Activities of Cu-containing proteins in Cu-depleted pea leaves

The effect of Cu deficiency on Cu-containing enzymes and on their activities was studied with two subsequent generations of Cu-deficient pea plants ( Pisum sativum L., cv. Progress) grown in low Cu²⁺ media. Cu deficiency caused growth inhibition and a decrease in photosynthesis as well as in the activities of 3 Cu-containing enzymes: diamine oxidase (EC 1.4.3.6), ascorbate oxidase (EC 1.10.3.3) and superoxide dismutase (EC 1.15.1.1). Determinations of photosynthetic electron-transport rates as well as the concentrations of several redox components showed that the target of Cu deprivation in the photosynthetic apparatus is the synthesis of Cu-containing plastocyanin which is positively correlated to the Cu content of the leaves. Inhibited formation of plastocyanin resulted in low activities of photosynthetic electron transport in photosystem I. Under Cu-deficient conditions, the activities of diamine oxidase and ascorbate oxidase were inhibited by about 50% in the first and 80% in the second generation of pea plants. Enzyme assays showed an inhibition of the activities of both the plastidic and cytoplasmic Cu/Zn-containing superoxide dismutases. An observed simultaneous increase of Mn-superoxide dismutase may be a compensation mechanism to partially maintain the total superoxide-dismutase activity under Cu-deficient conditions. This result indicates that the formation of superoxide-dismutase isoenzymes is interdependent and coordinated.     

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.     

Importance of oxidative electron transport over oxidative phosphorylation in optimizing photosynthesis in mesophyll protoplasts of pea (Pisum sativum L.)

The role of mitochondrial respiration in optimizing photosynthesis was assessed in mesophyll protoplasts of pea ( Pisum sativum L., cv. Arkel) by using low concentrations of oligomycin (an inhibitor of oxidative phosphorylation), antimycin A (inhibits cytochrome pathway of electron transport) and salicylhydroxamic acid (SHAM, an inhibitor of alternative oxidase). All three compounds decreased the rate of photosynthetic O₂ evolution in mesophyll protoplasts, but did not affect chloroplast photosynthesis. The inhibition of photosynthesis by these mitochondrial inhibitors was stronger at optimal CO₂ (1.0 m M NaHCO₃) than that at limiting CO₂ (0.1 m M NaHCO₃). We conclude that mitochondrial metabolism through both cytochrome and alternative pathways is essential for optimizing photosynthesis at limiting as well as at optimal CO₂. The ratios of ATP to ADP in whole protoplast extracts were hardly affected, despite the marked decrease in their photosynthetic rates by SHAM. Similarly, the decrease in the ATP/ADP ratio by oligomycin or antimycin A was more pronounced at limiting CO₂ than at optimal CO₂. The mitochondrial oxidative electron transport, through both cytochrome and alternative pathways, therefore akppears to be more important than oxidative phosphorylation in optimizing photosynthesis, particularly at limiting CO₂ (when ATP demand is expected to be low). Our results also confirm that the alternative pathway has a significant role in contributing to the cellular ATP, when the cytochrome pathway is limited.     

Resistance and resilience of Cu-polluted soil after Cu perturbation, tested by a wide range of soil microbial parameters

Copper (Cu)-polluted and unpolluted soils were used to study the effect of initial pollution on soil biological resistance and resilience by measuring the responses to perturbation using different parameters. Microbial biomass carbon, substrate-induced respiration and copy numbers of 16S rRNA gene were grouped as general parameters, while potential ammonia oxidation rate and copy numbers of amo A gene were grouped as specific functions. In addition, to illustrate how initial pollution affects soil biological resistance and resilience following secondary perturbation, the microbial community structure, together with free Cu²⁺ activities ([Cu²⁺]) in soil pore water and soil pH were also measured after secondary perturbation. Results showed that general parameters were more stable than specific ones. High [Cu²⁺] and low pH in soil pore water induced by Cu addition may lead to apparently low resistance and resilience, whereas the formation of a tolerant community after Cu pollution, secondary perturbation and Cu aging may contribute to resistance and resilience. Analysis of the phospholipid fatty acids profile showed that microbial community structure shifted along with the [Cu²⁺] gradient. The microbial community structure of the control soil was both resistant and resilient to 400 mg kg⁻¹ Cu perturbation, whereas other treatments were neither resistant nor resilient.     

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⁺.     

Chlorophyll fluorescence and photoacoustic characteristics in relationship to changes in chlorophyll and Ca2+ content of a Cu-tolerant Silene compacta ecotype under Cu treatment

The effect of Cu toxicity on photosynthetic function, chlorophyll and Ca²⁺ content of Cu-tolerant Silene compacta plants grown in nutrient solution was studied. Since, in plants grown under 8 μ M Cu, the chlorophyll and Ca²⁺ concentration as well as the photosystem II (PSII) photochemistry were increased, compared to the control, the development of an adaptive mechanism of the Cu-tolerant ecotype of S. compacta to 8 μ M Cu is suggested. Increased Cu tolerance of the S. compacta ecotype reflects modulation of the photosynthetic apparatus to optimize photosynthesis. However, exposure of plants to 160 μ M Cu resulted in a marked increase of the fraction of closed PSII centres and decreased quantum yield of PSII electron transport (Φ_PSU) which was accompanied by a significant decline of relative quantum yield for O₂ evolution (A_ox/A_pt). The concentration of chlorophyll and Ca²⁺ in leaves also decreased significantly under 160 μ M Cu treatment. Photochemical quenching (q_p) displayed a reduction as a result of perturbation of the photosynthetic electron transfer chain, while non-photochemical quenching (q_N) increased. High Cu treatment reduced photosynthetic productivity of S. compacta plants which can be attributed, in part, to pertubation of photosynthetic process and photosynthetic pigments as well as to Ca²⁺ loss.     

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.     

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).     

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.     

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.     

Direct and indirect effects of Cd2+ on photosynthesis in sugar beet (Beta vulgaris)

Sugar-beet plants ( Beta vulgaris L. cv. Monohill) were cultivated for 4 weeks in a complete nutrient solution. Indirect effects of cadmium were studied by adding 5, 10 or 20 μ M CdCl₂ to the culture medium while direct effects were determined by adding 1, 5, 20, 50 or 2 000 μ M CdCl₂ to the assay media. The photosynthetic properties were characterized by measurement of CO₂ fixation in intact plants, fluorescence emission by intact leaves and isolated chloroplasts, photosystem (PS) I and PSII mediated electron transport of isolated chloroplasts, and CO₂-dependent O₂ evolution by protoplasts. When directly applied to isolated leaves, protoplasts and chloroplasts. Cd²⁺ impeded CO₂ fixation without affecting the rates of electron transport of PSI or PSII or the rate of dark respiration. When Cd²⁺ was applied through the culture medium the capacity for, and the maximal quantum yield of CO₂ assimilation by intact plants both decreased. This was associated with: (1) decreased total as well as effective chlorophyll content (PSII antennae size), (2) decreased coupling of electron transport in isolated chloroplasts, (3) perturbed carbon reduction cycle as indicated by fluorescence measurements. Also, protoplasts isolated from leaves of Cd²⁺-cultivated plants showed an increased rate of dark respiration.     

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⁺.     

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.     

Growth in elevated CO2 enhances temperature response of photosynthesis in wheat

The temperature dependence of C₃ photosynthesis may be altered by the growth environment. The effects of long-term growth in elevated CO₂ on photosynthesis temperature response have been investigated in wheat ( Triticum aestivum L.) grown in controlled chambers with 370 or 700 μmol mol⁻¹ CO₂ from sowing through to anthesis. Gas exchange was measured in flag leaves at ear emergence, and the parameters of a biochemical photosynthesis model were determined along with their temperature responses. Elevated CO₂ slightly decreased the CO₂ compensation point and increased the rate of respiration in the light and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) V_cmax, although the latter effect was reversed at 15°C. With elevated CO₂, J_max decreased in the 15–25°C temperature range and increased at 30 and 35°C. The temperature response (activation energy) of V_cmax and J_max increased with growth in elevated CO₂. CO₂ enrichment decreased the ribulose 1,5-bisphosphate (RuBP)-limited photosynthesis rates at lower temperatures and increased Rubisco- and RuBP-limited rates at higher temperatures. The results show that the photosynthesis temperature response is enhanced by growth in elevated CO₂. We conclude that if temperature acclimation and factors such as nutrients or water availability do not modify or negate this enhancement, the effects of future increases in air CO₂ on photosynthetic electron transport and Rubisco kinetics may improve the photosynthetic response of wheat to global warming.     

The effect of a day at low irradiance of a maize crop

During the growth of maize ( Zea mays L.cv. INRA F₇× F₂) under constant climatic conditions, the effects of reductions in irradiance simulating a cloudy day were studied. Hourly and daily measurements made in an assimilation chamber (C₂ 3A) showed important and lasting effects in root activity. After reduction of photosynthesis, it took approximately 2 hours to start a lowering of the uptake of NO₃⁻ and NH₄⁺, three hours for K⁺ and four hours for phosphate. Root respiration started to fall after 3 hours. The level to which these activities were reduced also varied. Phosphate uptake was reduced by a mean of 27%, nitrate uptake by 47%, and K⁺ uptake by 55% while the root respiration was reduced by 55%. After return to the initial irradiance, root activities took 3 days to recover their initial rates. Shoot respiration was re-established after one day, while the effects on photosynthesis and transpiration were immediate. The delay of the effect of a change of photosynthesis on the activities of the root, indicates the existence of considerable metabolic reserves. Over longer periods, root metabolism depends on photosynthetic assimilates, but in the short term it is much more dependent on the level of metabolic reserves than on the direct flow of photosynthetic translocates.     

The kinetics of P-700+ reduction in leaves: a novel in situ probe of thylakoid functioning

Abstract. The half time (t_1/2) of the reduction of P-700⁺ in the millisecond time frame is known to be limited by the reaction between plastoquinol and the cytochrome cytb₆f complex. This is considered to be the rate limiting reaction of thylakoid electron transport and measurements of it provide a means of analysing how thylakoid election transport is regulated in vivo. The half time for the reduction of photochemically oxidized P-700 has been measured in vivo using absorbance changes around 820 nm. The results showed that t_1/2 is independent of irradiance and decreases as photosynthetic induction progresses. Even with a constant t_1/2 the quantum efficiency of PSI declined as irradiance increased. The significance of the concept of photosynthetic control of electron transport is discussed in the light of these observations.     

Copper deficiency inhibits Ca2+-induced swelling in rat cardiac mitochondria

Cu deficiency disrupts the architecture of mitochondria, impairs respiration, and inhibits the activity of cytochrome c oxidase - the terminal, Cu-dependent respiratory complex (Complex IV) of the electron transport chain. This suggests that perturbations in the respiratory chain may contribute to the changes in mitochondrial structure caused by Cu deficiency. This study investigates the effect of Cu deficiency on Ca2+-induced mitochondrial swelling as it relates to changes in respiratory complex activities in cardiac mitochondria of rats. Male weanling rats were fed diets containing either no added Cu (Cu0), 1.5 mg Cu/kg (Cu1.5), 3 mg Cu/kg (Cu3) or 6 mg Cu/kg (Cu6). The rate of Ca2+-induced mitochondrial swelling in the presence of succinate and oligomycin was reduced, and the time to reach maximal swelling was increased only in the rats consuming Cu0 diet. Cytochrome c oxidase activity was reduced 60% and 30% in rats fed Cu0 and Cu1.5, respectively, while NADH:cytochrome c reductase (Complex I+ComplexIII) activity was reduced 30% in rats consuming both Cu0 and Cu1.5. Mitochondrial swelling is representative of mitochondrial permeability transition pore (MPTP) formation and the results suggest that Ca2+-induced MPTP formation occurs in cardiac mitochondria of Cu-deficient rats only when cytochrome c oxidase activity falls below 30% of normal. Decreased respiratory complex activities caused by severe Cu deficiency may inhibit MPTP formation by increasing matrix ADP concentration or promoting oxidative modifications that reduce the sensitivity of the calcium trigger for MPTP formation.     

Photosynthesis in ozone-exposed duckweed (Lemna gibba)

The photosynthetic light saturation curve in duckweed was lowered by 20–25% after ozone exposure (300 nmol mol⁻¹, 1 h). The light flux and oxygen concentration during ozone-exposure had no effect on reduction of net photosynthesis. Net photosynthesis and photorespiration were both depressed by about 40% after exposure for 1 h to 360 nmol mol⁻¹ ozone. We could not find any change in dark respiration after ozone exposure below 300 nmol mol⁻¹. When the concentration of ozone was doubled from 150 nmol mol⁻¹ to 300 nmol mol⁻¹, the uptake of ozone in duckweed changed from 100 nmol m⁻² s⁻¹ to 170 nmol m⁻² s⁻¹. We found no differences in fluorescence (pattern) between ozone treated plants and the control plants during a period of 150 min after ozone treatment, but there was an increase in synthesis of the Dl-protein and a significant reduction in degradation after ozone treatment (300 nmol mol⁻¹, 1 h). These results, together with fluorescence measurements, indicate that photochemical electron transport was not responsible for the ozone-induced reduction in net photosynthesis.     

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.