Role of the alternative oxidase in limiting superoxide production by plant mitochondria

Mitochondria isolated from the pericarp tissue of green bell pepper ( Capsicum annuum L.) fruit and purified on a Percoll gradient produced superoxide in buffers aerated with oxygen. ADP and uncouplers of the electron transport chain reduced superoxide production. Disulfiram, an inhibitor of the alternative oxidase, enhanced superoxide production. Inhibitors of complex III had little effect on superoxide production by mitochondria which were insensitive to cyanide. Less superoxide was produced when dithiothreitol was used to reduce the sulfhydryl groups of the alternative oxidase protein and the enzyme was activated with pyruvate than when the sulfhydryl groups were oxidized with diamide. A role for the alternative oxidase in limiting the level of reactive oxygen species produced in stressed and senescing plant tissues is suggested.     

Inhibition of electron transport activities in mitochondria from avocado and pepper fruit by naturally occurring polyamines

The effects of the naturally occurring polyamines, spermine, putrescine, and spermidine were explored on mitochondrial state 3. state 4, and uncoupled respiration activities, ADP/O ratio, respiratory control ratio of pepper ( Capsicum annuum L. cv. Early Cal Wonder) and avocado ( Persea americana Mill. cv. Booth-8 or Simmonds) mitochondria oxidizing either succinate, external NADH, malate, α-ketoglutarate or tetramethyl- p -phenylenediamine. Abnormally high concentrations of spermine and spermidine such as might occur during chilling stress of these chilling-sensitive fruits were detrimental to several oxidase activities, especially to external NADH oxidase. State 3 respiration for NADH oxidase was inhibited more than 70% by 10 m M spermine. The spermine inhibition of uncoupled NADH oxidase was not reversed by the presence of divalent cations including Ca²⁺, Mg²⁺, Mn²⁺, and Sr²⁺ at concentrations up to 10 m M or by 100 m M KCl. The inhibition primarily affected the V_max. Other possible sites of polyamine interactions are discussed.     

Effects of paclobutrazol and chilling temperatures on lipids, antioxidants and ATPase activity of plasma membrane isolated from green bell pepper fruits

The effects of paclobutrazol treatment on plasma membrane lipid composition and ATPase activity of bell pepper fruit ( Capsicum annuum ) subjected to chilling temperatures were assessed. Application of the growth regulator paclobutrazol affected plant growth and fruit morphology. The plants were more compact and the fruits were less elongated than control fruits. There was about 60% more plasma membrane on a fresh weight basis from treated fruits. At harvest there was no difference in sterol to phospholipid ratio, or in phospholipid fatty acid composition of control compared with paclobutrazol treated fruit. However, plasma membrane ATPase acitivity of treated fruit was two times higher than that of control fruit. After storage at chilling temperature (2°C), the control fruit developed more chilling iniury, and had greater weight loss and a higher rate of K⁺ leakage than paclobulrazol treated fruit. Plasma membrane phospholipid content decreased and saturation of phospholipid fatty acids was higher than in control fruit. These two changes were largely absent in plasma membrane from treated fruit. At harvest antioxidant levels in the plasma membrane of paclobutrazol treated peppers were higher than in those of controls and changed little during storage, whereas levels in control fruit plasma membrane decreased 66%. ATPase activity increased and then decreased in control fruit held at low temperature, whereas in treated fruit activity was constant. The protective effect of paclobutrazol against chilling injury of pepper fruit may result from a combination of its effect on fruit morphology, and protection of the lipids against oxidative stress.     

Physiology of pepper fruit and the metabolism of antioxidants: chloroplasts,mitochondria and peroxisomes

Background and Aims Pepper (Capsicum annuum) contains high levels of antioxidants, such as vitamins A and C and flavonoids. However, information on the role of these beneficial compounds in the physiology of pepper fruit remains scarce. Recent studies have shown that antioxidants in ripe pepper fruit play a key role in responses to temperature changes, and the redox state at the time of harvest affects the nutritional value for human consumption. In this paper, the role of antioxidant metabolism of pepper fruit during ripening and in the response to low temperature is addressed, paying particular attention to ascorbate, NADPH and the superoxide dismutase enzymatic system. The participation of chloroplasts, mitochondria and peroxisomes in the ripening process is also investigated.Scope and Results Important changes occur at a subcellular level during ripening of pepper fruit. Chloroplasts turn into chromoplasts, with drastic conversion of their metabolism, and the role of the ascorbate–glutathione cycle is essential. In mitochondria from red fruits, higher ascorbate peroxidase (APX) and Mn-SOD activities are involved in avoiding the accumulation of reactive oxygen species in these organelles during ripening. Peroxisomes, whose antioxidant capacity at fruit ripening is substantially affected, display an atypical metabolic pattern during this physiological stage. In spite of these differences observed in the antioxidative metabolism of mitochondria and peroxisomes, proteomic analysis of these organelles, carried out by 2-D electrophoresis and MALDI-TOF/TOF and provided here for the first time, reveals no changes between the antioxidant metabolism from immature (green) and ripe (red) fruits.Conclusions Taken together, the results show that investigation of molecular and enzymatic antioxidants from cell compartments, especially chloroplasts, mitochondria and peroxisomes, is a useful tool to study the physiology of pepper fruit, particularly in the context of expanding their shelf-life after harvest and in maintaining their nutritional value.     

Disinfestations of the oriental tobacco budworm in green hot pepper by ultra high carbon dioxide: Implications for postharvest fruit quality

To develop environmentally amenable insect disinfestations, effects of a carbon dioxide (CO₂) controlled atmosphere (CA) on the control of the oriental tobacco budwormHelicoverpa assulta were investigated in green hot peppers. Green hot peppers (cv. Nokgwang) were exposed to CO₂. at 80% and 100% in 0.08-mm polyethylene film bags for 24 and 48 h at 20°C. Mortality percentages of oriental tobacco budworm larvae were determined after gas exposure. The CO₂-CA at both concentrations for 24 h greatly reduced survival of the larvae, showing approximately 65% mortality when compared with control fruit. Prolonged exposure at both concentrations up to 48 h completely disinfested the larvae. To evaluate plausible deleterious effects of the ultra high CO₂-CA on green hot peppers, the fruit were stored at 10°C, and postharvest quality was analyzed in terms of firmness, electrolyte leakage, respiration rate, and content of vitamin C and capsaicin. There were no significant differences in postharvest fruit quality up to 20 days of storage, compared with control fruit. Meanwhile, respiration rates of exposed pepper fruit were approximately half the control’s rate after 20 days of storage. These results suggested that ultra high CO₂ CA could disinfestH. assulta without significant differences in postharvest quality of green hot peppers, compared with control fruit. Exposure of 80% CO₂ for 24 h would be recommended as a reliable control means that is harmless to humans and can alleviate concern regarding pesticide residues.     

Impact of moderate and extreme climate change scenarios on growth,morphological features,photosynthesis, and fruit production of hot pepper

Horticultural crop production and changes in physiological aspects during the growing season may be affected by climate change factors (CC), which include increased temperature and the associated doubling or tripling of atmospheric CO₂ concentrations. However, the potential effects are complex and many parameters might impact on the observed effects. To evaluate the effects of CC, the growth, yield, fruit characteristics, photosynthetic traits, and morphological characteristics of hot peppers were investigated. The hot peppers were grown under two CC scenarios, with the Representative Concentration Pathway (RCP) of 4.5 (Temp.; +3.4°C, CO₂ conc.; 540 μmol/mol, Precipitation +17.3%) and RCP 8.5 (Temp.; +6.0°C and CO₂ conc.; 940 μmol/mol, Precipitation +20.3%), respectively, using extreme weather simulators. This was compared with existing weather conditions occurring in Jeonju, South Korea in terms of air temperature, relative humidity, radiation, and precipitation. Overall, the plant height showed the highest under moderate CC conditions (RCP 4.5) among all the treatments tested. The number of leaves in the RCP 8.5 condition showed 7,739/plants, which was 2.2 times higher than that of the control. In addition, fruit shape was shortened and percentage dry matter was also the highest. The yield of hot pepper in the CC RCP 4.5 and 8.5 conditions were decreased by 21.5% and 89.2% when compared with that of the control, respectively. The days to harvest in the condition of CC scenarios were shortened from 5 to 13 compared with that of control, predominantly due to the increased air temperature. The results indicated that the severe RCP CC scenarios made reduction in the yields and negative affection on the fruit qualities. Overall, hot pepper was tolerant of mild CC scenarios of temperature × CO₂ but was significantly affected by more extreme CC interacting parameter concentrations (or similar).     

Effect of electron-transport inhibitors on the generation of reactive oxygen species by pea mitochondria during succinate oxidation

The effect of inhibitors of the cytochrome pathway and alternative oxidase on the rate of respiration and generation of reactive oxygen species by pea mitochondria was studied. Respiration of mitochondria from pea cotyledons was inhibited by 70-80% by salicylhydroxamate (SHAM). The rate of hydrogen peroxide production by pea cotyledon mitochondria during succinate oxidation was 0.15 nmol/min per mg protein. SHAM considerably accelerated the hydrogen peroxide production. The SHAM-dependent H₂O₂ production was stimulated by 2 M antimycin A and inhibited by 5 mM KCN and 1 M myxothiazol. The study of the rate of generation by pea mitochondria using EPR spin traps and epinephrine oxidation showed that H₂O₂ accumulation can be accounted for by a significant increase in the rate of production.     

Zinc inhibition of cellular energy production: implications for mitochondria and neurodegeneration

An increasing body of evidence suggests that high intracellular free zinc promotes neuronal death by inhibiting cellular energy production. A number of targets have been postulated, including complexes of the mitochondrial electron transport chain, components of the tricarboxylic acid cycle, and enzymes of glycolysis. Consequences of cellular zinc overload may include increased cellular reactive oxygen species (ROS) production, loss of mitochondrial membrane potential, and reduced cellular ATP levels. Additionally, zinc toxicity might involve zinc uptake by mitochondria and zinc induction of mitochondrial permeability transition. The present review discusses these processes with special emphasis on their potential involvement in brain injury.     

Reduction of superoxide production by mitochondria oxidizing NADH in the presence of organic acids

Effects of multiple substrates on oxygen uptake and superoxide production by mitochondria isolated from the pericarp tissue of green bell pepper (Capsicum annuum L.) were studied. Mitochondria isolated from peppers stored at 4 °C for 5 and 6 days had higher rates of oxygen uptake and were less sensitive to cyanide than mitochondria isolated from freshly harvested peppers. Succinate enhanced state 2 and state 4 rates of oxygen uptake with exogenous NADH in the absence of cytochrome path inhibitors, but not state 3 rates by mitochondria isolated from either freshly harvested or cold-stored bell peppers. The sensitivity of NADH oxidation to cyanide was reduced by both malate and succinate in mitochondria from cold-stored bell peppers, whereas only succinate was effective in mitochondria from freshly harvested peppers.Mitochondria isolated from both freshly harvested peppers and those stored at 4 °C for 5 and 6 days produced superoxide in the absence of exogenous substrates. Superoxide production by mitochondria from freshly harvested bell peppers increased when the mitochondria were supplied with malate, succinate or NADH, but only NADH enhanced superoxide production by mitochondria from cold-stored peppers. Both succinate and malate reduced the production of superoxide by mitochondria isolated from cold-stored bell peppers. Succinate and malate as second substrates also reduced the production of superoxide with NADH by mitochondria from both freshly harvested and cold-stored bell peppers. Malonate, a competitive inhibitor of succinate dehydrogenase, was inhibitory to oxygen uptake and to superoxide production.Mitochondria isolated from cold-stored bell peppers converted succinate to pyruvate at 25 °C at considerably higher rates than those of mitochondria from freshly harvested bell peppers. Since pyruvate has been shown to activate the alternative oxidase and the presence of pyruvate is essential for continued alternative oxidase activity, we suggest that pyruvate limits superoxide production by enhancing the flow of electrons through the alternative path. A direct scavenging of superoxide by succinate, malate and pyruvate, however, cannot be ruled out.     

Does the alternative pathway ameliorate chilling injury in sensitive plant tissues?

Free radical processes have been observed in senescence and several membrane-associated disorders of plants including chilling, freezing, and desiccation injuries. The mitochondria of plant tissues exposed to low temperatures, and other abiotic and biotic stresses, produce superoxide and/or hydrogen peroxide when electron transport through the cytochrome pathway is impaired due to the energy state of the cell or to stress-induced physical changes in the membrane components. The superoxide and/or hydrogen peroxide produced can diffuse throughout the cell causing peroxidation of membrane lipids which results in membrane disruption, increased permeability and metabolic disturbances, and eventually the visible symptoms of chilling injury. The alternative pathway of electron transport in the mitochondria, which is induced by low temperatures in some plant tissues, can mediate these degradative processes by reducing the level of superoxide generated by the mitochondria.     

The role of UCP 1 in production of reactive oxygen species by mitochondria isolated from brown adipose tissue

We provide evidence that ablation or inhibition of, uncoupling protein 1 increases the rate of reactive oxygen containing species production by mitochondria from brown adipose tissue, no matter what electron transport chain substrate is used (succinate, glycerol-3-phosphate or pyruvate/malate). Consistent with these data are our observations that (a) the mitochondrial membrane potential is maximal when uncoupling protein 1 is ablated or inhibited and (b) oxygen consumption rates in mitochondria from uncoupling protein 1 knock-out mice, are significantly lower than those from wild-type mice, but equivalent to those from wild-type mice in the presence of GDP. In summary, we show that uncoupling protein 1 can affect reactive oxygen containing species production by isolated mitochondria from brown adipose tissue.     

Low rates of hydrogen peroxide production by isolated heart mitochondria associate with long maximum lifespan in vertebrate homeotherms

An inverse correlation between free radical production by isolated mitochondria and longevity in homeotherms has been reported, but previous comparative studies ignored possible confounding effects of body mass and phylogeny. We investigated this correlation by comparing rates of hydrogen peroxide (H(2)O(2)) production by heart mitochondria isolated from groups or pairs of species selected to have very different maximum lifespans but similar body masses (small mammals, medium-sized mammals, birds). During succinate oxidation, H(2)O(2) production rates were generally lower in the longer-lived species; the differences arose at complex I of the electron transport chain during reverse electron transport. Additional data were obtained from large species and the final dataset comprised mouse, rat, white-footed mouse, naked mole-rat, Damara mole-rat, guinea pig, baboon, little brown bat, Brazilian free-tailed bat, ox, pigeon and quail. In this dataset, maximum lifespan was negatively correlated with H(2)O(2) production at complex I during reverse electron transport. Analysis of residual maximum lifespan and residual H(2)O(2) production revealed that this correlation was even more significant after correction for effects of body mass. To remove effects of phylogeny, independent phylogenetic contrasts were obtained from the residuals. These revealed an inverse association between maximum lifespan and H(2)O(2) production that was significant by sign test, but fell short of significance by regression analysis. These findings indicate that enhanced longevity may be causally associated with low free radical production by mitochondria across species over two classes of vertebrate homeotherms.     

Production of reactive oxygen species by isolated mitochondria of the Antarctic bivalve Laternula elliptica (King and Broderip) under heat stress

Formation of reactive oxygen species (ROS) in mitochondrial isolates from gill tissues of the Antarctic polar bivalve Laternula elliptica was measured fluorimetrically under in vitro conditions. When compared to the rates measured at habitat temperature (1 degrees C), significantly elevated ROS formation was found under temperature stress of 7 degrees C and higher. ROS formation correlated significantly with oxygen consumption in individual mitochondrial preparations over the entire range of experimental temperatures (1-12 degrees C). ROS generation per mg of mitochondrial protein was significantly higher in state 3 at maximal respiration and coupling to energy conservation, than in state 4+, where ATPase-activity is inhibited by oligomycin and only proton leakage is driving the residual oxygen consumption. The percent conversion of oxygen to the membrane permeant hydrogen peroxide amounted to 3.7% (state 3) and 6.5% (state 4+) at habitat temperature (1 degrees C), and to 7% (state 3) and 7.6% (state 4+) under experimental warming to 7 degrees C. This is high compared to 1-3% oxygen to ROS conversion in mammalian mitochondrial isolates and speaks for a comparatively low control of toxic oxygen formation in mitochondria of the polar bivalve. However, low metabolic rates at cold Antarctic temperatures keep absolute rates of mitochondrial ROS production low and control oxidative stress at habitat temperatures. Mitochondrial coupling started to fall beyond 3 degrees C, closely to pejus temperature (4 degrees C) of the bivalve. Accordingly, the proportion of state 4 respiration increased from below 30% at 1 degrees C to over 50% of total oxygen consumption at 7 degrees C, entailing reduced ADP/O ratios under experimental warming. Progressive mitochondrial uncoupling and formation of hazardous ROS contribute to bias mitochondrial functioning under temperature stress in vitro. Deduced from a pejus temperature, heat stress commences already at 5 degrees C, and is linked to progressive loss of phosphorylation efficiency, increased mitochondrial oxygen demand and elevated oxidative stress above pejus temperatures.