Cyanide-insensitive Respiration Research Articles

Leaf mitochondria modulate whole cell redox homeostasis, set antioxidant capacity, and determine stress resistance through altered signaling and diurnal regulation.

To explore the role of plant mitochondria in the regulation of cellular redox homeostasis and stress resistance, we exploited a Nicotiana sylvestris mitochondrial mutant. The cytoplasmic male-sterile mutant (CMSII) is impaired in complex I function and displays enhanced nonphosphorylating rotenone-insensitive [NAD(P)H dehydrogenases] and cyanide-insensitive (alternative oxidase) respiration. Loss of complex I function is not associated with increased oxidative stress, as shown by decreased leaf H(2)O(2) and the maintenance of glutathione and ascorbate content and redox state. However, the expression and activity of several antioxidant enzymes are modified in CMSII. In particular, diurnal patterns of alternative oxidase expression are lost, the relative importance of the different catalase isoforms is modified, and the transcripts, protein, and activity of cytosolic ascorbate peroxidase are enhanced markedly. Thus, loss of complex I function reveals effective antioxidant crosstalk and acclimation between the mitochondria and other organelles to maintain whole cell redox balance. This reorchestration of the cellular antioxidative system is associated with higher tolerance to ozone and Tobacco mosaic virus.

Alternative Respiration Pathways and Secondary Metabolism in Plants with Different Adaptive Strategies under Mineral Deficiency

The costs of adaptation respiration, the contribution of different biochemical pathways of respiration and alternative oxidases to the adaptation to mineral deficiency were compared in plant species representing different types of adaptive strategies: ruderal (Ru) (Amaranthus retroflexus L. and Leonurus quinquelobatus Gilib.) and predominantly stress-tolerant type (St) (Dactylis glomerata L. and Medicago sativa L.). The adaptation component of the total dark respiration was calculated on the basis of the relative growth rate and the ratio of dark respiration to gross photosynthesis. Adaptation costs were greater in less tolerant species. The inhibitory analysis showed that the contribution of glycolytic pathway decreased and the proportion of oxidative pentose phosphate pathway and residual respiration increased in the intolerant Ru-species under the conditions of abiotic stress. Conversely, in the tolerant St-species, the contribution of glycolysis increased. In all species studied, the contribution of the cytochrome pathway of substrate oxidation was reduced and the contribution of the alternative cyanide-insensitive and residual respiration increased under conditions of mineral deficiency; this phenomenon was particularly pronounced in a less tolerant species L. quinquelobatus. In the species characterized by Ru-strategy, stress increased the content of phenolic compounds and shikimic acid. We suppose that the adaptation respiration costs are associated with alternative biochemical pathways and alternative cyanide-resistant oxidase. The role of these pathways in the plants with St-strategy consists in maintaining the active state of oxidative pathways, whereas in the plants with Ru-strategy, they serve to burn excess metabolites and enhance the synthesis of secondary metabolites, which perform protective functions.

Regulation of alternative oxidase gene expression in soybean.

Soybean (Glycine max cv. Stevens) suspension cells were used to investigate the expression of the alternative oxidase (Aox) multigene family. Suspension cells displayed very high rates of cyanide-insensitive respiration, but Aox3 was the only isoform detected in untreated cells. Incubation with antimycin A, citrate, salicylic acid or at low temperature (10 degrees C) specifically induced the accumulation of the Aox1 isoform. Aox2 was not observed under any conditions in the cells. Increases in Aox1 protein correlated with increases in Aox1 mRNA. Treatment of soybean cotyledons with norflurazon also induced expression of Aox1. Reactive oxygen species (ROS) were detected upon incubation of cells with antimycin, salicylic acid or at low temperature, but not during incubation with citrate. Aox1 induction by citrate, but not by antimycin, was prevented by including the protein kinase inhibitor staurosporine in the medium. The results suggest that multiple pathways exist in soybean to regulate expression of Aox genes and that Aox1 specifically is induced by a variety of stress and metabolic conditions via at least two independent signal transduction pathways.

Alternative respiratory system and formamide hydro-lyase activity as the key components of the cyanide-resistance mechanism inFusarium oxysporum

A strain of Fusarium oxysporum, isolated from an industrial effluent containing a high cyanide concentration, detoxifies cyanide via formamide hydro-lyase (FHL). The importance of the coexistence of the alternative (cyanide insensitive) respiratory system and the synthesis of FHL was assessed. This enzyme, induced by cyanide, converts this compound to formamide and is partially responsible for the tolerance of F. oxysporum to high cyanide concentration. The FHL induction for cyanide detoxification depends on the alternative respiratory system when the terminal oxidase of the electron transport chain is blocked by cyanide used during the induction process. The respiratory metabolism of a F. oxysporum strain was studied. Whole cells exhibited a cyanide-sensitive respiration but developed a partially cyanide-resistant respiration under certain physiological conditions, namely, in the stationary phase of growth, in the presence of chloramphenicol in the growth medium, or upon aeration in the absence of nutrients (starvation conditions). Cycloheximide prevented the appearance of cyanide-insensitive respiration when the cells were aerated under starvation conditions. This fact suggested some form of induction involving de novo protein synthesis.Key words: cyanide tolerance, cyanide-resistant respiration, formamide hydro-lyase, Fusarium oxysporum.

Function and Expression of Flavohemoglobin in Saccharomyces cerevisiae: EVIDENCE FOR A ROLE IN THE OXIDATIVE STRESS RESPONSE

We have studied the function and expression of the flavohemoglobin (YHb) in the yeast Saccharomyces cerevisiae. This protein is a member of a family of flavohemoproteins, which contain both heme and flavin binding domains and which are capable of transferring electrons from NADPH to heme iron. Normally, actively respiring yeast cells have very low levels of the flavohemoglobin. However, its intracellular levels are greatly increased in cells in which the mitochondrial electron transport chain has been compromised by either mutation or inhibitors of respiration. The expression of the flavohemoglobin gene, YHB1, of S. cerevisiae is sensitive to oxygen. Expression is optimal in hyperoxic conditions or in air and is reduced under hypoxic and anaerobic conditions. The expression of YHB1 in aerobic cells is enhanced in the presence of antimycin A, in thiol oxidants, or in strains that lack superoxide dismutase. All three conditions lead to the accumulation of reactive oxygen species and promote oxidative stress. To study the function of flavohemoglobin in vivo, we created a null mutation in the chromosomal copy of YHB1. The deletion of the flavohemoglobin gene in these cells does not affect growth in either rhoo or rho+ genetic backgrounds. In addition, a rho+ strain carrying a yhb1(-) deletion has normal levels of both cyanide-sensitive and cyanide-insensitive respiration, indicating that the flavohemoglobin does not function as a terminal oxidase and is not required for the function or expression of the alternative oxidase system in S. cerevisiae. Cells that carry a yhb1(-)deletion are sensitive to conditions that promote oxidative stress. This finding is consistent with the observation that conditions that promote oxidative stress also enhance expression of YHB1. Together, these findings suggest that YHb plays a role in the oxidative stress response in yeast.

Enhancement and repression of cyanide-insensitive respiration in Aspergillus niger.

The biosynthesis of the alternative oxidase protein consisting of cyanide-insensitive and salicylhydroxamic acid-sensitive respiration in Aspergillus niger was investigated. Cyanide-insensitive respiration was enhanced by addition of antimycin A to the incubation mixture, but repressed by the addition of cycloheximide, emetine, puromycin (cytosolic translation inhibitors), carbonylcyanide-m-chlorophenylhydrazone (uncoupler) and actinomycin D (transcription inhibitor). These results show that the alternative oxidase protein of A. niger was synthesized de novo in cytosol and transported in mitochondria.

Enhancement and repression of cyanide-insensitive respiration in Aspergillus niger

The biosynthesis of the alternative oxidase protein consisting of cyanide-insensitive and salicylhydroxamic acid-sensitive respiration in Aspergillus niger was investigated. Cyanide-insensitive respiration was enhanced by addition of antimycin A to the incubation mixture, but repressed by the addition of cycloheximide, emetine, puromycin (cytosolic translation inhibitors), carbonylcyanide- m-chlorophenylhydrazone (uncoupler) and actinomycin D (transcription inhibitor). These results show that the alternative oxidase protein of A. niger was synthesized de novo in cytosol and transported in mitochondria.

Paradoxical Increase in Peroxisomal Cyanide-Insensitive Respiration Following Dietary Exposure to WY-14,643 in the Perfused Liver

WY-14,643, a lipid-lowering drug, increases basal rates of oxygen uptake in perfused livers. Because peroxisomes consume oxygen for H2O2production and are induced by WY-14,643 treatment, it is possible that peroxisomal β-oxidation can account for some of this increase in cellular respiration. Therefore, cyanide, an inhibitor of mitochondrial cytochrome oxidase, was infused into livers of WY-14,643-fed rats (0.1% WY-14,643 in laboratory rat chow for 1, 21, and 105 days) to assess peroxisomal cyanide-insensitive respiration. As expected, the addition of cyanide abolished oxygen uptake nearly completely; however, after approximately 20 min oxygen consumption unexpectedly returned to basal levels in 105-day WY-14,643-treated animals but not in untreated controls. Urea synthesis, a process dependent upon ATP, was decreased and remained low during cyanide infusion in livers from both groups, indicating that mitochondria were not responsible for this unusual increase in oxygen uptake in the presence of cyanide. Methanol metabolism, which requires oxygen to form H2O2, was decreased from 37 ± 5 to 6 ± 1 μmol/g/hr in all groups treated with cyanide; however, it was increased significantly about 20 min later to 25 μmol/g/hr in livers from WY-14,643-treated rats, indicating that oxygen for peroxisomal H2O2production is involved in cellular respiration in the presence of cyanide. Fasting abolished the recovery of both oxygen uptake and methanol metabolism in WY-14,643-fed rats, suggesting that ATP for acyl CoA synthetase, an enzyme which metabolizes fatty acids to acyl CoA compounds, is provided by glycolysis. Indeed, oleate significantly increased methanol metabolism in fed control rats from 8 ± 4 to 26 ± 3 μmol/g/hr in the presence of cyanide, indicating that fatty acid supply is necessary for peroxisomal respiration. Taken together, [bnthese experiments demonstrate that when mitochondrial respiration is inhibited, livers from rats fed WY-14,643 chronically have the unique ability of metabolizing fatty acids through the peroxisome using glycolytic ATP.

Cyanide-insensitive Respiration of Phytopathogenic Fungi Demonstrated by Antifungal Joint Action of Respiration Inhibitors

植物病原菌のシアン耐性呼吸の有無を調べる目的で, シアン耐性呼吸の阻害剤であるサリチルヒドロキサム酸 (SHAM) と呼吸鎖シトクローム電子伝達系の阻害剤であるメトキシアクリレート系化合物やアンチマイシンAとの抗菌連合作用を調べた. Botrytis cinerea, Monilinia fructicola および Pyricularia oryzae において顕著な抗菌協力作用が認められた. このことから, シトクローム系阻害剤によるシアン耐性呼吸の誘導とSHAMによるその阻害が考えられた. それを確かめる目的で, シトクローム系阻害剤で処理したこれら3病原菌の菌糸体の酸素消費と, その酸素消費に及ぼすシアン耐性呼吸阻害剤の影響を調べた. その結果, これら3病原菌におけるシアン耐性呼吸の誘導が確認された. 抗菌協力作用は, Cochliobolus miyabeanus においても小さいながら認められ, シアン耐性呼吸の存在が示唆された. Rhizoctonia solani では, メトキシアクリレート系化合物では協力作用が認められたが, アンチマイシンAにおいては認められず, シアン耐性呼吸の誘導が, アンチマイシンAでは起こりにくいと考えられた. Gibberella fujikuroi, Pythium sp., Valsa ceratosperma における抗菌協力作用は, 本実験では顕著ではなかった. 病原細菌の Xanthomonas campestris pv. citri および酵母 Sacchamyces cerevisiae においては, 抗菌協力作用はまったく認められなかった.

Characteristics of Mitochondrial Particles in Relation to Flooding Tolerance in Pear Rootstocks.

Flooding-tolerant pear rootstocks, Pyrus calleryana, No. 6 and No. 8 strains and nontolerant rootstocks, P. betulaefolia and P. pyrifolia were used in the experiment.The rootstocks were flooded, and the change in the density of mitochondrial particles in the fine roots was checked by sucrose density gradient centrifugation. There was no marked change in the distribution of density of mitochondrial particles in roots of P. betulaefolia and P. pyrifolia before and after the flooding treatments; the mitochondrial particles (Fraction I) were detected at 1.221.29 M of the sucrose concentrations in the gradient centrifugation. Conversely, the heavier mitochondrial particles (Fraction II), which were detected at the 1.421.49 M in the gradient, appeared in the roots of P. calleryana, No. 6 and No. 8 after the flooding treatment.The level of cyanide-insensitive respiration was measured in the mitochondrial fractions, Fraction I and II. Fractions I prepared from P. calleryana, No. 6 and No. 8 were less sensitive to cyanide than were those from P. betulaefolia and P. pyrifolia. Fraction II, induced in the roots of P. calleryana by the flooding treatment, was markedly insensitive to cyanide.These results suggest that the changes in density and respiration chain of mitochon-drial particles may be closely related to flooding tolerance in pear rootstocks.

Characterization of the respiratory chain of Leishmania donovani promastigotes

Inhibition analysis of respiration of Leishmania donovani promastigotes in resting, starved and permeabilized cells in the presence of classical electron transfer complex inhibitors such as rotenone, thenoyltrifluoroacetone and antimycin demonstrated the absence of complex I component of the respiratory chain in this organism. Cyanide failed to completely block the oxygen uptake (residual 25–30%) even at high concentrations. The alternative oxidase inhibitor for Trypanosoma brucei, salicylhydroxamic acid (SHAM) had no effect on respiration while the cytochrome o inhibitor orthohydroxydiphenyl (OHD) could block cyanide-insensitive respiration at low concentrations. Succinate-dependent O 2 uptake in permeabilized cells follows the classical pathway. Oxidation of NADH by a membrane-rich fraction produced H 2O 2 as the end product and was insensitive to repiratory chain inhibitors. The presence of NADH-fumarate reductase was demonstrated in membrane-rich fraction and fumarate could reduce H 2O 2 production from NADH indicating fumarate to be an endogenous substrate for accepting electrovs from NADH. A differential route for NADH oxidation was further confirmed by NADH cytochrome c reductase insensitivity to antimycin. A tentative scheme for electron transfer pathway in this organism is proposed in which a reversal of Krebs cycle enzymes occur producing succinate that can be excreted or oxidized depending upon the energy demands of the cell. Inhibition studies also suggest bifurcations of the respiratory chain that can be of minor importance for the organism.

Existence of Cyanide-Insensitive Respiration in the Yeast Pichia stipitis and Its Possible Influence on Product Formation during Xylose Utilization

A cyanide-insensitive and salicyl hydroxamic acid-sensitive respiration (CIR) was found in the yeast Pichia stipitis in contrast to Candida utilis, Pachysolen tannophilus, and Saccharomyces cerevisiae. During xylose utilization in the presence of either salicyl hydroxamic acid or cyanide, P. stipitis formed xylitol, arabitol, and ribitol. The existence of CIR is discussed in terms of a redox sink preventing xylitol formation in P. stipitis.

Isolation and characterization of mutants defective in the cyanide-insensitive respiratory pathway of Pseudomonas aeruginosa.

The branched respiratory chain of Pseudomonas aeruginosa contains at least two terminal oxidases which are active under normal physiological conditions. One of these, cytochrome co, is a cytochrome c oxidase which is completely inhibited by concentrations of the respiratory inhibitor potassium cyanide as low as 100 microM. The second oxidase, the cyanide-insensitive oxidase, is resistant to cyanide concentrations in excess of 1 mM as well as to sodium azide. In this work, we describe the isolation and characterization of a mutant of P. aeruginosa defective in cyanide-insensitive respiration. This insertion mutant was isolated with mini-D171 (a replication-defective derivative of the P. aeruginosa phage D3112) as a mutagen and by screening the resulting tetracycline-resistant transductants for the loss of ability to grow in the presence of 1 mM sodium azide. Polarographic studies on the NADH-mediated respiration rate of the mutant indicated an approximate 50% loss of activity, and titration of this activity against increasing cyanide concentrations gave a monophasic curve clearly showing the complete loss of cyanide-insensitive respiration. The mutated gene for a mutant affected in the cyanide-insensitive, oxidase-terminated respiratory pathway has been designated cio. We have complemented the azide-sensitive phenotype of this mutant with a wild-type copy of the gene by in vivo cloning with another mini-D element, mini-D386, carried on plasmid pADD386. The complemented cio mutant regained the ability to grow on medium containing 1 mM azide, titration of its NADH oxidase activity with cyanide gave a biphasic curve similar to that of the wild-type organism, and the respiration rate returned to normal levels. Spectral analysis of the cytochrome contents of the membranes of the wild type, the cio mutant, and the complemented mutant suggests that the cio mutant is not defective in any membrane-bound cytochromes and that the complementing gene does not encode a heme protein.

Regulation of the Alternative Oxidase in Plants and Fungi.

The alternative oxidase of the inner mitochondrial membrane catalyses cyanide-insensitive respiration in plants and fungi. The molecular biology and regulation of this oxidase have been intensively studied over the past 10 years. Genes have been isolated, expression has been investigated and novel mechanisms for the regulation of activity have been discovered. This paper reviews these recent advances, focusing on the regulation of gene expression and activation by protein modification and organic acids, and possible roles of the alternative oxidase are discussed.

Regulation of Alternative Oxidase Activity by Pyruvate in Soybean Mitochondria.

The regulation of alternative oxidase activity by the effector pyruvate was investigated in soybean (Glycine max L.) mitochondria using developmental changes in roots and cotyledons to vary the respiratory capacity of the mitochondria. Rates of cyanide-insensitive oxygen uptake by soybean root mitochondria declined with seedling age. Immunologically detectable protein levels increased slightly with age, and mitochondria from younger, more active roots had less of the protein in the reduced form. Addition of pyruvate stimulated cyanide-insensitive respiration in root mitochondria, up to the same rate, regardless of seedling age. This stimulation was reversed rapidly upon removal of pyruvate, either by pelleting mitochondria (with succinate as substrate) or by adding lactate dehydrogenase with NADH as substrate. In mitochondria from cotyledons of the same seedlings, cyanide-insensitive NADH oxidation was less dependent on added pyruvate, partly due to intramitochondrial generation of pyruvate from endogenous substrates. Cyanide-insensitive oxygen uptake with succinate as substrate was greater than that with NADH, in both root and cotyledon mitochondria, but this difference became much less when an increase in external pH was used to inhibit intramitochondrial pyruvate production via malic enzyme. Malic enzyme activity in root mitochondria declined with seedling age. The results indicate that the activity of the alternative oxidase in soybean mitochondria is very dependent on the presence of pyruvate: differences in the generation of intramitochondrial pyruvate can explain differences in alternative oxidase activity between tissues and substrates, and some of the changes that occur during seedling development.

Cyanide-insensitive oxygen uptake and pyridine nucleotide dehydrogenases in the cyanobacterium Anabaena PCC 7120

The nature and subcellular location of cyanide-insensitive respiration and the pyridine nucleotide dehydrogenases were investigated in the cyanobacterium Anabaena PCC 7120. Oxygen uptake by isolated thylakoid membranes in the presence of either NADH, NADPH or duroquinol as substrates, was partially insensitive to KCN at concentrations which completely inhibited plasma membrane-mediated oxygen uptake. The cyanide-insensitive oxidase was not inhibited by salicyl hydroxamic acid (SHAM) or by n-propyl gallate (n-PG), which are inhibitors of the plant mitochondrial alternative oxidase; nor did total membrane proteins cross-react with antibodies raised against the alternative oxidase. We conclude that cyanide-insensitive oxidation is confined to the thylakoid membrane in Anabaena PCC 7120 and is not catalysed by the alternative oxidase responsible for cyanide-insensitive respiration in plants. Both plasma membranes and thylakoid membranes oxidised NADH and NADPH. Isolated plasma membrane was shown to contain a single type-1 dehydrogenase that is antigenically homologous to complex I of mitochondria and sensitive to the respiratory poison rotenone, but which oxidises both NADH and NADPH. The thylakoid membrane was shown to contain two NAD(P)H dehydrogenases: that responsible for NADH oxidation has inhibition characteristics of a type-2 dehydrogenase while NADPH oxidation may be catalysed by ferredoxin NADP +-oxidoreductase, the last enzyme in the photosynthetic electron transport chain.

Involvement of the Cyanide-Insensitive Respiration in Bacterivorous Growth in the Myxamoebae of a True Slime Mold, Didymium sp. Strain H-1.

The aerophilic growth of the myxamoebae of a true slime mold, Didymium sp. strain H-1, was studied. Two pathways of oxygen uptake, the cyanide-sensitive and cyanide-insensitive pathways, existed in the myxamoebae. Superoxide anions were produced by the cyanide-insensitive respiration. The cyanide-insensitive respiration had a low affinity for oxygen and the growth yield vs. oxygen concentration was the same pattern as the respiratory activity. The growth of myxamoebae is concluded to be controlled by the activity of the cyanide-insensitive respiration. The importance of the cyanide-insensitive respiration of the myxamoebae in a microenvironment was discussed.

Tissue-Specific Expression of the Alternative Oxidase in Soybean and Siratro

Alternative oxidase activity (cyanide-insensitive respiration) was measured in mitochondria from the shoots, roots, and nodules of soybean (Glycine max L.) and siratro (Macroptilium atropurpureum) plants. Activity was highest in the shoots and lowest in the nodules. Alternative oxidase activity was associated with one (roots) or two (shoots) proteins between 30 and 35 kilodaltons that were detected by western blotting with a monoclonal antibody against Sauromatum guttatum alternative oxidase. No such protein was detected in nodule mitochondria. Measurements of oxygen uptake by isolated soybean root and nodule cells in the presence of cyanide and salicylhydroxamic acid indicated that alternative oxidase activity was confined to the uninfected cortex cells of the nodule. Immunoprecipitation of translation products of mRNA isolated from soybean shoots revealed a major band at 43 kilodaltons that is assumed to be the precursor of an alternative oxidase protein. This band was not seen when mRNA from nodules was treated in the same fashion. The results indicate that tissue-specific expression of the alternative oxidase occurs in soybean and siratro.

Response of Pseudomonas aeruginosa to pyocyanin: mechanisms of resistance, antioxidant defenses, and demonstration of a manganese-cofactored superoxide dismutase

Pseudomonas aeruginosa produces a blue pigment, pyocyanin. Pyocyanin is a redox-active phenazine compound that kills mammalian and bacterial cells through the generation of reactive oxygen intermediates. We examined the mechanisms by which P. aeruginosa resists pyocyanin. [14C]pyocyanin was taken up by both Escherichia coli and P. aeruginosa, though more slowly by the latter. Cyanide-insensitive respiration, used as an indicator of intracellular superoxide and/or hydrogen peroxide production, was 50-fold less in pyocyanin-treated P. aeruginosa than in E. coli. P. aeruginosa showed less cyanide-insensitive respiration than E. coli upon exposure to other redox-active compounds (paraquat, streptonigrin, and plumbagin). Electron paramagnetic resonance spectrometry and spin trapping showed that P. aeruginosa generated less pyocyanin radical and superoxide than E. coli. Cell extracts from E. coli contained an NADPH:pyocyanin oxidoreductase which increased the rate of reduction of pyocyanin by NADPH. Conversely, cell extracts from P. aeruginosa contained no NADPH:pyocyanin oxidoreductase activity and actually decreased the rate of pyocyanin-mediated NADPH oxidation. Antioxidant defenses could also reduce the sensitivity of P. aeruginosa to pyocyanin. Under culture conditions of limited phosphate, both pyocyanin production and catalase activity were enhanced. Superoxide dismutase activity was also increased under low-phosphate conditions. When cells were grown in a high-phosphate succinate medium, P. aeruginosa formed a previously described iron-superoxide dismutase as well as a manganese-cofactored superoxide dismutase. These results demonstrate that P. aeruginosa resists pyocyanin because of limited redox cycling of this compound and that under conditions favoring pyocyanin production, catalase and superoxide dismutase activities increase.

The effects of infection by powdery mildew ( Erysiphe graminis f.sp. hordei) and low temperature on the respiratory activity of winter barley

The rapid changes in respiratory metabolism that follow exposure of plants grown at cold temperatures to warm conditions have been investigated in barley infected by powdery mildew ( Erysiphe graminis f.sp. hordei D. C. Marchal). Total rates of dark respiration, measured between 4 and 20 °C, were higher in cold-grown plants (9 ± 2 °C day, 4 ± 2 °C night) than in warm-grown plants (20 ± 2 °C). Infection caused an increase in respiration at all temperatures. Leaves from healthy cold-grown plants showed a marked change in the contribution of the individual components of total respiration in response to increasing temperature; cyanide-sensitive respiration dropped from 71·2 to 42·8%, while cyanide-insensitive respiration increased from 16·4% to 44·0%, over the temperature range 4−20 °C. Such changes were inhibited by mildew infection. Mlitochondrial respiration exhibited similar trends in response to temperature and infection. Mitochondria isolated from infected cold-grown barley exhibited a higher respiratory control index and a lower ADP:O ratio than those from healthy cold-grown plants. With the exception of a 16% reduction in the fructan fraction in infected barley, there were no significant ( P < 0·05) differences in the carbohydrate contents of healthy and infected cold-grown plants. Temperature and infection-induced changes in the pattern of respiration are discussed with respect to plant carbohydrate status and modified mitochondrial efficiency.