Respiratory activities in chloramphenicol-treated tobacco cells

Chloramphenicol (CAP) inhibited tobacco cell growth as shown by a reduction (34%) of cell mass 4 days after treatment. The rates of cell respiration were slightly higher than control under coupled conditions. However, CAP-treated cells showed a decreased maximal capacity of the cytochrome pathway (48%) and an increased maximal capacity of alternative path (56%) 4 days after treatment. In purified mitochondria, the rates of NADH or malate oxidation under state 4 conditions were not significantly changed by CAP treatment. However, the state 3 rates were 34–40% lower in CAP-treated than in control mitochondria. Succinate oxidation decreased by 31–46% under both state 4 and state 3 conditions after CAP treatment. The activities of complexes I, III, and IV, which contain mitochondrially encoded subunits, decreased by about 50% in CAP-treated mitochondria. There was also a decrease in the contents of mitochondrial cytochromes. Unexpectedly, the activities of complex II and the matrix-facing rotenone-insensitive NADH dehydrogenase, which are thought to be nuclear-encoded, also declined. The activities of external NADH dehydrogenase, NAD-linked malic enzyme, and fumarase remained unchanged after CAP treatment. There was a slight increase in the activity and protein level of alternative oxidase. An electrochemical gradient across the mitochondrial membranes was observed by Rhodamine 123 staining in CAP-treated cells. However, the morphology of most of the mitochondria changed from spherical to vermicular. A method for purifying a high yield of intact mitochondria from tobacco cell suspension cultures is described.     

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.     

Measurements of in Vivo Ubiquinone Reduction Levels in Plant Cells

A method is described for the determination of in vivo ubiquinone (UQ) reduction levels in nongreen tissues by extraction and subsequent detection of ubiquinone-10 and ubiquinol-10 with high-performance liquid chromatography. In Petunia hybrida cell suspensions UQ reduction remained at a stable level of about 60%, despite the changing conditions during the batch culture (from excess sugar to starvation) and the concomitant variations in respiration. Also, in the presence of uncoupler, which causes a large increase in respiration via both the cytochrome pathway and the alternative pathway, UQ reduction levels stayed at 60%. In mitochondria isolated from these cells, activity of the alternative pathway was only observed at UQ reduction levels higher than 80%. It is proposed that in vivo the relationship between UQ reduction and the activity of the alternative oxidase is modulated by mechanisms such as thiol modifications and accumulation of organic acids. Accordingly, pyruvate concentration in P. hybrida cells increased in the presence of uncoupler.     

Chloramphenicol Induced Changes in Some Respiratory Characteristics of Potato Tuber Callus

Chloramphenicol (CAP), an inhibitor of the mitochondrial proteinsynthesis inhibits callus induction and subsequent growth ofpotato tuber tissue discs. Tissue respiration increase did notoccur in the presence of CAP. Both with and without CAP theinitially CN^–-sensitive tissue becomes totally CN^–-resistantin 1–2 weeks. CAP blocks the development of mitcohondrial cytochrome oxidase.A gradual decrease in the activities of cytochrome oxidase andof cytochrome pathway-mediated mitochondrial respiration isfound in CAP-tissue. The mitochondrial alternative pathway whichis absent in mitochondria from freshly sliced tissue developsduring incubation both in the absence and presence of CAP. Thealternative pathway is only operative in uninhibited state IIIrespiration in mitochondria from CAP-tissue. Cycloheximide, an inhibitor of the cytoplasmic protein synthesisinhibits the developments of the alternative pathway and ofthe cytochrome pathway. Alcohol dehydrogenase activity increasestenfold in the tissue during two weeks of incubation on mediawith and without CAP. Alcohol production in the tissue did nottake place in the controls nor in the CAP-treated tissue. (Received April 18, 1981; Accepted July 17, 1981)     

The effect of aluminium on respiration of wheat roots

The effects of aluminium ions on respiration of excised root apices from wheat (Triticum aestivum L. cv. Vulcan) and on isolated mitochondria have been investigated. Addition of 75μ M aluminium to the growth medium of 4-day-old seedlings inhibited O₂ uptake by excised root apices by 23 and 35% after 12 and 24 h, respectively. This decreased rate of respiration was initially caused by inhibition of the cytochrome pathway of mitochondrial electron transport. The cyanide-insensitive, alternative pathway was inhibited only after more prolonged exposure to aluminium. Mitochondria isolated from roots of aluminium-treated seedlings had reduced oxidative capacity with substrates that supply electrons to Complexes I and II, compared with mitochondria from roots of untreated control seedlings. The state 3 and state 4 rates of O₂ uptake and the uncoupled rates with these substrates were also inhibited when aluminium was added directly to reaction mixtures containing mitochondria isolated from untreated plants. In contrast, when aluminium was added to reaction mixtures oxidizing exogenous NADH, state 4 O₂ uptake was stimulated, whereas no effect was observed on the state 3 rate or the rate in the presence of uncoupler. The results suggest that aluminium initially affects electron flow through Complexes I and II, and that after more prolonged exposure, aluminium may also interact with other sites in mitochondria.     

The effect of aluminum exposure on root respiration in an aluminum-sensitive and an aluminum-tolerant cultivar of Triticum aestivum

The effects of aluminum (Al) exposure on intact root respiration of an Al-sensitive (Scout-66) and an Al-tolerant (Atlas-66) cultivar of Triticum aestivum were investigated. Exposure to a wide range of Al concentrations (0–900 μmol) for 4 days stimulated respiration along the energy-conserving cytochrome pathway in both cultivars and increased the ratio of maintenance respiration to growth respiration. The maximum rate of Scout-66 root respiration occurred after exposure to 100–200 μmol Al. Atlas-66 root respiration peaked after exposure to 300–400 μmol Al. Similarly, calculations of theoretical adenosine 5'-triphosphate (ATP) production indicated that maximum daily rate of ATP production also increased upon exposure to Al in both cultivars, with peak ATP production occurring during peak respiration. Maximum root respiration rates in both cultivars were related to the Al concentration that inhibited root growth. Temporal exposure to 200 μmol Al quickly stopped root growth and stimulated cytochrome pathway respiration in Scout-66 after 4 days. Atlas-66 root growth and respiration were unaffected by 200 μmol Al. These results suggest that Al exposure imposes a demand for additional metabolic energy. A model describing Al effects on root respiration is presented     

Genetic differentiation in Plantago major: Growth and root respiration and their role in phenotypic adaptation

The root respiration and the growth of plants of four inbred lines of Plantago major L. were followed at two levels, of mineral nutrition. In addition the response to a transfer of plants from one condition to the other was studied. The activities of the cytochrome and of the alternative pathway wee determined and used to distinguish between genetic differences among the inbred lines and the plasticity within each inbred line. Root respiration and growth parameters differed significantly between the lines and were directly related to seed number per capsule (low respiratory activity and slow growth – 11 seeds per capsule; high respiratory activity and fast growth – 33 seeds per capsule).
Plasticity of respiration and growth parameters is expressed as differences in the total respiration, the activities of the cytochrome and of the alternative pathway, and in the shoot to root ratio, as a response to nutritional level or to changes of the strength of the nutrient solution. Differences in this plasticity in the four selected lines and in quickness of response to a change in mineral nutrition were directly related to the ecological strategy. These results are discussed in relation to the strategy of the genotypes for survival in the field. The high growth rate and the presence of plasticity in line 4 (ssp. Pleiosperma ) make this genotype act like an annual, following a ruderal strategy. The lower growth rate and the absence of plasticity in line 1 (ssp. major ) fit a more competitive strategy.     

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.     

Short-term effects of two aphid species on plant growth and root respiration of three leguminous species

The short-term effects of cowpea aphids ( Aphis craccivora Koch) and pea aphids ( Acyrthosiphon pisum Harris), both Homoptera: Aphididae, on plant growth and respiration of excised, intact roots of cowpea [ Vigna unguiculata (L.) Walp. cv. Caloona], broadbean ( Vicia faba L. cv. Aquadulce) and garden pea ( Pisum sativum L. cv. Victory Freezer) seedlings were investigated, but not all plant-aphid combinations were used. Plant and root mean relative growth rates were significantly reduced within 10 days in the infested plants. Rates of total root respiration were was also significantly reduced in all infested plants within 10 days, presumably because of the reduced availability of translocate to the roots. The contribution of the cytochrome pathway to root respiration was significantly greater in control than in infested plants. The activity and engagement of the alternative respiratory pathway was also greater in control plants, and was absent in infested plants after 10 days infestation in all cases but one. These data indicate that the roots of aphid-infested plants were more efficient, in terms of energy conversion, than their respective controls.     

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.     

Activities of plasma membrane-bound enzymes isolated from roots of spruce (Picea abies) grown in the presence of aluminium

In batch cultures of Petunia hybrida cv. Rosy Morn Fertile. one respiratory peak is usually observed shortly after subculturing. However, two types of peak respiration could be distinguished, one connected with the dilution process and one with sugar addition at low biomass concentrations. The dilution peak was observed when cells were diluted in medium without sugar, in the presence or absence of mannitol. The sugar peak occurred only after previous dilution of the cells and not when sugar is added at high biomass concentrations Apparently the existence of a dilute suspension is a prerequisite for the induction of the peak. The presence of sugar is not a prerequisite for the increased respiratory activity but it is necessary lor growth: however, growth is possible without the increase in respiration, as was shown by the addition of sugar to a culture with a high biomass concentration. The peak caused by dilution either in the presence or absence of sugar showed no significant differences in height. The height of the peak caused by sugar addition to a previously diluted cell suspension was correlated with the sugar concentration. The respiratory peak disappeared long before the end of the growth period; this decline of the respiratory rates was not connected to sugar or oxygen limitation. In a continuous culture of Petunia hybrida growing at low biomass concentration, the respiration was always at the high level as observed during the peak of batch culture. Growing at lower biomass concentrations might be more expensive for plant cell suspensions.     

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 relationship between phosphate status and cyanide-resistant respiration in bean roots

Bean ( Phaseolus vulgaris L.) seedlings were cultured on complete or phosphate-deficient nutrient medium. After 14 days of culture on phosphate-deficient medium the visible symptoms of P_i deficiency were observed only in the shoot, the fresh and dry weights of the roots were slightly higher than in control plants. The decreased P_i content in the roots had little effect on total respiration rate but had an effect on the level of inhibition of respiration by cyanide. The high resistance of respiration to cyanide observed in P_i-deficient roots was the result of the suppression of cytochrome path activity and an increased participation of the alternative, cyanide-resistant pathway. The cytochrome pathway activity increased when inorganic phosphate was supplied to P_i-deficient roots for 1 or 3.5 h. It is speculated that the suppression of cytochrome pathway in P_i-deficient roots may result from restriction of the phosphorylating capacity or a partial inhibition of cytochrome oxidase activity.     

LIGHT RESPIRATION IN SIX ESTUARINE PHYTOPLANKTON SPECIES: CONTRASTS UNDER PHOTOAUTOTROPHIC AND MIXOTROPHIC GROWTH CONDITIONS1

The photoresponse of respiration was examined in six estuarine phytoplankton species grown at a limiting irradiance in the presence or absence of glucose. Cellular respiration rates in the dark, at the growth irradiance, and at saturating light levels were higher in glucose-grown cultures than in photoautotrophic cultures. Glucose uptake also affected the relationship between incident irradiance and gross respiration rate, and the specific respiratory pathways that contributed to the respiration versus irradiance (R vs. I) response. The energy-conseming Mehler reaction contributed largely to light-stimulated respiration in photoautotrophic (energy-limited) cultures. However, the energy-dissipating alternative pathway was the predominant form of respiration measured in mixotrophic (energy-sufficient) cultures. Based on these and previous observations, a model of phytoplankton R us. I response is presented, in which the relative contributions of different light respiratory processes (cytochrome pathway respiration, alternative pathway respiration, chlororespiration, Mehler reaction) are dependent on the incident irradiance and cellular energy content.     

Short-term effects of infestation by two aphid species on plant growth and shoot respiration of three legumes

The short-term effects of infestation by cowpea aphids ( Aphis craccivora Koch) and pea aphids [ Acyrthosiphon pisum (Harris)], both Homoptera: Aphididae, on plant growth and respiration of excised, intact shoots of cowpea [ Vigna unguiculata (L.) Walp. cv. Caloona], broad bean ( Vicia faba L. cv. Aquadulce) and garden pea ( Pisum sativum L. cv. Victory Freezer) seedlings were investigated, but not all plant-aphid combinations were utilized. Root, shoot and plant dry weights were significantly reduced within 10 days in the infested plants. Rates of total shoot respiration were significantly greater in infested plants within 10 days, and the increase was not due to increased alternative pathway activity but, rather, to increased cytochrome pathway activity. It is suggested that the aphid-induced increase in shoot respiration may be due to increased rates of photosynthesis, to substances injected into the phloem by the aphids and/or delayed senescence. These data indicate that aphid-infested shoots had a decreased carbon use efficiency.     

The rate of development of water deficits affects Saxifraga cernua leaf respiration

We allowed plant water deficits to develop at two different rates following the cessation of watering in order to investigate the effects of water stress on cytochrome pathway and alternative pathway respiration in the leaves of the arctic herb Saxifraga cernua. Plants were pretreated by growth in either a commercial organic (CO) mixture or a vermiculite-perlite (VP) mixture, which allowed the complete development of water deficits in 19 and 8 days, respectively. The rate of water potential reduction was approximately 0.11 MPa day⁻¹ in the leaves of CO plants, compared to a reduction of 0.21 MPa day⁻¹ in leaves of VP plants. Osmotic adjustment occurred to a greater extent in leaves of CO plants and corresponded with an increase in ethanol-soluble sugars. In leaves of CO plants, cytochrome pathway activity gradually declined from that of control rates until day 11, and then declined more rapidly. In contrast, cytochrome pathway activity significantly increased in response to water deficits in leaves of VP plants. In leaves of both CO and VP plants, alternative pathway activity declined as water stress progressed. Relatively severe water deficits reduced alternative pathway capacity in leaves of both CO and VP plants. We also investigated the effect of previous exposure to water deficits on leaf respiration. In plants that had previously experienced three cycles of water stress, the increase in cytochrome pathway activity during the fourth water stress cycle was small compared to the increase observed in leaves of plants experiencing water stress for the first time. These results suggest that cytochrome pathway activity is differentially sensitive to the rate of development of plant water deficits and that respiratory responses to acute water stress are not necessarily similar to the responses to chronic water stress.     

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.     

Respiratory pathways in aged soybean seeds

Respiratory activities in soybean seeds (Glvcine max (L.) Merr. cv. Chippewa 64) have been examined in the first minutes after water imbibition and after three hours of imbibition, using either particles or intact axes. Cyanide and azide were utilized as inhibitors of the cytochrome oxidase pathway of respiration, and SHAM inhibitions were interpreted as effects on the alternative pathway, since in unaged axes inhibitions by SHAM were obtained only when respiratory activity had either been restricted with inhibitors of the cytochrome oxidase pathway or expanded by an uncoupler (CCCP). From the experiments with these inhibitors, it is suggested that unaged seeds utilize both respiration pathways in the cotyledon but only the cytochrome pathway in the axis. Accelerated aging causes a marked deterioration of respiration, especially that through the cytochrome pathway, and there is an associated engagement of the alternative pathway in the seed axis. It is suggested that the lowering of respiratory activity and the shift in respiratory pathways may play a major role in the decline of germinability and vigor.     

Possible role of adenylates in the engagement of the cyanideresistant pathway in nutrient-starved Catharanthus roseus Cells

In Cathuranthus roseus (L.) G. Don cells the cyanide-resistant pathway is engaged after phosphate or nitrogen starvation. Re-addition of these nutrients disengaged it again. Re-addition of phosphate leads to a transient disengagement which becomes only permanent after a second addition of phosphate. Disengagement after re-addition of nitrogen is slow: it takes 9 days before the activity has disappeared. In this system the mechanism of engagement of the cyanide-resistant pathway was studied. Addition of phosphate to phosphate-starved cells induced cell division within 24 h. The disengagement of the cyanide-resistant pathway was probably only an indirect effect of phosphate because the cellular P, content, which increased rapidly after addition, was low again before the cyanide-resistant pathway was disengaged. A better correlation was observed between high ADP and adenylate content of the cells and disengagement of the cyanide-resistant pathway. In addition it appeared that the engagement of the cyanide-resistant pathway was not the result of a limited carrier capacity of the cytochrome pathway. It is tentatively concluded that the engagement of the cyanide-resistant pathway in phosphate-starved cells was the result of a limited adenylate content. After nitrogen addition to N-starved cells, it took 5 days until the first growth occurred. Before the cyanide-resistant pathway was disengaged, its activity increased with the increased respiration rate which preceded growth. Within 72 h a higher ADP content was observed, which was still high after 10 days. The stimulation of the cytochrome pathway by uncoupler was small and more or less the same with and without added nitrogen, as long as the cyanide-resistant pathway was engaged. After disengagement the stimulation by uncoupler was significantly larger. It is suggested that the engagement during N-starvation was the result of a limited carrier capacity of the cytochrome pathway. Stimulation of the metabolism by re-addition of phosphate, nitrogen or sucrose resulted in a rapid increase in the levels of uracil nucleotides and uridine diphosphoglucose (UDPG) which are involved in sucrose metabolism.     

The regulation of respiration and growth of potato tuber callus by endogenous ethylene. Suppression of ethylene action by 2,5-norbornadiene

The growth (fresh and dry weight increase) of potato tuber ( Solanum tuberosum L. cv. Bintje) callus discs was stimulated by incubation in air with 500 ppm 2,5-norbornadiene (NBD, a competitive inhibitor of ethylene action) and inhibited by incubation in air with 4 000 ppm NBD. Ethylene formation by the callus was stimulated by NBD. The development of the alternative pathway, measured in isolated mitochondria was inhibited by NBD in a concentration-dependent way. The alternative pathway capacity, measured in vivo, was inhibited by 4 000 ppm NBD, but not by 500 ppm. Uninhibited in vivo respiration, which consists of cytochrome path activity and alternative path activity, was stimulated by the treatment with 500 ppm NBD. The main contribution to this stimulation was made by the cytochrome pathway. In 4 000 ppm NBD-treated callus, uninhibited respiration seemed to be unaffected as a consequence of an inhibited cytochrome path activity, which was compensated by a stimulated alternative path activity. Both in 500 and 4 OIK) ppm NBD-treated callus the alternative path activity in vivo was stimulated.
The regulatory role for endogenous ethylene in potato tuber callus is discussed in relation to: 1) The induction of respiratory pathways, 2) the supply of reduction equivalents in vivo and 3) growth.