Levels Of Alternative Oxidase Protein Research Articles

Differential modulation of photosynthesis, ROS and antioxidant enzyme activities in stress-sensitive and -tolerant rice cultivars during salinity and drought upon restriction of COX and AOX pathways of mitochondrial oxidative electron transport

Drought and salt stresses are two major abiotic stress factors that hamper crop growth and productivity. Three rice cultivars with different sensitivity and tolerance towards abiotic stress were used in the current study. While cultivar Aiswarya is salt- and drought-sensitive, cultivar Vyttila is salt-tolerant and cultivar Vaisakh is drought-tolerant. We compared the physiological and biochemical responses of these rice cultivars under salt and drought stress conditions after restricting their cytochrome oxidase (COX) and alternative oxidase (AOX) pathways using antimycin A and salicylhydroxamic acid treatment. Further, changes in their expression of AOX genes and corresponding protein levels were compared and analysed. The sensitive and tolerant rice cultivars subjected to drought and salt stress showed differential responses in physiological and biochemical traits. Whereas Aiswarya showed clear phenotypic differences, such as stunted growth, leaf curling, and loss of greening in leaf tissues, with increase in salt content and progressive drought stress, Vyttila and Vaisakh showed no remarkable changes. Moreover, the drought-tolerant cultivar rehydrated after 10 days of drought exposure, whereas the sensitive variety did not show any rehydration of leaf tissue. The leaves of the tolerant cultivars showed lower reactive oxygen species (ROS) production than that of the sensitive plants under drought and salt stress conditions because of the activation of a stronger antioxidant defence. Although, the restriction of COX and AOX pathways increased the susceptibility of sensitive cultivars, it affected the tolerant varieties moderately. Higher photosynthetic rates, an efficient antioxidant system comprising higher superoxide dismutase, ascorbate peroxidase, and catalase activity along with higher AOX1a gene expression levels during drought and salt stress were observed in tolerant cultivars. The results suggest that an efficient antioxidant system and increased transcription of the AOX1a gene along with higher AOX protein levels are important for tolerant rice cultivars to maintain higher photosynthesis rates, lower ROS, and stress tolerance. Restriction of COX and AOX pathways impact the photosynthesis, ROS, and antioxidant enzymes in both sensitive and tolerant cultivars. The restriction of COX and AOX pathways have a stronger impact on gas exchange and fluorescence parameters of the sensitive cultivar than on that of the tolerant cultivars owing to the higher photosynthetic rates in tolerant cultivars.

Cytochrome oxidase and alternative oxidase pathways of mitochondrial electron transport chain are important for the photosynthetic performance of pea plants under salinity stress conditions

The flexible plant mitochondrial electron transport chain with cytochrome c oxidase (COX) and alternative oxidase (AOX) pathways is known to be modulated by abiotic stress conditions. The effect of salinity stress on the mitochondrial electron transport chain and the importance of COX and AOX pathways for optimization of photosynthesis under salinity stress conditions is not clearly understood. In the current study, importance of COX and AOX pathways for photosynthetic performance of pea plants (Pisum sativum L. Pea Arkel cv) was analysed by using the mitochondrial electron transport chain inhibitors Antimycin A (AA) and salicylhydroxamic acid (SHAM) which restrict the electron flow through COX and AOX pathways respectively. Salinity stress resulted in decreased CO2 assimilation rates, leaf stomatal conductance, transpiration and leaf intercellular CO2 concentration in a stress dependent manner. Superimposition of leaves of salt stressed plants with AA and SHAM caused cellular H2O2 and O2− accumulation along with cell death. Additionally, aggravation in decrease of CO2 assimilation rates, leaf stomatal conductance, transpiration and leaf intercellular CO2 concentration upon superimposition with AA and SHAM during salinity stress suggests the importance of mitochondrial oxidative electron transport for photosynthesis. Increased expression of AOX1a and AOX2 transcripts along with AOX protein levels indicated up regulation of AOX pathway in leaves during salinity stress. Chlorophyll fluorescence measurements revealed enhanced damage to Photosystem (PS) II in the presence of AA and SHAM during salinity stress. Results suggested the beneficial role of COX and AOX pathways for optimal photosynthetic performance in pea leaves during salinity stress conditions.

The alternative respiratory pathway mediates carboxylate synthesis in white lupin cluster roots under phosphorus deprivation

Plant adaptations associated with a high efficiency of phosphorus (P) acquisition can be used to increase productivity and sustainability in a world with a growing population and decreasing rock phosphate reserves. White lupin (Lupinus albus) produces cluster roots that release carboxylates to efficiently mobilize P from P-sorbing soils. It has been hypothesized that an increase in the activity of the alternative oxidase (AOX) would allow for the mitochondrial oxidation of NAD(P)H produced during citrate synthesis in cluster roots at a developmental stage when there is a low demand for ATP. We used the oxygen-isotope fractionation technique to study the in vivo respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) in different root sections of white lupins grown hydroponically with and without P. In parallel, AOX protein levels and internal carboxylate concentrations were determined in cluster and non-cluster roots. Higher in vivo AOP activity was measured in cluster roots when malate and citrate concentrations were also high, thus confirming our hypothesis. AOX protein levels were not always correlated with in vivo AOP activity, suggesting post-translational regulation of AOX.

Alternative respiration as a primary defence during cadmium-induced mitochondrial oxidative challenge in Arabidopsis thaliana

Plant growth and development can be highly restricted by environmental stressors such as cadmium (Cd) pollution. The mitochondrial non-phosphorylating alternative respiratory pathway, mediated by alternative oxidase (AOX), alternative NAD(P)H dehydrogenases (NDs) and uncoupling protein (UCP), was suggested to be crucial in the acclimation of plants to fluctuating environmental conditions. Therefore, we examined the effects of environmentally realistic Cd exposure (5 and 10μM) on the alternative respiratory chain in Arabidopsis thaliana using a kinetic exposure setup. We demonstrated that during exposure to Cd, Arabidopsis seedlings show a mitochondrial oxidative challenge to which they acutely respond by increasing the transcript level of several AOX, ND and UCP isoforms in both roots and leaves. In addition, AOX protein levels increased during acute Cd exposure (2 and 24h). Based on our data, we suggest the formation of a condensed non-phosphorylating electron transport chain (ETC) functioning through cytosolic NDs and AOX, with co-regulation of ND and AOX expression during Cd stress. Therefore, both enzymes might cooperate in the potential acclimation of Arabidopsis seedlings to environmentally realistic Cd exposure by modulating the extent of mitochondrial ROS production.

Impact of oxidative stress on Acanthamoeba castellanii mitochondrial bioenergetics depends on cell growth stage

Addition of a moderate (1.4mM) concentration of H(2)O(2) to protozoon Acanthamoeba castellanii cell cultures at different growth phases caused a different response to oxidative stress. H(2)O(2) treatment of exponentially growing cells significantly delayed their growth; however, in mitochondria isolated from these cells, no damage to their bioenergetic function was observed. In contrast, addition of H(2)O(2) to A. castellanii cells approaching the stationary phase did not influence their growth and viability while seriously affecting mitochondrial bioenergetic function. Although mitochondrial integrity was maintained, oxidative damage was revealed in the reduction of cytochrome pathway activity, uncoupling protein activity, and the efficiency of oxidative phosphorylation as well as the membrane potential and the endogenous ubiquinone reduction level of the resting state. An increase in the alternative oxidase protein level and activity as well as an increase in the membranous ubiquinone content were observed in mitochondria isolated from late H(2)O(2)-treated cells. For the first time, the regulation of ubiquinone content in the inner mitochondrial membrane is shown to play a role in the response to oxidative stress. A physiological role for the higher activity of the alternative oxidase in response to oxidative stress in unicellular organisms, such as amoeba A. castellanii, is discussed.

Distribution of thermogenic activity in floral tissues of Nelumbo nucifera

Thermogenesis in Nelumbo nucifera (Gaertn.) has been known to scientists for many years; however, the extent of heating by different floral parts remains unclear. We present evidence that the receptacle, stamens and petals produce heat independently, and that the source of heating in these tissues is most likely the alternative oxidase (AOX). The temperatures of the receptacle, petals and stamens were significantly higher than non-thermogenic leaf tissue. After removal from the pedicel, the receptacle retained the most heat (8.1 ± 1.9°C above non-thermogenic tissue temperature) and the petals the least (2.8 ± 4.2°C), with the stamens intermediate. High AOX protein levels and flux through the AOX pathway (in all tissues) during the thermogenic period are consistent with AOX being the mechanism used for thermogenesis. Lipids and carbohydrates were investigated as possible substrates for thermogenesis. There was little change in total lipids during floral development; however, soluble carbohydrate levels decreased by 70% with the onset of thermogenesis. These sugars may fuel thermogenesis in the stamens. The localisation of AOX protein in the various floral parts and the evolutionary significance of its heating role are discussed.

Chilling stress and mitochondrial genome rearrangement in the MSC16 cucumber mutant affect the alternative oxidase and antioxidant defense system to a similar extent

The mosaic MSC16 cucumber (Cucumis sativus L.) mutant, which houses a rearranged mitochondrial genome, has altered respiratory chain activity, with a dysfunctional Complex I, increased external NADH dehydrogenases (ND(ex)) activity, and a higher alternative oxidase (AOX) capacity and AOX protein level. In the present study, changes in oxidative defense metabolism resulting from the respiratory chain dysfunction in the MSC16 mutant were compared with those induced by chilling. Chilling increased the enzymatic and non-enzymatic antioxidant defense systems in the wild-type (WT) but not in MSC16, which displays elevated antioxidant defenses as a result of the mitochondrial mutation. The high AOX capacity and protein level in MSC16 were unchanged as a result of chilling, whereas chilling increased these parameters in WT leaves. In mitochondria isolated from WT plants, superoxide was produced to a similar extent in the matrix and the intermembrane space, but in MSC16 mitochondria superoxide was produced largely within the intermembrane space. Mitochondria isolated from both genotypes after chilling showed increased superoxide production within the intermembrane space. Cytochemical detection revealed an increased abundance of H2O2 in the mitochondrial membrane in mesophyll cells of MSC16 leaves. The mitochondrial mutation also resulted in changes in the antioxidative defense system, including AOX, which were similar to those observed following chilling. The results presented here support the hypothesis that AOX is an effective marker of the cellular reprogramming resulting from stress. Moreover, we propose a role for reactive oxygen species (ROS) generated within the mitochondria in signal transduction.

Effects of Salicylic Acid on Alternative Pathway Respiration and Alternative Oxidase Expression in Tobacco Calli

The alternative pathway (AP) respiration of plants is a cyanide-resistant and non-phosphorylating electron transport pathway in mitochondria. Alternative oxidase (AOX) is the terminal oxidase of the AP and exists in plant mitochondria as two states: the reduced, noncovalently linked state or the oxidized, covalently cross-linked state. In the present study, the effects of 20 microM exogenous salicylic acid (SA) on both AP activity and AOX expression in mitochondria of tobacco (Nicotiana rustica L. cv. yellow flower) calli were investigated. The results showed that SA treatment enhanced the AP activity. During the process of SA treatment, the AP activity increased dramatically and achieved the peak value after 8 h of treatment. Then it declined until 16 h, and maintained a steady level between 16 and 24 h. Changes in both the total AOX protein level and the reduced state were in accordance with the AP activity, but the oxidized state changed differently. The aox1 gene transcript level also showed a similar change as the AP activity and AOX protein level. The induction of AOX expression by low concentrations of SA was inferred through a reactive oxygen species (ROS)-independent pathway. These results indicate that the enhancement of AP activity in response to SA is correlated to the expression of AOX, and the reduced, non-covalently linked state of AOX plays an important role during this process.

Enhanced high temperature tolerance in transgenic rice seedlings with elevated levels of alternative oxidase, OsAOX1a

The cyanide-insensitive alternative oxidase (AOX) respiratory pathway can reduce the excessive production of reactive oxygen species. Here we showed that transgenic rice seedlings transformed with OsAOX1a for rice alternative oxidase under the control of ubiquitin promoter exhibited thermotolerance after acute exposure, 41–45°C for 10 min, or chronic exposure, 37°C for up to 8 days. In contrast, these high temperature stresses resulted in significant growth inhibition in wild-type and transgenic plants with antisense OsAOX1a. The enhanced tolerance was significant in shoot growth, suggesting that the increased levels of AOX protein would dissipate the excess reductants produced in the chloroplasts suffering from oxidative damage due to high temperature stress.

Effect of mitochondrial genome rearrangement on respiratory activity, photosynthesis, photorespiration and energy status of MSC16 cucumber (Cucumis sativus) mutant

The effects of changes in mitochondrial DNA in cucumber (Cucumis sativus L.) mosaic mutant (MSC16) on respiration, photosynthesis and photorespiration were analyzed under non-stressed conditions. Decreased respiratory capacity of complex I in MSC16 mitochondria was indicated by lower respiration rates of intact mitochondria with malate and by rotenone-inhibited NADH or malate oxidation in the presence of alamethicin. Moreover, blue native PAGE indicated decreased intensity of protein bands of respiratory chain complex I in MSC16 leaves. Concerning the redox state, complex I impairment could be compensated to some extent by increased external NADH dehydrogenases (ND(ex)NADH) and alternative oxidase (AOX) capacity, the latter presenting differential expression in the light and in the dark. Although MSC16 mitochondria have a higher AOX protein level and an increased capacity, the AOX activity measured in the dark conditions by oxygen discrimination technique is similar to that in wild-type (WT) plants. Photosynthesis induction by light followed different patterns in WT and MSC16, suggesting changes in feedback chloroplast DeltapH caused by different adenylate levels. At steady-state, net photosynthesis was only slightly impaired in MSC16 mutants, while photorespiration rate (PR) was significantly increased. This was the result of large decreases in both stomatal and mesophyll conductance to CO2, which resulted in a lower CO2 concentration in the chloroplasts. The observed changes on CO2 diffusion caused by mitochondrial mutations open a whole new view of interaction between organelle metabolism and whole tissue physiology. The sum of all the described changes in photosynthetic and respiratory metabolism resulted in a lower ATP availability and a slower plant growth.

Partitioning of respiratory electrons in the dark in leaves of transgenic tobacco with modified levels of alternative oxidase

This study examined tobacco (Nicotiana tabacum cv. Petit Havana SR1) leaf respiration in the dark, utilizing both wild‐type plants and transgenic plants with increased or decreased levels of alternative oxidase (AOX) protein. AOX represents a non‐energy‐conserving branch in mitochondrial electron transport. Inhibitor studies showed that the maximum possible flux of electrons to AOX (AOX capacity) correlated with the level of AOX protein present in the different plant lines. A comparison of the plants using online 18O isotope discrimination was done to determine whether AOX protein level would impact the actual steady‐state partitioning of electrons to AOX (AOX engagement). Under a range of pretreatment and measurement conditions, there was little if any effect of AOX protein level on the degree of engagement. This suggests that the metabolic conditions inherent to a particular growth condition and/or the biochemical regulatory properties of AOX itself are the critical factors that control partitioning. Interestingly, we found that measurement temperature and water status are parameters that may have some influence over AOX engagement.

Interaction between photorespiration and respiration in transgenic potato plants with antisense reduction in glycine decarboxylase

Potato (Solanum tuberosum L. cv. Désirée) plants with an antisense reduction in the P-protein of the glycine decarboxylase complex (GDC) were used to study the interaction between respiration and photorespiration. Mitochondria isolated from transgenic plants had a decreased capacity for glycine oxidation and glycine accumulated in the leaves. Malate consumption increased in leaves of GDC deficient plants and the capacity for malate and NADH oxidation increased in isolated mitochondria. A lower level of alternative oxidase protein and decreased partitioning of electrons to the alternative pathway was found in these plants. The adenylate status was altered in protoplasts from transgenic plants, most notably the chloroplastic ATP/ADP ratio increased. The lower capacity for photorespiration in leaves of GDC deficient plants was compensated for by increased respiratory decarboxylations in the light. This is interpreted as a decreased light suppression of the tricarboxylic acid cycle in GDC deficient plants in comparison to wild-type plants. The results support the view that respiratory decarboxylations in the light are restricted at the level of the pyruvate dehydrogenase complex and/or isocitrate dehydrogenase and that this effect is likely to be mediated by mitochondrial photorespiratory products.

Developmental physiology of cluster-root carboxylate synthesis and exudation in harsh hakea. Expression of phosphoenolpyruvate carboxylase and the alternative oxidase.

Harsh hakea (Hakea prostrata R.Br.) is a member of the Proteaceae family, which is highly represented on the extremely nutrient-impoverished soils in southwest Australia. When phosphorus is limiting, harsh hakea develops proteoid or cluster roots that release carboxylates that mobilize sparingly soluble phosphate in the rhizosphere. To investigate the physiology underlying the synthesis and exudation of carboxylates from cluster roots in Proteaceae, we measured O2 consumption, CO2 release, internal carboxylate concentrations and carboxylate exudation, and the abundance of the enzymes phosphoenolpyruvate carboxylase and alternative oxidase (AOX) over a 3-week time course of cluster-root development. Peak rates of citrate and malate exudation were observed from 12- to 13-d-old cluster roots, preceded by a reduction in cluster-root total protein levels and a reduced rate of O2 consumption. In harsh hakea, phosphoenolpyruvate carboxylase expression was relatively constant in cluster roots, regardless of developmental stage. During cluster-root maturation, however, the expression of AOX protein increased prior to the time when citrate and malate exudation peaked. This increase in AOX protein levels is presumably needed to allow a greater flow of electrons through the mitochondrial electron transport chain in the absence of rapid ATP turnover. Citrate and isocitrate synthesis and accumulation contributed in a major way to the subsequent burst of citrate and malate exudation. Phosphorus accumulated by harsh hakea cluster roots was remobilized during senescence as part of their efficient P cycling strategy for growth on nutrient impoverished soils.

Possible involvement of the mitochondrial alternative pathway in ethylene-induced apoptosis in tomato protoplasts

In this study, a monoclonal antibody to the terminal oxidase of the alternative pathway from Sauromatum guttatum was used to detect the expression of alternative oxidase (AOX) protein in tomato mitochondria. The results show that there was an obvious correlation between the ethylene-induced apoptosis and the levels of AOX protein in tomato cells undergoing ethylene-induced apoptosis. In addition, when tomato protoplasts were preincubated with 2 mM salicylhydroxamic acid (SHAM), an inhibitor of the alternative pathway, before their exposure to ethylene, the TUNEL positive reaction and DNA fragmentation were obviously accelerated. We suggest that AOX may play an important role in protecting tomato protoplasts against ethylene-induced apoptosis.

Alternation of the Accumulation of Alternative Oxidase by Introducing Sense and Antisense OsAOX1a in Rice

Sense and antisense DNA constructs of rice (Oryza sativa L.) alternative oxidase (AOX) gene, OsAOX1a, under the control of maize Ubiquitin promoter were introduced into rice. Leaves of transgenic plants with sense OsAOX1a were shown to produce high levels of AOX protein, which was not usually observed in wild-type leaves. Transgenic plants with decreased levels of AOX protein were identified by the investigation of AOX protein in calli derived from mature seeds with antisense OsAOX1a. Transgenic plants with increased and decreased levels of AOX will be useful for studies on the role of AOX.

Metabolic regulation of leaf respiration and alternative pathway activity in response to phosphate supply

In this study the question whether the alternative respiratory pathway acts as an electron bypass for the cytochrome pathway under conditions of growth on limited phosphorus in leaves of bean (Phaseolus vulgaris L.), tobacco (Nicotiana tabacum L.) and Gliricidia sepium Walp was investigated. The oxygen isotope fractionation technique was used to assess the in vivo activities of the cytochrome and alternative respiratory pathways in the absence of added inhibitors. The response of respiration to low phosphorus supply varied among species. Growth at low phosphorus reduced cytochrome pathway activity in bean and tobacco. Alternative pathway activity increased only in bean leaves in response to low phosphorus and not in tobacco. In the case of G. sepium, cytochrome pathway activity remained unchanged whereas the alternative pathway activity increased with low nutritional phosphorus. At low phosphorus, alternative oxidase protein levels increased in the leaves of bean and G. sepium but not in tobacco, suggesting a dependence of alternative pathway activity on protein level. Alternative pathway activity was also not correlated with soluble carbohydrate concentration in bean or tobacco at any phosphorus level. These results show that the alternative pathway does not always act as an electron bypass in response to the downstream restriction of the cytochrome pathway imposed by low phosphorus supply. These results suggest that factors in addition to cellular carbohydrate level and adenylate control can act to regulate alternative pathway activity.

Effects of ageing and salicylate on beetroot mitochondria

Root tissue slices of Beta vulgaris were aged by aerated mixing for 5 d in 0.1 mM CaSO4 solution with and without 0.5 mM salicylate (pH 7.0). Mitochondria, isolated from treated and fresh beetroot tissue, were investigated, and the activities of the NAD(P)H oxidising pathways of the mitochondrial inner and outer membranes, together with the activity of the alternative pathway, were measured. From these we determined which non-phosphorylating pathways of mitochondrial respiration were induced by the treatments. The presence of salicylate in the ageing treatment was shown to increase the activity of the alternative pathway as well as the level of alternative oxidase protein. This increase was above that seen in the ageing treatment alone. Investigation of the other non-phosphorylating pathways of mitochondrial respiration indicated that while they also increased during ageing, they were not specifically affected by salicylate treatment. The specific induction of the alternative pathway increased activity by almost 4-fold from the fresh tissue and 2-fold from the aged tissue rates.

In Organello and in Vivo Evidence of the Importance of the Regulatory Sulfhydryl/Disulfide System and Pyruvate for Alternative Oxidase Activity in Tobacco.

After isolation of tobacco (Nicotiana tabacum) leaf mitochondria, alternative oxidase (AOX) is predominantly present as the disulfide-linked, less-active "oxidized" form. In an in organello assay, significant AOX activity was dependent upon both the reduction of the regulatory disulfide bond (such as occurs by dithiothreitol) and upon the presence of the activator pyruvate. However, AOX activity in these assays was substantially affected when mitochondria were isolated in the presence of pyruvate. First, pyruvate protects against the oxidation of the regulatory sulfhydryl during isolation, such that subsequent in organello AOX activity is not dependent upon dithiothreitol. Second, pyruvate stabilizes AOX activity, such that mitochondria kept in the presence of pyruvate have higher maximum rates of AOX activity than mitochondria kept for some time in the absence of pyruvate. The ability of pyruvate to protect against AOX oxidation was exploited to assess the in vivo status of the regulatory sulfhydryl/disulfide system. In both tobacco suspension cells and tobacco leaves with high levels of AOX protein, the protein is predominantly present as the "reduced" active form in vivo under a range of respiratory conditions. Experiments also indicate that, while the presence of reduced protein may be a necessary prerequisite for significant AOX activity, it is not sufficient for activity and other factors must also be critical.

The effect of growth and measurement temperature on the activity of the alternative respiratory pathway

A postulated role of the CN-resistant alternative respiratory pathway in plants is the maintenance of mitochondrial electron transport at low temperatures that would otherwise inhibit the main phosphorylating pathway and prevent the formation of toxic reactive oxygen species. This role is supported by the observation that alternative oxidase protein levels often increase when plants are subjected to growth at low temperatures. We used oxygen isotope fractionation to measure the distribution of electrons between the main and alternative pathways in mung bean (Vigna radiata) and soybean (Glycine max) following growth at low temperature. The amount of alternative oxidase protein in mung bean grown at 19 degrees C increased over 2-fold in both hypocotyls and leaves compared with plants grown at 28 degrees C but was unchanged in soybean cotyledons grown at 14 degrees C compared with plants grown at 28 degrees C. When the short-term response of tissue respiration was measured over the temperature range of 35 degrees C to 9 degrees C, decreases in the activities of both main and alternative pathway respiration were observed regardless of the growth temperature, and the relative partitioning of electrons to the alternative pathway generally decreased as the temperature was lowered. However, cold-grown mung bean plants that up-regulated the level of alternative oxidase protein maintained a greater electron partitioning to the alternative oxidase when measured at temperatures below 19 degrees C supporting a role for the alternative pathway in response to low temperatures in mung bean. This response was not observed in soybean cotyledons, in which high levels of alternative pathway activity were seen at both high and low temperatures.

Cyanide-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration during postharvest ripening of tomato fruit

Tomato (Lycopersicon esculentum) mitochondria contain both alternative oxidase (AOX) and uncoupling protein as energy-dissipating systems that can decrease the efficiency of oxidative phosphorylation. We followed the cyanide (CN)-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration of isolated mitochondria, as well as the immunologically detectable levels of uncoupling protein and AOX, during tomato fruit ripening from the mature green stage to the red stage. The AOX protein level and CN-resistant respiration of isolated mitochondria decreased with ripening from the green to the red stage. The ATP-synthesis-sustained respiration followed the same behavior. In contrast, the level of uncoupling protein and the total uncoupling-protein-sustained respiration of isolated mitochondria decreased from only the yellow stage on. We observed an acute inhibition of the CN-resistant respiration by linoleic acid in the micromolar range. These results suggest that the two energy-dissipating systems could have different roles during the ripening process.