photosynthesis compared to cultures with sufficient Cu²⁺.
The inhibitory influence of low Cu²⁺ on respiration was located at the site of cytochrome oxidase. In contrast to blue-green algae, a strong coordination of the biosynthesis of the cytochrome oxidase complex was evident. During restricted Cu²⁺ supply the formation of cytochiome aa₃ , another component besides Cu, was stalled. The resulting low activities of cytochrome oxidase are responsible for decreased respiratory electron transfer activity from NADPH to oxygen. At Cu²⁺ concentrations which exert only moderate effects on Dunalietla , the cytochrome oxidase reaction was more strongly affected than the photosystem I reaction.     

Acute toxicology to an estuarine teleost of mixtures of cadmium, copper and zinc salts

Effects of mixtures of chloride salts of cadmium, copper and zinc on survival, whole body residues, and histopathology of mummichog, Fundulus heteroclitus (L.), were investigated in synthetic sea water at 20‰ salinity and 20°C. Mixtures of Cu²⁺ and Zn²⁺ as indicated by 96 h bioassay studies produced more deaths than expected on the basis of toxicities of individual components. Concentrations of Cd²⁺ not ordinarily lethal exerted a negative effect on survival of fish intoxicated by salts of copper, zinc, or both.
Atomic absorption determinations of Cd, Cu, and Zn residues in mummichog which survived 96 h exposures to each of these toxicants provided useful indices of total body burdens for these metals. Residues from survivors held in mixtures, especially Cd²⁺ and Zn²⁺ mixtures, did not conform to patterns observed for single elements. Whole body aggregates of Cd, Cu, and Zn from dead mummichogs were of limited worth owing to possible accumulation of these metals from the medium after death.
Renal and lateral line canal lesions were noted in all fish subjected to copper concentrations of 1 mg/1 and higher. Renal lesions observed in fish immersed in mixtures of Cu²⁺ and Cd²⁺ assumed a damage pattern characteristic of Cd²⁺; with mixtures of Cu²⁺ and Zn²⁺, lesion were typical of Cu²⁺-induced damage. Lesions induced in lateral line epithelium by Cu²⁺ were not affected by either Cd²⁺ or Zn²⁺. Epithelia lining the oral cavity were necrotized by the caustic action of high levels of Zn²⁺ (60 mg/1) and of Cu²⁺ (8 mg/1).     

The regulation of Mn2+ and Cu2+ uptake in cells of the yeast Candida utilis grown in continuous culture

Abstract Transport of Mn²⁺ was repressed in Candida utilis cells grown in continuous culture in high-Mn²⁺ (100 μM Mn²⁺) medium as compared to cells grown in basic (0.45 μM Mn²⁺) and low-Mn²⁺ (< 0.05 μM Mn²⁺) media. In contrast, no repression of Cu²⁺ uptake occurred in high-Cu²⁺-grown (25 μM Cu²⁺) cells as compared to cells grown in basic medium (0.54 μM Cu²⁺). Cu²⁺-limited cells did not hyperaccumulate Cu²⁺ and there was not significant difference in initial uptake rates for all 3 Cu²⁺ conditions. Mn²⁺ uptake appears to be regulated by a mechanism sensitive to the external Mn²⁺ concentration, whereas Cu²⁺ transport is not governed in this way by the external Cu²⁺.     

Activity of methanotrophic bacteria in Green Bay sediments

Abstract Sediment pore water samples obtained from a 19 m station in Green Bay in Lake Michigan were examined for levels of ambient dissolved methane and copper, and for the potential for in situ methane oxidation by methanotrophs found within surface sediments. The in situ methane concentration in the upper oxic sediment layer ranged from 20–150 μmol · 1⁻¹ at this station. The activity of methanotrophs and the kinetics of methane oxidation in these sediments were demonstrated by the uptake of radiolabeled methane. K_s values varied between 4.1–9.6 nmol · cm³ of sediment slurry. High V_max values (12.7–35.2 nmol · cm⁻³ · h⁻¹) suggest a large population of methanotrophs in the sediments. An average methane flux to the oxic sediments of 0.24 mol · m⁻² · year⁻¹ was calculated from the pore water methane gradients. Pore water concentrations of copper in the upper sediment layer ranged from 10–120 nmol · 1⁻¹. Based upon the copper concentration, other measured parameters, and equilibrium conditions defined by WATEQF4, an estimate for dissolved free Cu²⁺ concentration of 5–38 nmol · 1⁻¹ pore water was obtained. Several factors control the rate of methane oxidation, including oxygen, methane, and the bioavailability of free Cu²⁺.     

THE RELEASE OF COPPER-COMPLEXING LIGANDS BY THE BROWN ALGA FUCUS VESICULOSUS (PHAEOPHYCEAE) IN RESPONSE TO INCREASING TOTAL COPPER LEVELS

The growth of Fucus vesiculosus L. germlings in chemically defined culture media containing a range of Cu concentrations (20–1000 nM) was monitored simultaneously with measurement of the Cu speciation in the media by competitive equilibrium-adsorptive cathodic stripping voltammetry. Fucus vesiculosus germlings were found to exude Cu-complexing ligands with conditional stability constants of the order of 1.6 × 10¹¹. Ligand concentrations increased with increasing total dissolved Cu concentrations (Cu_T) until a concentration of 500–800 neq Cu·L⁻¹ was reached. Concentrations of the ligand exceeded Cu_T in treatments containing 20 and 100 nM Cu, were similar to Cu_T in the 500-nM Cu treatment, but were less than Cu_T in the 1000-nM treatment. Therefore, [Cu²⁺] were calculated to be at concentrations of 10⁻¹¹− 10⁻¹⁰ M in the 20- and 100-nM treatments, 10⁻⁹ M in the 500-nM treatment, and 10⁻⁷ M in the 1000-nM treatment. Growth rates were lowest at Cu²⁺ concentration > 10⁻⁹. These results are discussed within the context of the potential roles for exuded copper-complexing ligands.     

The effect of hydrogen peroxide on spores of Clostridium perfringens

Dithiothreitol (DTT)-treated spores of Clostridium perfringens were much more sensitive to lysis by H₂O₂ in the presence of Cu²⁺ than untreated spores. Lysis was greatly inhibited by hydroxyl radical (.OH) scavengers such as thiourea, dimethylthiourea and dimethylsulfoxide, suggesting that lysis of spores by H₂O₂ involves formation of OH by Cu²⁺-catalysed decomposition of the peroxide. DTT-treated spores took up Cu²⁺ at almost the same rate and extent as did isolated cortical fragments. Hydrogen peroxide caused both the decrease in optical density and the hexosamine solubilization of cortical fragments which bound Cu²⁺.     

Enzymatic desulfurization of dibenzothiophene by a cell-free system of Rhodococcus erythropolis D-1

Abstract Pseudomonas syringae cells were exposed to Cu²⁺ alone or in the precence of acetate, proline or cysteine, at concentrations that reduced free Cu²⁺ to 1/10 of the total copper. Ligand concentrations (designated as isoeffective) were determined experimentally using a Cu²⁺-selective electrode and confirmed by computer calculations using published stability constants. Exposure of P. syringae cells to Cu²⁺ alone resulted in rapid and pronounced cell death, and binding of most of the copper in solution. The addition of acetate, proline or cysteine, a few minutes after Cu²⁺ treatment, resulted in a significant reduction in cell death, and in the amount of copper bound to the cells. For short exposures to Cu²⁺, cysteine was more effective than acetate or proline, but after 60 min of treatment, similar results were observed with these ligands. The addition of ligands before Cu²⁺ resulted in even more reduced copper toxicity. The results showed that, at isoeffective concentrations, weak and moderate copper-ligands can effectively antagonize copper toxicity, and that this protective effect does not require previously equilibrated copper-ligand solutions and is not very dependent of the nature of the ligand.     

Characterization of Wild-Type and Amyotrophic Lateral Sclerosis-Related Mutant Cu,Zn-Superoxide Dismutases Overproduced in Baculovirus-Infected Insect Cells

Abstract: We describe the use of a baculovirus expression system to overproduce human Cu,Zn-superoxide dismutase (SOD). Spodoptera frugiperda (Sf21) insect cells infected with a baculovirus carrying the Cu,Zn-SOD cDNA synthesized a large amount of Cu,Zn-SOD apoprotein in the conventional medium. The SOD activity of the apoprotein, which was initially very low, increased in a dose-dependent manner when Cu²⁺ and Zn²⁺ were added to the medium. Cells grown in media supplemented with Cu²⁺ alone exhibited nearly maximal SOD activity. SOD activity reached 40% of the maximal level within 2 h after addition of Cu²⁺ to postinfected cells cultivated for 3 days in the conventional medium, and the activity gradually increased thereafter. The protein produced by the infected cells was purified by a simple procedure involving two chromatographic steps, DE52 ion exchange and ACA54 gel filtration. Identification of the recombinant Cu,Zn-SOD with the human erythrocyte enzyme was confirmed by immunochemical reactivity to anti-human Cu,Zn-SOD antibody and by partial amino acid sequencing of peptides from purified protein (50 amino acid residues in total). We constructed three mutant enzymes, which have been found in familial amyotrophic lateral sclerosis and are overproduced in Sf21 cells, and purified them. Mutant enzymes Gly⁴¹Asp, His⁴³Arg, and Gly⁸⁵Arg exhibited 47, 66, and 99% of wild-type SOD activity, respectively. The availability of this protein will facilitate investigation of the relationship between the structure and function of the mutant enzymes found in familial amyotrophic lateral sclerosis.     

Bactericidal activity of catechin-copper (II) complexes on Escherichia coli ATCC11775 in the absence of hydrogen peroxide

Washed Escherichia coli ATCC11775 cells were killed by (–)-epigallocatechin (EGC) in the presence of a non- lethal concentration of Cu²⁺ (1 μmol l⁻¹) without additional H₂O₂, but not by (–)-epicatechin (EC). EGC alone (< 0·1 mmol l⁻¹) did not reduce the viability of the cells. The survival curve obtained in the presence of EGC and Cu²⁺ was similar to that obtained in the presence of (–)-adrenaline (EN) and Cu²⁺.     

Cadmium and copper change root growth and morphology of Pinus pinea and Pinus pinaster seedlings

Heavy metal loads in forest soils have been increasing over time due to atmospheric inputs. Accumulation in the upper soil layers could affect establishment of seedlings and forest regeneration. Mediterranean species show a high initial root development, allowing seedlings to reach the moisture of deeper soil layers. In the present work seedlings of stone pine ( Pinus pinea L.) and maritime pine ( Pinus pinaster Ait.), were grown in culture solution supplied with 0.0, 0.1, 1 or 5 μ M CdSO₄ or with 1 μ M CdSO₄ and 1 μ M CuSO₄ combined. In both species tap-root elongation was drastically reduced in the 5 μ M Cd²⁺ and in the (Cd²⁺+ Cu²⁺) treatments. A supply of 0.1 or 1 μ M Cd²⁺, however, enhanced root elongation in Pinus pinea without significantly influencing root elongation in Pinus pinaster . In both species the root density (weight per unit length) and the width of the cortex increased in response to Cd²⁺ exposure. In Pinus pinaster the mitotic index decreased at the higher Cd²⁺ concentrations and when Cd²⁺ and Cu²⁺ were combined. The data suggest that cell elongation is more sensitive to Cd²⁺ than cell division. The number and length of the lateral roots were also affected by Cd²⁺ treatment to a higher degree in Pinus pinaster than in Pinus pinea, reflecting the different Cd- tolerance of the two species.     

Mechanism of copper-enhanced photoinhibition in thylakoid membranes

The effect of copper on photoinhibition of photosystem II (PSII) in vitro was studied in bean ( Phaseolus vulgaris L. cv. Dufrix) and pumpkin ( Cucurbita pepo L.) thylakoids. The thylakoids were illuminated at 200–2 000 μmol photons m⁻² s⁻¹ in the presence of 70–1 830 added Cu²⁺ ions per PSII. Three lines of evidence show that the irreversible damage of PSII caused by illumination of thylakoids in the presence of Cu²⁺ was mainly due to donor-side photoinhibition resulting from inhibition of the PSII donor side by Cu²⁺. First, addition of an artificial electron donor partially restored PSII activity of thylakoids that had been illuminated in the presence of Cu²⁺. Second, already moderate light was enough to cause rapid inhibition of PSII, and the inhibition could be saturated by light. Third, the extrinsic polypeptides of the oxygen-evolving complex were found to become oxidized by the combined effect of Cu²⁺ and light. The presence of oxygen was not necessary for the copper-induced enhancement of photoinhibition of PSII. When the illumination was prolonged, copper caused a gradual collapse of the thylakoid structure by increasing degradation of thylakoid proteins.     

New insights into the metal specificity of the Pseudomonas aeruginosa pyoverdine–iron uptake pathway

Pyoverdine (PvdI) is the major siderophore secreted by Pseudomonas aeruginosa PAOI in order to get access to iron. After being loaded with iron in the extracellular medium, PvdI is transported across the bacterial outer membrane by the transporter, FpvAI. We used the spectral properties of PvdI to show that in addition to Fe³⁺, this siderophore also chelates, but with lower efficiencies, all the 16 metals used in our screening. Afterwards, FpvAI at the cell surface binds Ag⁺, Al³⁺, Cd²⁺, Co²⁺, Cu²⁺, Fe³⁺, Ga³⁺, Hg²⁺, Mn²⁺, Ni²⁺ or Zn²⁺ in complex with PvdI. We used Inductively Coupled Plasma-Atomic Emission Spectrometry to monitor metal uptake in P. aeruginosa : TonB-dependent uptake, in the presence of PvdI, was only efficient for Fe³⁺. Cu²⁺, Ga³⁺, Mn²⁺ and Ni²⁺ were also transported into the cell but with lower uptake rates. The presence of Al³⁺, Cu²⁺, Ga³⁺, Mn²⁺, Ni²⁺ and Zn²⁺ in the extracellular medium induced PvdI production in P. aeruginosa . All these data allow a better understanding of the behaviour of the PvdI uptake pathway in the presence of metals other than iron: FpvAI at the cell surface has broad metal specificity at the binding stage and it is highly selective for Fe³⁺ only during the uptake process.     

氮添加诱导的磷限制改变了亚热带黄山松林土壤微生物群落结构

土壤微生物在陆地生态系统的生物地球化学循环中起着重要作用。然而目前尚不清楚氮(N)添加量及其持续时间如何影响土壤微生物群落结构,以及微生物群落结构变化与微生物相对养分限制状况是否存在关联。本研究在亚热带黄山松林开展了N添加试验以模拟N沉降,并设置3个处理:对照(CK, 0 kg N·hm^-2·a^-1)、低N(LN, 40 kg N·hm^-2·a^-1)和高N(HN, 80 kg N·hm^-2·a^-1)。在N添加满1年和3年时测定土壤基本理化性质、磷脂脂肪酸含量和碳(C)、N、磷(P)获取酶活性,并通过生态酶化学计量分析土壤微生物的相对养分限制状况。结果表明: 1年N添加对土壤微生物群落结构无显著影响,3年LN处理显著提高了革兰氏阳性菌(G⁺)、革兰氏阴性菌(G^-)、放线菌(ACT)和总磷脂脂肪酸(TPLFA)含量,而3年HN处理对微生物的影响不显著,表明细菌和ACT对N添加可能更为敏感。N添加加剧了微生物C和P限制,而P限制是土壤微生物群落结构变化的最佳解释因子。这表明,N添加诱导的P限制可能更有利于部分贫营养菌(如G⁺)和参与P循环的微生物(如ACT)的生长,从而改变亚热带黄山松林土壤微生物群落结构。     

5'-Methylthioadenosine nucleosidase and 5-methylthioribose kinase activities in relation to stress-induced ethylene biosynthesis

The activities of 5'-methylthioadenosine (MTA) nucleosidase (EC 2.2.2.28) and 5-methylthioribose (MTR) kinase (EC 2.7.1.100) were related to changes in ethylene biosynthesis in tomato ( Lycopersicon esculentum Mill. cv. Rutgers) and cucumber ( Cucumis sativus Mill. cv. Poinsett 76) fruit following wounding and chemically induced stresses. Stress ethylene formation in wounded tomato and cucumber tissue continued to increase after wounding, reached its peak by 3h, and then declined. The activities of MTA nucleosidase and MTR kinase increased parallel to stress ethylene in both tissues. At peak ethylene formation, MTA and MTR kinase activities were 2- to 4-fold higher in wounded than in intact tissue. Wounded, mature-green tomato tissue treated with specific inhibitors of MTA nucleosidase and MTR kinase showed a significant reduction in the activities of these enzymes, which was concomitant with a decline in stress ethylene biosynthesis. When mature-green tomato discs were infiltrated with [¹⁴CH₃] MTA and wounded, radioactive MTR and methionine were formed. Incubation of mature-green tomato discs with Cu²⁺ and Li⁺ in the presence of kinetin increased ethylene biosynthesis. MTA nucleosidase activity was higher than that of the control in the presence of Cu²⁺ but not in the presence of Li⁺, while MTR kinase activity was lower than that of the control in both Cu²⁺ and Li⁺ treatments. Data indicate that MTA nucleosidase and MTR kinase are required for wound-induced ethylene biosynthesis but not for chemical stress-induced ethylene by Cu²⁺ or Li⁺ treatments.     

Ascorbate-Induced Oxidative Inactivation of Zn2+-Glycerophosphocholine Cholinephosphodiesterase

Abstract: Zn²⁺-glycerophosphocholine cholinephosphodiesterase, responsible for the conversion of glycerophosphocholine into glycerol and phosphocholine, was inactivated during incubation with ascorbic acid at 38°C. The inclusion of copper ions or Fe²⁺ accelerated the ascorbate-induced inactivation, with Cu²⁺ or Cu⁺ being much more effective than Fe²⁺, suggestive of ascorbate-mediated oxidation. Dehydroascorbic acid had no effect on the phosphodiesterase, but H₂O₂ inactivated the enzyme in a concentration-dependent manner. Also, the enzyme was inactivated partially by a superoxide anion-generating system but not an HOCl generator. In support of involvement of H₂O₂ in the ascorbate action, catalase and superoxide dismutase expressed a complete and a partial protection, respectively. However, hydroxy radical scavengers such as mannitol, benzoate, or dimethyl sulfoxide were incapable of preventing the ascorbate action, excluding the participation of extraneous ^•OH. Although p -nitrophenylphosphocholine exhibited a modest protection against the ascorbate action, a remarkable protection was expressed by amino acids, especially by histidine. In addition, imidazole, an electron donor, showed a partial protection. Separately, when Cu²⁺-induced inactivation of the phosphodiesterase was compared with the ascorbate-mediated one, the protection and pH studies indicate that the mechanism for the ascorbate action is different from that for the Cu²⁺ action. Here, it is proposed that Zn²⁺-glycerophosphocholine cholinephosphodiesterase is one of brain membrane proteins susceptible to oxidative inactivation.     

Effects of soil frost on nitrogen net mineralization, soil solution chemistry and seepage losses in a temperate forest soil

Freezing and thawing may alter element turnover and solute fluxes in soils by changing physical and biological soil properties. We simulated soil frost in replicated snow removal plots in a mountainous Norway spruce stand in the Fichtelgebirge area, Germany, and investigated N net mineralization, solute concentrations and fluxes of dissolved organic carbon (DOC) and of mineral ions (NH₄⁺, NO₃⁻, Na⁺, K⁺, Ca²⁺, Mg²⁺). At the snow removal plots the minimum soil temperature was −5 °C at 5 cm depth, while the control plots were covered by snow and experienced no soil frost. The soil frost lasted for about 3 months and penetrated the soil to about 15 cm depth. In the 3 months after thawing, the in situ N net mineralization in the forest floor and upper mineral soil was not affected by soil frost. In late summer, NO₃⁻ concentrations increased in forest floor percolates and soil solutions at 20 cm soil depth in the snow removal plots relative to the control. The increase lasted for about 2–4 months at a time of low seepage water fluxes. Soil frost did not affect DOC concentrations and radiocarbon signatures of DOC. No specific frost effect was observed for K⁺, Ca²⁺ and Mg²⁺ in soil solutions, however, the Na⁺ concentrations in the upper mineral soil increased. In the 12 months following snowmelt, the solute fluxes of N, DOC, and mineral ions were not influenced by the previous soil frost at any depth. Our experiment did not support the hypothesis that moderate soil frost triggers solute losses of N, DOC, and mineral ions from temperate forest soils.     

Influence of pulsing and postpulsing media tonicity on electrotransformation of intact yeast cells

Abstract The carboxylesterases from Proteus vulgaris, Salmonella enterica and Citrobacter amalonaticus were purified 104-, 95- and 120-fold, respectively by chromatography. The enzymes had similar catalytic activities but differed considerably in their inactivation by heat, di-isopropyl fluorophosphate and Cd²⁺, Zn²⁺, Hg²⁺ and Cu²⁺. Quantitative neutralization of hydrolytic activity with specific immunoglobulins indicated that the three enzymes were antigenically distinct.     

Variation in the relationship between nitrogen fixation, leghemoglobin, nodule numbers and plant biomass in alfalfa (Medicago sativa) caused by treatment with arsenate, heavy metals and fluoride

Potential relationships between alfalfa ( Medicago sativa L. cv. Vernal) acetylene reduction activity (ARA) and leghemoglobin content, nodule numbers, shoot biomass, root biomass, or total plant biomass were estimated using linear regression analysis after treatment with a range of concentrations of arsenate, Cd²⁺, Cu²⁺, Pb²⁺, Zn²⁺, and F^–. There were highly significant positive linear regressions between ARA and all the other plant values in the control plants, but this linear relationship was significantly altered between ARA and one or more of the other parameters in all treatments. There was also evidence for an alteration of the slope in some of the treatments when compared to control treatments even though a linear model was still applicable.