Cytochrome Pathway Research Articles

Growth Inhibition Occurs Independently of Cell Mortality in Tomato (Solanum lycopersicum) Exposed to High Cadmium Concentrations

In order to analyze the adaptation potential of tomato shoots to a sudden increase in Cd concentration, tomato plants (Solanum lycopersicum L. var. Ailsa Craig) were exposed under controlled environmental conditions to a high dose of this heavy metal (250 microM CdCl2) in nutrient solution for 7 and 14 d. Both root and shoot growth was completely inhibited but all plants remained alive until the end of the treatment. Cell viability remained unaffected but the activity of the mitochondrial alternative pathway was stimulated by Cd stress at the expense of the cytochrome pathway. Cadmium concentration was higher in roots than in shoots and a decrease in the rate of net Cd translocation was noticed during the second week of stress. Cadmium decreased both leaf conductance (g(l)) and chlorophyll concentration. However, the effect on net CO2 assimilation remained limited and soluble sugars accumulated in leaves. Photochemical efficiency of PSII (Fv/Fm) was not affected despite a decrease in the number of reaction centers and an inhibition of electron transfer to acceptors of PSII. It is concluded that tomato shoot may sustain short term exposure to high doses of cadmium despite growth inhibition. This property implies several physiological strategies linked to both avoidance and tolerance mechanisms.

Expression profiles of respiratory components associated with mitochondrial biogenesis during germination and seedling growth under normal and restricted conditions in wheat

Germination of plant embryo is a dynamic phase-changing process that is driven by a rapid increase in mitochondrial respiration. We studied the development of respiratory electron transport pathways and the profiles of their transcript and protein components during this critical period using wheat embryos. Oxygen consumption through both the cytochrome and alternative pathways increased rapidly upon imbibition. Quantitative RT-PCR analysis using specific primers and western blot analysis using specific antibodies suggested that this respiratory burst was supported both by the stored mRNA and protein components and ones synthesized de novo at least in the cytochrome pathway. Dry embryos also contained transcript and protein of alternative oxidase (AOX), but their levels remained constant during the studied period. By contrast, the alternative pathway capacity showed a marked increase when the cytochrome pathway was inhibited by antimycin A and this increase was associated with increased levels of AOX transcript and protein. Our results suggest that mitochondrial biogenesis is accompanied by sequential and differential gene expression and protein accumulation, and that AOX allows the complex I to continue to conserve energy thus to support embryo germination and initial seedling growth in wheat when the cytochrome pathway is restricted.

The Cytotoxic Lipid Peroxidation Product 4-Hydroxy-2-nonenal Covalently Modifies a Selective Range of Proteins Linked to Respiratory Function in Plant Mitochondria

Plants encounter a variety of environmental stresses that affect their cellular machinery and that they adapt to on a daily basis. Lipid peroxidation is one consequence, at the cellular level, of such stresses and yields cytotoxic lipid aldehydes, including 4-hydroxy-2-nonenal (HNE), that react with specific sites in proteins, leading to diverse changes in protein function and/or stability. We have assessed the sensitivity of plant mitochondrial proteins to HNE modification, using one-dimensional and two-dimensional denaturing PAGE and blue native-PAGE coupled to immunological detection and tandem mass spectrometry identification. A select range of proteins was modified by exogenous application of HNE to mitochondria isolated from Arabidopsis cell cultures. These included a number of proteins that directly interact with the ubiquinone pool, as well as a number of soluble matrix proteins. Mitochondria isolated from cell cultures following hydrogen peroxide, antimycin A, or menadione treatment had significantly reduced respiratory capacity and elevated levels of HNE adduction to specific subsets of proteins. Targets identified included the proteins affected by direct application of HNE but also some new proteins, including a number of matrix dehydrogenases, the inner membrane adenine nucleotide translocator, and the outer membrane voltage-dependent anion channel. Degradation products of some proteins were also found to be HNE adducted, suggesting a link between HNE adduction and protein turnover. Some of the major enzyme complexes that were HNE adducted did not show demonstrable changes in their maximal activity measured with artificial acceptors, but changes did occur in associations between respiratory chain complexes following stress treatments.

Branching in the sequential folding pathway of cytochrome c

Previous results indicate that the folding pathways of cytochrome c and other proteins progressively build the target native protein in a predetermined stepwise manner by the sequential formation and association of native-like foldon units. The present work used native state hydrogen exchange methods to investigate a structural anomaly in cytochrome c results that suggested the concerted folding of two segments that have little structural relationship in the native protein. The results show that the two segments, an 18-residue omega loop and a 10-residue helix, are able to unfold and refold independently, which allows a branch point in the folding pathway. The pathway that emerges assembles native-like foldon units in a linear sequential manner when prior native-like structure can template a single subsequent foldon, and optional pathway branching is seen when prior structure is able to support the folding of two different foldons.

Changes in respiratory mitochondrial machinery and cytochrome and alternative pathway activities in response to energy demand underlie the acclimation of respiration to elevated CO2 in the invasive Opuntia ficus-indica.

Studies on long-term effects of plants grown at elevated CO(2) are scarce and mechanisms of such responses are largely unknown. To gain mechanistic understanding on respiratory acclimation to elevated CO(2), the Crassulacean acid metabolism Mediterranean invasive Opuntia ficus-indica Miller was grown at various CO(2) concentrations. Respiration rates, maximum activity of cytochrome c oxidase, and active mitochondrial number consistently decreased in plants grown at elevated CO(2) during the 9 months of the study when compared to ambient plants. Plant growth at elevated CO(2) also reduced cytochrome pathway activity, but increased the activity of the alternative pathway. Despite all these effects seen in plants grown at high CO(2), the specific oxygen uptake rate per unit of active mitochondria was the same for plants grown at ambient and elevated CO(2). Although decreases in photorespiration activity have been pointed out as a factor contributing to the long-term acclimation of plant respiration to growth at elevated CO(2), the homeostatic maintenance of specific respiratory rate per unit of mitochondria in response to high CO(2) suggests that photorespiratory activity may play a small role on the long-term acclimation of respiration to elevated CO(2). However, despite growth enhancement and as a result of the inhibition in cytochrome pathway activity by elevated CO(2), total mitochondrial ATP production was decreased by plant growth at elevated CO(2) when compared to ambient-grown plants. Because plant growth at elevated CO(2) increased biomass but reduced respiratory machinery, activity, and ATP yields while maintaining O(2) consumption rates per unit of mitochondria, we suggest that acclimation to elevated CO(2) results from physiological adjustment of respiration to tissue ATP demand, which may not be entirely driven by nitrogen metabolism as previously suggested.

Low O 2 supply is involved in the poor growth in root-restricted plants of tomato ( Lycopersicon esculentum Mill.)

To investigate whether O 2 deficit is involved in the depressed shoot growth under root restriction condition, tomato ( Lycopersicon esculentum Mill.) plants were grown hydroponically and subjected to root restriction with or without supplemental aeration. Plant biomass, leaf nutrient concentration, dissolved O 2 concentration in nutrient solution, root respiration, root hydrolytic ATPase activities, xylem sap abscisic acid (ABA) concentration, water relations, leaf gas exchange, and root cell viability were investigated throughout the experiment. Root restriction significantly depressed root and shoot growth as early as 15 days after treatment and this depressive effect was alleviated by vigorous aeration around the restricted root zone. Growth suppression by root restriction occurred concomitantly with sharp decreases in dissolved O 2 concentration in solution together with significant decreases in root total respiration, cytochrome pathway capacity, hydrolytic ATPase activities, and root cell viability. However, no such decreases were found in well aerated root restriction plants. Root restriction-induced growth suppression was independent of nutrients level in leaves and was not primarily related to the decline of leaf water potential or the gas exchange. Root restriction resulted in an increase in xylem sap ABA concentration from day 15 to the end of the experiment but no such effect was observed in well aerated plants. It is likely therefore, that O 2 was one of the main limiting factors to the reduced shoot growth under root restriction condition.

Mitochondrial function plasticity in Acanthamoeba castellanii during growth in batch culture

The alterations in mitochondrial bioenergetics during growth in a batch culture of Acanthamoeba castellanii were studied. The capacity of cytochrome pathway-dependent respiration measured in vitro decreased from the intermediary phase, when cell division slowed down. The pattern of the cytochrome pathway capacity changes was paralleled from the intermediary phase by alterations in the amount of total (and reducible) membranous ubiquinone. These changes were accompanied by a decrease in mitochondrial reactive oxygen species production in vitro (when no energy-dissipating system was active), and almost no change in superoxide dismutase activity and protein level, thus indicating an equivalent need for this enzyme in oxidative stress defence in A. castellanii culture. On the other hand, a decrease in the activity and protein level of alternative oxidase and uncoupling protein was observed in vitro, when cells shifted from the exponential growth phase to the stationary phase. It turned out that the contribution of both energy-dissipating systems in the prevention of mitochondrial reactive oxygen species generation in vivo could lead to its constant level throughout the growth cycle of A. castellanii batch culture. Hence, the observed functional plasticity insures survival of high quality cysts of A. castellanii cells.

Lack of Respiratory Chain Complex I Impairs Alternative Oxidase Engagement and Modulates Redox Signaling during Elicitor-Induced Cell Death in Tobacco

Alternative oxidase (AOX) functions in stress resistance by preventing accumulation of reactive oxygen species (ROS), but little is known about in vivo partitioning of electron flow between AOX and the cytochrome pathway. We investigated the relationships between AOX expression and in vivo activity in Nicotiana sylvestris and the complex I-deficient CMSII mutant in response to a cell death elicitor. While a specific AOX1 isoform in the active reduced state was constitutively overexpressed in CMSII, partitioning through the alternative pathway was similar to the wild type. Lack of correlation between AOX content and activity indicates severe metabolic constraints in nonstressed mutant leaves. The bacterial elicitor harpin N(Ea) induced similar timing and extent of cell death and a twofold respiratory burst in both genotypes with little change in AOX amounts. However, partitioning to AOX was increased twofold in the wild type but remained unchanged in CMSII. Oxidative phosphorylation modeling indicated a twofold ATP increase in both genotypes. By contrast, mitochondrial superoxide dismutase activity and reduced forms of ascorbate and glutathione were higher in CMSII than in the wild type. These results demonstrate genetically programmed flexibility of plant respiratory routes and antioxidants in response to elicitors and suggest that sustained ATP production, rather than AOX activity by itself or mitochondrial ROS, might be important for in planta cell death.

A Honglian CMS line of rice displays aberrant F0 of F0F1-ATPase

Honglian (HL) is one of the three major types of cytoplasmic male sterility (CMS) of rice (Oryza sativa L) that has been commercially used in hybrid production. In previous studies, the CMS in HL is shown to be associated with a chimeric gene orfH79 that is cotranscribed with an extra atp6 in mitochondria. This study demonstrated that the intact F0F1-ATPase in HL CMS line was specifically reduced in both of its protein quantity and enzyme activity, whereas its F1 sector was not affected. It implies that the F0 sector presents a labile linkage with F1. In the presence of fertility restorer gene, F0F1-ATPase can be recovered. Furthermore, orfH79 transcripts were preferentially polyadenylated, and consequently degraded rapidly in florets of the restored hybrid plants, indicating that the atp6-orfH79 is involved in the sterile phenotype. With inhibition of cytochrome pathway of electron transfer chain, the biomass of the sterile plants grown in dark was significantly lower than that of the fertile lines. However, the respiration measurements showed an increase in the electron transferring capacity in the sterile plants, suggesting that the reduction of biomass in sterile line was caused by the disruption of F0F1-ATPase.

Mitochondrial biogenesis as revealed by mitochondrial transcript profiles during germination and early seedling growth in wheat

Germination of imbibed embryos is the initial stage of plant development that is accompanied by the burst of mitochondrial respiration. To understand the process of mitochondrial biogenesis during this critical stage in wheat development, we monitored changes in mitochondrial transcript profiles during the first 3 days by adopting a newly devised macroarray system. The whole experiment was conducted in the dark to avoid influences of photosynthesis. Dry quiescent embryos started respiration rapidly after imbibition and the rate of oxygen uptake increased to peak at the first day followed by a continuous decrease until the third day under this condition. Both the cytochrome and alternative electron transport pathways appeared to contribute to this initial burst. Shoot and root growth was also remarkable during this period. Mitochondrial transcriptome was studied by macroarray analysis using 28 mitochondrial protein-coding genes, 4 nuclear encoded mitochondria-targeted genes and 2 nuclear genes as control. All transcripts were present in dry embryos at different initial levels, and a large variability was observed in their abundance among individual genes throughout the tested period. Gene expression was categorized into four clusters according to the profiles of individual transcript accumulation. A majority of the genes encoding subunits of the respiratory complexes belonged to two major clusters, the time course of transcript accumulation of one cluster agreeing with that of respiratory development and the other remaining at high constant levels. The macroarray system devised in this study should be useful in monitoring mitochondrial biogenesis under various growth conditions and at different developmental stages in cereals.

Alteration of respiration capacity and transcript accumulation level of alternative oxidase genes in necrosis lines of common wheat

Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for cyanide-insensitive and salicylhydroxamic acid-sensitive respiration in plants. AOX is a key enzyme of the alternative respiration pathway. To study the effects of necrotic cell death on the mitochondrial function, production of reactive oxygen species (ROS), respiration capacities and accumulation patterns of mitochondria-targeted protein-encoding gene transcripts were compared between wild-type, lesion-mimic mutant and hybrid necrosis wheat plants. Around cells with the necrosis symptom, ROS accumulated abundantly in the intercellular spaces. The ratio of the alternative pathway to the cytochrome pathway was markedly enhanced in the necrotic leaves. Transcripts of a wheat AOX gene, Waox1a, were more abundant in a novel lesion-mimic mutant of common wheat than in the wild-type plants. An increased level of the Waox1a transcripts was also observed in hybrid plants containing Ne1 and Ne2 genes. These results indicated that an increase of the wheat AOX transcript level resulted in enhancement of respiration capacity of the alternative pathway in the necrotic cells.

Temperature‐dependent changes in respiration rates and redox poise of the ubiquinone pool in protoplasts and isolated mitochondria of potato leaves

In many environments, leaves experience large diurnal variations in temperature. Such short‐term changes in temperature are likely to have important implications for respiratory metabolism in leaves. Here, we used intact leaf, protoplasts and isolated mitochondria to determine the impact of short‐term changes in temperature on respiration rates (R), adenylate concentrations and the redox poise of the ubiquinone (UQ) pool in mitochondria of potato leaves. The Q10 (i.e. proportional change in R for each 10°C rise in temperature) of respiration was 1.8, both for intact leaves and protoplasts. In protoplasts, the redox poise of the extracted UQ pool (UQR/UQT) increased from 0.33 at 22°C, to 0.76 at 15°C. Further decreases in temperature (from 15 to 5°C) resulted in UQR/UQT decreasing to 0.40. Adenylate ratios in protoplasts were also temperature dependent. At high adenosine 5′‐triphosphate (ATP) adenosine 5′‐diphosphate (ADP) ratios (i.e. low ADP concentrations), UQR/UQT values were low, suggesting that adenylates restricted flux via the UQ‐reducing pathways more than they restricted flux via pathways that oxidized UQH2. To assess whether high rates of alternative oxidase (AOX) activity could have uncoupled respiratory flux (and thus UQR/UQT) from adenylate restriction of the cytochrome (Cyt) pathway, we constructed kinetic curves of O2 uptake (via the two pathways) vs UQR/UQT in isolated mitochondria, measured at two temperatures (15 and 25°C); measurements were made for mitochondria operating under state 3 (i.e. +ADP) and state 4 (i.e. −ADP) conditions. In contrast to the Cyt pathway, flux via the AOX was temperature insensitive, with maximal rates of AOX activity representing 21–57% of total O2 uptake in isolated mitochondria. We conclude that temperature‐dependent variations in UQR/UQT are largely dependent on temperature‐dependent changes in adenylate ratios, and that flux via the AOX could in some circumstances help reduce maximal UQ values.

Cytochrome and alternative pathway activity in roots of thermal and non‐thermal Agrostis species in response to high soil temperature

Partitioning of respiration between the cytochrome pathway (CP) and the alternative pathway (AP) may play an important role in plant adaptation to extreme environments. We examined changes in partitioning between CP and AP, and viability of roots associated with plant exposure to high soil temperature for two Agrostis species: Agrostis scabra Willd., a species adapted to high‐temperature soils in geothermal areas in Yellowstone National Park, and Agrostis stolonifera L. (cv. Penncross) a heat‐sensitive grass widely used in cool‐climate regions. Roots of A. scabra and A. stolonifera were exposed to soil temperature of 37 or 20°C, while shoots were exposed to 20°C for 28 days. Root viability decreased, and total root respiration increased for both species at 37°C. The decline in root viability and the increase in respiration rates were less pronounced for A. scabra than for A. stolonifera. A larger proportion of total root respiration was attributed to the AP in A. scabra compared with that in A. stolonifera when both species were exposed to 37°C. At 7 and 14 days at 37°C, the relative proportion of respiration passing through AP increased by 12 and 10%, respectively, in A. scabra, whereas in A. stolonifera, AP increased by 4 and 1%, respectively. Our results suggest that maintaining a higher proportion of AP at a high soil temperature may contribute to root thermo‐tolerance in A. scabra in comparison with A. stolonifera, and alternative respiration may play an important role in plant adaptation to high soil temperature.

Contribution of the cytochrome and alternative pathways to growth respiration and maintenance respiration in Arabidopsis thaliana

The activities of the cytochrome and alternative respiratory pathways were measured during the growth cycle in Arabidopsis thaliana using a newly developed Isotope Ratio Mass Spectrometer (IRMS) dual‐inlet system that allows very precise measurements of oxygen‐isotope fractionation under low oxygen consumption rates. Under optimum growth conditions, the relative growth rate was highly dependent on the activity of the cytochrome pathway. The activity of the alternative pathway was almost constant irrespective of the growth rate and appeared mostly involved in the maintenance respiration component, although the alternative pathway also played a role under optimum growth conditions. This is the first time that the contribution of the two respiratory pathways to the two main respiratory components (growth and maintenance) has been analyzed with the use of the oxygen‐isotope fractionation technique. The respiration efficiency of the specific costs for growth and maintenance (adenosine triphosphate/O2 ratio) was determined by a modified regression model. The ability to measure oxygen‐isotope fractionation during respiration in A. thaliana opens the door to a wider set of studies that are discussed.

Different effects of chilling on respiration in leaves and roots of cucumber ( Cucumis sativus)

The effects of chilling on respiration (SHAM-resistant, cytochrome pathway and KCN-resistant, alternative pathway), temperature sensitivity, relative electrolyte conductivity, and degrees of oxidative stress (H 2O 2 and malonaldehyde (MDA) contents) were separately examined in leaves and roots of cucumber ( Cucumis sativus L.). After chilling at 8 °C for 4 days, both total respiration and KCN-resistant respiration in roots increased at different measurement temperatures. In contrast, SHAM-resistant respiration remained unchanged. In comparison, chilling significantly decreased the total respiration in leaves and this decrease was mostly due to a decrease in SHAM-resistant respiration. Chilling apparently decreased the sensitivity of KCN-resistant respiration to changes of temperature. The reduction levels of ubiquinone pool (UQr/UQt) increased both in chilled leaves and roots whilst pyruvate content increased only in chilled roots, but not in chilled leaves. Furthermore increases of H 2O 2 and MDA contents were much greater in leaves than in roots. The same trend was also observed for ion leakage from tissues. Taken together, the results suggested that the higher chilling tolerance of roots was associated with their high total respiration and KCN-resistant respiration.

The physiologic role of alternative oxidase in Ustilago maydis

Alternative oxidase (AOX) is a ubiquitous respiratory enzyme found in plants, fungi, protists and some bacterial species. One of the major questions about this enzyme is related to its metabolic role(s) in cellular physiology, due to its capacity to bypass the proton-pumping cytochrome pathway, and as a consequence it has great energy-wasting potential. In this study, the physiological role and regulatory mechanisms of AOX in the fungal phytopathogen Ustilago maydis were studied. We found evidence for at least two metabolic functions for AOX in this organism, as a major part of the oxidative stress-handling machinery, a well-described issue, and as part of the mechanisms that increase the metabolic plasticity of the cell, a role that might be valuable for organisms exposed to variations in temperature, nutrient source and availability, and biotic or abiotic factors that limit the activity of the cytochrome pathway. Experiments under different culture conditions of ecological significance for this organism revealed that AOX activity is modified by the growth stage of the culture, amino acid availability and growth temperature. In addition, nucleotide content, stimulation of AOX by AMP and respiratory rates obtained after inhibition of the cytochrome pathway showed that fungal/protist AOX is activated under low-energy conditions, in contrast to plant AOX, which is activated under high-energy conditions. An estimation of the contribution of AOX to cell respiration was performed by comparing the steady-state concentration of adenine nucleotides, the mitochondrial membrane potential, and the respiratory rate.

Quantum mechanical study of the proton transfer via a peptide bond in the novel proton translocation pathway of cytochrome c oxidase

The validity of the proton transfer via a peptide bond in the novel proton translocation pathway of cytochrome c oxidase has been assessed with quantum mechanical calculations. When the proton affinity of heme a propionate decreases, a proton could be passed to an aspartate side-chain through a peptide bond, forming imidic acid followed by the conversion to an enol peptide bond. The enol tautomer could be converted to the keto state by a proton wiring mechanism. This proton wiring keto–enol tautomerism is the rate-determining step and is responsible for the unidirectional character of the proton translocation.

Expression analysis of alternative oxidase gene (aox1) with enhanced green fluorescent protein as marker in citric acid-producing Aspergillus niger

In a citric acid-producing filamentous fungus Aspergillus niger WU-2223L, a cyanide- and antimycin A-insensitive and salicylhydroxamic acid-sensitive respiratory pathway functions in the mitochondria besides the cytochrome pathway and is catalyzed by alternative oxidase (AOX). We constructed an A. niger transformant strain AOXEGFP-1 expressing a fusion gene, aox1-egfp, encoding AOX and enhanced green fluorescent protein (EGFP) to visually analyze the expression levels of aox1 without disruption of mycelia. In strain AOXEGFP-1, the localization of the fusion protein AOX-EGFP in the mitochondria was clearly confirmed because the sites of the green fluorescence by AOX-EGFP were in agreement with those of the red fluorescence of the mitochondria stained with MitoTracker Red CMXRos. When strain AOXEGFP-1 was cultivated with antimycin A, which inhibits the cytochrome pathway at the level of cytochrome bc(1) to cytochrome c and increases the expression level of aox1, EGFP fluorescence intensity increased with an increase in AOX activity measured as duroquinol oxidase activity. Moreover, EGFP fluorescence was detected in strain AOXEGFP-1 regardless of the glucose concentration in the cultivation media: for example, when cultivations were performed with 10, 30, 60 and 120 g/l glucose, EGFP fluorescence was usually detected in the mitochondria. These results indicate that aox1 was constitutively expressed regardless of the glucose concentration in A. niger.

Remote control of photosynthetic genes by the mitochondrial respiratory chain

In this paper, we study whether mitochondrial respiration has an impact on the biogenesis of photosynthetic apparatus in the unicellular alga, Chlamydomonas reinhardtii. When respiration was activated by acetate in the dark, mRNAs of nuclear-encoded photosynthetic genes were induced. This induction did not occur in the cells treated with respiration inhibitors or in respiration mutants. An uncoupler of oxidative phosphorylation did not inhibit this mRNA induction; rather, it enhanced it in response to the increase in respiratory electron transport (RET). Plant and algal mitochondria have two RET pathways: the cytochrome pathway and the alternative pathway. Inhibitors of the former pathway inhibited mRNA induction, but inhibitors of the latter enhanced it. Taken together, these indicate that photosynthetic gene mRNAs are induced in response to activation of the cytochrome pathway. This RET-responsive induction is analogous to the photosynthetic electron transport (PET)-responsive induction of photosynthetic gene mRNAs (Matsuo and Obokata, Plant Cell Physiol. 43, 1189). PET-responsive induction occurred in photo-autotrophic and mixotrophic conditions, while RET-responsive induction occurred in mixotrophic and dark heterotrophic conditions. These results indicate that the regulatory system of photosynthetic genes changes between chloroplastic PET-dependent type and mitochondrial RET-dependent type in response to shifts in the dominant energy source between photosynthesis and respiration.

Effect of Osmotic Stress in Early Stages of Ontogenesis on Root Respiration, Growth, Sugar Content, and Cell Injury in Maize Seedlings Differing in Drought Sensitivity

AbstractCultivars of maize (Zea mays L.) with different sensitivity to drought were exposed to 0.3 mol/L sorbitol (‐1.4 MPa water potential) for 24 h. Exposure to water deficiency significantly reduced the growth of both shoots (coleoptile and hypocotyl) and roots. Shoot growth was inhibited more than the growth of roots. Osmotic stress enhanced accumulation of soluble sugars. Electrolyte leakage, a cell injury index, was slightly increased after 0.3 mol/L sorbitol. Respiration was measured in the presence and absence of 2,6‐dichlorophenol indophenol. 2, 6‐Dichlorophenol indophenol did not influence respiration rates, because statistically equal results were observed under both conditions. Total respiration (vT) decreased after osmoticum treatment. There were no significant differences in the vT among the cultivars analysed. The decrease in vT was caused by a decline in the activities and capacities of both cytochrome (vcyt, Vcyt) and alternative pathway (valt, Valt) of respiration. A high residual respiration (vres) was observed, up to 27% of total uninhibited respiration. The result of uncoupler use clearly indicated that coupling was maintained after 24 h of osmotic stress. The recovery of the respiration rate was comparable with that of non‐stressed control rates. According to these observations, no possible mitochondrial damage is expected. Water deficiency did not induce a stimulation of the alternative oxidase, so we assume that the stimulation of the alternative pathway is not related to drought stress resistance; rather, the function of the alternative pathway is to balance carbon metabolism and electron transport in a response to a changing environment.(Managing editor: Ping He)