Changes in Mitochondrial Respiratory Chain Components of Petunia Cells during Culture in the Presence of Antimycin A

When petunia (Petunia hybrida Vilm, cv Rosy Morn) cells are cultured in the presence of 2 [mu]M antimycin A (AA), respiration proceeds mainly via the cyanide-resistant pathway. Cyanide-resistant respiratory rates were higher in mitochondria from AA cells than in control mitochondria. Compared with control cells, an increase in alternative oxidase protein was observed in AA cells, as well as an increase in ubiquinone (UQ) content. A change in the kinetics of succinate dehydrogenase was observed: there was a much higher activity at high UQ reduction in mitochondria from AA cells compared with control mitochondria. No changes were found for external NADH dehydrogenase kinetics. In AA cells in vivo, UQ reduction was only slightly higher than in control cells, indicating that increased electron transport via the alternative pathway can prevent high UQ reduction levels. Moreover, O2 consumption continues at a similar rate as in control cells, preventing O2 danger. These adaptations to stress conditions, in which the cytochrome pathway is restricted, apparently require, in addition to an increase in alternative oxidase protein, a new setup of the relative amounts and/or kinetic parameters of all of the separate components of the respiratory network.     

The regulation of respiration in cell suspensions of Petunia hybrida, studied by inhibiting mitochondrial protein synthesis with chloramphenicol

When Petunia hybrida L. cv. Rosy Morn Fertile suspension cells were inoculated in fresh medium with chloramphenicol (CAP), the activity of cytochrome C oxidase (EC 1. 9. 3. 1), and the respiration via the cytochrome pathway of isolated mitochondria decreased, while in untreated cells these parameters more than doubled in 2–3 days. However, the in vivo respiratory activity of the cytochrome pathway of CAP-treated cells showed a similar course in time as that of untreated cells, even in the presence of an uncoupler, a large rise during the first 2–3 days followed by a decline. This leads to the conclusion that the respiration via the cytochrome pathway, even when measured in the presence of an uncoupler, is not the capacity of this pathway. Furthermore, the results suggest that, although new-synthesis of proteins occurs directly after in-oculation, a large overcapacity must be present of cytochrome pathway elements (at least of those that are mitochondrial encoded). CAP had little effect on the uninhibited respiration and the cyanide-resistant, alternative pathway of the Petunia cells. However, the engagement of the alternative pathway (in the presence or absence of uncoupler) was increased in CAP-treated cells, especially after day 3 of the batch cycle, possibly as an effect of higher sugar degradation in combination with substrate phosphorylation to compensate the loss of ATP-synthesizing ability of the cytochrome pathway. It will be discussed that in general one should be careful using the term 'capacity' for the respiratory pathways.     

Effect of Temperature on Rates of Alternative and Cytochrome Pathway Respiration and Their Relationship with the Redox Poise of the Quinone Pool

We investigated the effect of short-term changes in temperature on alternative (Alt) and cytochrome (Cyt) pathway respiration, both in intact tissues and isolated mitochondria of 14-d-old cotyledons of soybean (Glycine max L. cv Stevens). We also established the extent to which temperature alters the interaction between the oxidizing pathways and the level of ubiquinone (UQ) reduction (UQ(r)/UQ(t)). No difference was found between the temperature coefficient of respiration (Q(10); proportional change per 10 degrees C) of Alt and Cyt pathway respiration in cotyledon slices (Q(10) = 1.92 and 1.86, respectively). In isolated mitochondria, the Q(10) of the fully activated Alt pathway (Q(10) = 2.24-2.61) was always equal to, or higher than, that of Cyt c oxidase (COX) alone (Q(10) = 2.08) and the complete Cyt pathway (Q(10) = 2.40-2.55). This was true regardless of substrate or whether ADP was present. There was little difference in the Q(10) of the Cyt pathway with or without ADP; however, the Q(10) of COX was substantially lower in the presence of an uncoupler (Q(10) = 1.61) than its absence (Q(10) = 2.08). The kinetics of Alt and Cyt pathway activity in relation to UQ(r)/UQ(t) were not affected by temperature. For a given UQ(r)/UQ(t) value, the proportion of maximum flux taking place was similar at all temperatures for both pathways (+/-ADP). However, the Q(10) of the Alt and the Cyt pathways (+ADP) increased with increasing UQ(r)/UQ(t). We conclude that the Alt pathway is not less temperature sensitive than the Cyt pathway or COX per se and that changes in the degree of control exerted by individual steps in the respiratory apparatus could result in changes in the Q(10) of mitochondrial O(2) uptake.     

In vivo redox state of the ubiquinone pool in the spadices of the thermogenic skunk cabbage, <Emphasis Type="Italic">Symplocarpus renifolius</Emphasis>

In vivo ubiquinone (UQ) reduction levels were determined in thermogenic stigma and post-thermogenic male stages of spadices of the skunk cabbage, Symplocarpus renifolius. In contrast to Arum maculatum, in which the UQ pool is almost fully reduced during thermogenesis, the reduction levels of UQ9 and UQ10 were not affected by the thermogenic status or developmental stage of individual S. renifolius spadices. Moreover, these levels were controlled within the ranges 40–75% and 35–60%, respectively. These results suggest that the reduction state of the UQ pool per se is not primarily involved in thermoregulation in S. renifolius.     

Respiratory Shifts in Developing Petals of Saxifraga cernua

Changes in the oxygen uptake of petal slices by the cytochrome and alternative respiratory pathways were monitored during petal development in the arctic herb Saxifraga cernua. As the petals developed, rates of total respiration increased to a maximum rate during petal unfolding (day 4.5), and thereafter declined. Respiration in petals of all ages was at least partially resistant to cyanide, indicating the capacity for the alternative pathway. In all, except day 1 and senescing day 8 petals, respiration was inhibited by salicylhydroxamic acid, indicating engagement of the alternative pathway. In general, temporal changes in the respiratory activity along each pathway were similar and in parallel with changes in total respiration, although maximum rates along each pathway occurred at different times. Maximum cytochrome pathway activity occurred during petal expansion (day 4) whereas the alternative pathway peaked during petal unfolding at day 4.5. The control of respiration was also investigated. In the presence of salicylhydroxamic acid, the addition of the uncoupler carbonyl cyanide m-chlorophenylhydrazone was never stimulatory, suggesting that the cytochrome pathway was not restricted by adenylate levels. The addition of sucrose stimulated respiration only in day 1 petals, suggesting substrate limitation at this developmental stage. Since the rate of alternative pathway respiration peaked during petal unfolding, a time of high energy requirement, we suggest that the alternative pathway may have been used as an inefficient energy source during petal development.     

Alternative pathway respiration in vivo of potato tuber callus grown at various temperatures

During incubation of potato tuber discs ( Solanum tuberosum L. cv. Bintje) on a callus-inducing medium at 28°C, a high capacity of in vivo alternative pathway respiration develops (75% of uninhibited respiration is azide-resistant). When callus induction takes place at 8°C, only 45% of respiration is resistant to azide. In the lag phase of growth the activity of alternative pathway is low. during the exponential growth phase the activity reaches its maximal rate. This in vivo activity is of the same size at both culture temperatures. As a consequence a greater part of alternative pathway capacity is operating in uninhibited respiration during growth at low temperatures.     

Involvement of the alternative oxidase in respiration of Yarrowia lipolytica mitochondria is controlled by the activity of the cytochrome pathway

The degree of involvement of cyanide-resistant alternative oxidase in the respiration of Yarrowia lipolytica mitochondria was evaluated by comparing the rate of oxygen consumption in the presence of cyanide, which shows the activity of the cyanide-resistant alternative oxidase, and the oxidation rate of cytochrome c by ferricyanide, which shows the activity of the main cytochrome pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was associated with oxygen consumption due to the functioning of the alternative oxidase. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation) completely inhibited oxygen consumption by the mitochondria. Under these conditions, the inhibition of the alternative oxidase by benzohydroxamic acid (BHA) failed to affect the reduction of ferricyanide at the level of cytochrome c. BHA did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These data indicate that the alternative system is unable to compete with the cytochrome respiratory chain for electrons. The alternative oxidase only transfers the electrons that are superfluous for the cytochrome respiratory chain.     

Growth and respiration of Petunia hybrida cells in chemostat cultures: A comparison of glucose-limited and nitrate-limited cultures

Nitrate-limited and glucose-limited chemostat cultures of Petunia hybrida cells were compared at a specific biomass (+extracellular product) formation rate of 0.0042 C.mol/C.mol h. The composition of the biomass differed considerably in both culture types. The N/C (mol/mol) ratio in the biomass was almost four times lower in the nitrate-limited than in the glucose-limited cultures. On a dry weight basis (g/g DW) the biomass in the nitrate-limited cultures contained about 2.5 times less ions and protein N and about 2.5 times more carbohydrates than the biomass in the glucose-limited cultures. On a fresh weight basis (mmol/g FW) the biomass in nitrate-limited and glucose-limited cultures differed mainly in carbohydrate content. The yields of biomass on glucose and oxygen were generally higher in the nitrate-limited than in the glucose-limited cultures. Average values for these parameters were 0.27 C . mol biomass/C . mol glucose and 0.42 C . mol biomass/mol O(2) in the glucose-limited cultures and 0.34 C . mol biomass/C . mol glucose and 0.55 C . mol biomass/mol O(2) in the nitrate-limited cultures. On a C . mol basis the total respiration was about 25% and the maximally attainable cytochrome pathway activity (measured in the presence of hydroxamate) about 30% higher in the glucose-limited than in the nitrate-limited cultures. The maximally attainable activity of the alternative pathway (measured in the presence of KCN) was significantly lower in the glucose-limited cultures. On an organic N ( approximately protein) basis all respiratory parameters were significantly higher in the nitrate-limited cultures. In the presence of the respiratory uncoupler carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP) and excess glucose, cellular respiratory activity shows its maximal activity; under these conditions the total respiration increased more than 150% in the glucose-limited and only 30% in the nitrate-limited cultures. It is suggested that glucose-limited cultures are able to react more flexibly to changes in the environmental conditions than nitrate-limited cultures. (c) 1996 John Wiley & Sons, Inc.     

Ubiquinone is necessary for mouse embryonic development but is not essential for mitochondrial respiration

Ubiquinone (UQ) is a lipid found in most biological membranes and is a co-factor in many redox processes including the mitochondrial respiratory chain. UQ has been implicated in protection from oxidative stress and in the aging process. Consequently, it is used as a dietary supplement and to treat mitochondrial diseases. Mutants of the clk-1 gene of the nematode Caenorhabditis elegans are fertile and have an increased life span, although they do not produce UQ but instead accumulate a biosynthetic intermediate, demethoxyubiquinone (DMQ). DMQ appears capable to partially replace UQ for respiration in vivo and in vitro. We have produced a vertebrate model of cells and tissues devoid of UQ by generating a knockout mutation of the murine orthologue of clk-1 (mclk1). We find that mclk1-/- embryonic stem cells and embryos accumulate DMQ instead of UQ. As in the nematode mutant, the activity of the mitochondrial respiratory chain of -/- embryonic stem cells is only mildly affected (65% of wild-type oxygen consumption). However, mclk1-/- embryos arrest development at midgestation, although earlier developmental stages appear normal. These findings indicate that UQ is necessary for vertebrate embryonic development but suggest that mitochondrial respiration is not the function for which UQ is essential when DMQ is present.     

The alternative oxidase in roots of poa annua after transfer from high-light to low-light conditions

The activity of the alternative pathway can be affected by a number of factors, including the amount and reduction state of the alternative oxidase protein, and the reduction state of the ubiquinone pool. To investigate the importance of these factors in vivo, we manipulated the rate of root respiration by transferring the annual grass Poa annua L. from high-light to low-light conditions, and at the same time from long-day to short-day conditions for four days. As a result of the low-light treatment, the total respiration rate of the roots decreased by 45%, in vitro cytochrome c oxidase capacity decreased by 49%, sugar concentration decreased by 90% and the ubiquinone concentration increased by 31%, relative to control values. The absolute rate of oxygen uptake via the alternative pathway, as determined using the 18O-isotope fractionation technique, did not change. Conversely, the cytochrome pathway activity decreased during the low-light treatment; its activity increased upon addition of exogenous sugars to the roots. Interestingly, no change was observed in the concentration of the alternative oxidase protein or in the reduction state of the protein. Also, there was no change in the reduction state of the ubiquinone pool. In conclusion, the concentration and activity of the alternative oxidase were not changed, even under severe light deprivation.     

Respiration in the filarial nematode Brugia pahangi

Glucose-supported O2 uptake in the filarial nematode Brugia pahangi was partially inhibited by antimycin A (30-40%), with the remaining activity being sensitive to o-hydroxydiphenyl or salicylhydroxamic acid (SHAM). The production of CO2 by B. pahangi in the presence of D-glucose was stimulated by O2; the stimulation of CO2; the stimulation of CO2 production was sensitive to antimycin A. The O2 dependencies of respiration showed that the apparent O2 affinity for B. pahangi was diminished in the presence of antimycin A; O2 thresholds for inhibition of respiration were observed which showed that the alternative electron transport pathway was less sensitive to inhibition at elevated O2 concentrations. H2O2 production and its excretion could be detected in whole B. pahangi; higher rates were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. The effects of inhibitors on H2O2 production suggest two sites of H2O2 production, one associated with the classical antimycin A-sensitive pathway, the other with the alternative respiratory pathway. The similarity in the O2 dependencies of H2O2 production and respiration may indicate that H2O2 production is involved in O2-mediated toxicity. Succinate and malate respiring sub-mitochondrial particles of B. pahangi produced O2.- radicals at a site on the antimycin A-sensitive respiratory pathway. Inhibition of the alternative electron pathway by SHAM was unusual; sub-millimolar concentrations markedly stimulated respiration, H2O2 production and O2.- production by 30, 20 and 25%, respectively, whereas higher concentrations (greater than 2.5 mM) inhibited respiration by 75% and H2O2 and O2.- production by up to 85%.     

REGULATION OF ALTERNATIVE PATHWAY RESPIRATION IN CHLAMYDOMONAS REINHARDTH (CHLOROPHYCEAE)1

The inhibitor propyl gallate was used to estimate partitioning of respiratory electron flow between the cytochrome amd alternative pathways in Chlamydomonas reinhardtii Dangeard. Nutrient limitation (nitrogen or phosphorus resulted in a large increase in alternative pathway capacity relative to cytochrome pathway activity, without regulating in engagement of the alternative pathway. High rates of respiration, which could be induced in phosphate-starved cells by a combination of phosphate addition and uncoupler, resulted in alternative pathway activity. Osmotic stress resulted in decreased electron flow through the cytochrome pathway and increased flow through the alternative pathway, while high temperature also resulted in alternative pathway engagement. Incubation with exogenous carbon sources could increase the rate of respiratory O₂ consumption; the increase was mediated entirely by the alternative pathway. We suggest that the alternative pathway functions in these cells both to maintain respiration during environmentally induced stress and as on energy overflow.     

The appearance and characterization of cyanide-resistant respiration in the fungus Candida albicans

The respiration of yeast-form cells of the dimorphic fungus Candida albicans became resistant to cyanide during aging treatment in the resting state. An alternative, cyanide-resistant respiratory pathway was found to develop fully in cells aged at a concentration of 0.75 X 10(9)/ml or more at 25 C, but did not appear at 5 C. Chloramphenicol did not prevent the appearance of the alternative respiratory pathway. The effects of inhibitors, salicylhydroxamic acid (SHAM) and disulfiram (tetraethylthiuram disulfide), on respiration of aged cells were examined, and results indicated that SHAM binds at a site on the alternative respiratory pathway whereas disulfiram binds at two sites, one on the conventional respiratory pathway and the other on the alternative pathway. Thus, SHAM is a more selective inhibitor of the alternative respiration of C. albicans cells. SHAM-titration of the alternative respiration revealed that less than 10% of the maximal activity of the alternative respiratory pathway was utilized under normal conditions, indicating that the alternative respiratory pathway makes a small contribution to the total respiration. It was therefore concluded that the alternative, cyanide-resistant respiratory pathway operates fully when the cyanide-sensitive, cytochrome pathway is blocked although aged cells possess both respiratory pathways.     

The alternative oxidase of Yarrowia lipolytica mitochondria is unable to compete with the cytochrome pathway for electrons

The activity of the cyanide-resistant alternative oxidase (pathway) of Y. lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.     

Inhibition and stimulation of root respiration in pisum and plantago by hydroxamate : its consequences for the assessment of alternative path activity

The contribution of the alternative pathway in root respiration of Pisum sativum L. cv Rondo, Plantago lanceolata L., and Plantago major L. ssp major was determined by titration with salicylhydroxamate (SHAM) in the absence and presence of cyanide. SHAM completely inhibited the cyanide-resistant component of root respiration at 5 to 10 millimolar with an apparent K_i of 600 micromolar. In contrast, SHAM enhanced pea root respiration by 30% at most, at concentrations below 15 millimolar. An unknown oxidase appeared to be responsible for this stimulation. Its maximum activity in the presence of low SHAM concentrations (1-5 millimolar) was 40% of control respiration rate in pea roots, since 25 millimolar SHAM resulted in 10% inhibition. In plantain roots, the maximum activity was found to be 15%. This hydroxamate-activated oxidase was distinct from the cytochrome path by its resistance to antimycin. The results of titrations with cyanide and antimycin indicated that high SHAM concentrations (up to 25 millimolar) block the hydroxamate-activated oxidase, but do not affect the cytochrome path and, therefore, are a reliable tool for estimating the activity of the alternative path in vivo. A considerable fraction of root respiration was mediated by the alternative path in plantain (45%) and pea (15%), in the latter because of the saturation of the cytochrome path.     

Influence of chloramphenicol on growth and respiration of soybean (Glycine max L.) suspension cultures

By addition of chloramphenicol (CAP) to the growth medium of green soybean ( Glycine max L.) cells in batch culture, growth is inhibited and the activity of the cytochrome oxidase decreases to 60% of the value found in control cells. The presence of CAP induces an enhancement of the contribution of the alternative pathway to total respiration. This enlarged contribution results both from a higher capacity of the alternative pathway and from a greater part of this capacity being used. Also in mitochondria isolated from cells treated with CAP, a higher capacity of the alternative pathway has been found, while the part of this capacity which is really used is comparable with the values found in control cells.     

Dark respiration in the marine macroalga Chondrus crispus (Rhodophyceae)

Dark respiration in the red macroalga Chondrus crispus was studied under a variety of conditions. The components of respiration were examined using selective inhibitors in order to characterise pathways of respiration and examine regulation of respiration in marine macroalgae.In comparison to respiration rates generally reported for higher-plant leaves and roots, the steady-state rate of O₂ consumption by this alga, after 30 min dark pretreatment, was found to be quite low (three- to sixfold lower than in higher plants). The addition of uncoupler had only a slight effect on the basal respiration rate, indicating that in these conditions, substrate supply could be limiting respiration. The addition of KCN inhibited respiration by approx. 60%, indicating the presence of alternative oxidase activity. The coefficient of engagement of the alternative pathway (calculated from the data herein) showed that under normal conditions there was little participation of the alternative pathway in O₂ consumption. The response of respiration to O₂ tension was examined with and without inhibitors and the apparent K _m was 17 to 21 M. The addition of KCN plus salicylhydroxamic acid almost completely blocked respiration in C. crispus. The hypothesis that respiratory substrate limits respiration in this alga was investigated by measuring respiration rates immediately after periods of photosynthetic activity. It was found that the respiration rate was dependent on the duration of the light period and could increase up to twofold. This stimulated rate of respiration declined in a first-order fashion during the next 20 to 30 min, finally reaching the basal, zero-order rate measured before illumination. These results strongly indicate a change in the nature of the respiratory substrates during this period. No change in the contribution of the alternative pathway of respiration could be detected following light pretreatment.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - SHAM salicyl hydroxamic acid     

The Alternative Oxidase of Yarrowia lipolytica Mitochondria Is Unable To Compete with the Cytochrome Pathway for Electrons

The activity of the cyanide-resistant alternative oxidase (pathway) of Yarrowia lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.     

Hydrogen peroxide production in uncoupled mitochondria of the parasitic nematode worm Nippostrongylus brasiliensis.

1. Mitochondria from the parasitic nematode worm Nippostrongylus brasiliensis produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 2. Antimycin A inhibits respiration and H2O2 production by 70 and 65% respectively; the residual activities can be attributed to alternative electron-transport pathway(s). 3. o-Hydroxydiphenyl and 1,3,5-trihydroxybenzene, inhibitors of alternative electron transport, inhibit respiration by 37% and H2O2 production by 26%. 4. Another inhibitor of alternative electron transport, salicylhydroxamic acid, shows a complex mode of action; low concentrations (less than 0.5 mM) stimulate respiration and H2O2 production, whereas 2 mM-salicylhydroxamic acid inhibited respiration by 35% and stopped H2O2 production completely. 5. O2 thresholds were observed for the inhibition of respiration at O2 concentrations greater than 57.7 microM and inhibition of H2O2 production (greater than 20.5 microM-O2); apparent Km values for oxygen were 5.5 microM and 3.0 microM respectively. 6. In the presence of antimycin A the O2-inhibition thresholds and apparent Km values for O2 of respiration and H2O2 production matched closely, suggesting that the alternative oxidase is a likely site of H2O2 production. 7. These results are discussed in relation to O2 toxicity to N. brasiliensis.     

The effects of abscisic acid on growth and respiratory characteristics of potato tuber tissue discs

Incubation of potato tuber tissue discs on B5 medium supplemented with 1-naphtyl-acetic acid (NAA) led to callus formation, irrespective of the presence of kinetin; without NAA no callus formation occurred. Incubation in the presence of abscisic acid (ABA) reduced the increases in fresh weight and dry weight both in callus-forming and in non-callus-forming tissue. Mitochondrial respiration was lowered by ABA as well. The induction of the alternative, CN-resistant pathway was inhibited by the presence of ABA, especially in mitochondria from non-callus-forming tissue.The in vivo respiration of the callus-forming tissue was higher than that of the non-callus-forming tissue. Total respiration, cytochrome pathway activity and the capacity of the alternative pathway were all lowered in callus-forming tissue by treatment with ABA. The in vivo activity of the alternative pathway was low in all tissue types, especially after ABA-treatment. The slight stimulation by hydroxamates of the oxygen uptake of callus-forming tissue incubated on medium with NAA and ABA indicates the involvement of a hydroxamate-activated peroxidase in the oxygen uptake of this tissue; this peroxidase seemed not to participate in the oxygen uptake of the other tissues types.In non-callus-forming tissue the oxygen uptake of ABA-treated tissue was very low and almost completely resistant to the combined addition of inhibitors of both the cytochrome and the alternative pathway, indicating that the in vivo activity of the mitochondria in the oxygen uptake of the tissue was very low. The possible causes for this ABA-effect are discussed. In non-callus-forming tissue the treatment with ABA creates a situation which is comparable with that observed in intact potato tubers. This situation is characterized by a tissue respiration lower than that of the isolated mitochondria and an alternative pathway capacity that is low or absent.