Cyanide-resistant respiration in photosynthetic organs of freshwater aquatic plants

The rate and sensitivity to inhibitors (KCN and salicylhydroxamic acid[SHAM]) of respiratory oxygen uptake has been investigated in photosynthetic organs of several freshwater aquatic plant species: six angiosperms, two bryophytes, and an alga. The oxygen uptake rates on a dry weight basis of angiosperm leaves were generally higher than those of the corresponding stems. Leaves also had a higher chlorophyll content than stems. Respiration of leaves and stems of aquatic angiosperms was generally cyanide-resistant, the percentage of resistance being higher than 50% with very few exceptions. The cyanide resistance of respiration of whole shoots of two aquatic bryophytes and an alga was lower and ranged between 25 and 50%. These results suggested that the photosynthetic tissues of aquatic plants have a considerable alternative pathway capacity. The angiosperm leaves generally showed the largest alternative path capacity. In all cases, the respiration rate of the aquatic plants studied was inhibited by SHAM alone by about 13 to 31%. These results were used for calculating the actual activities of the cytochrome and alternative pathways. These activities were generally higher in the leaves of angiosperms. The basal oxygen uptake rate of Myriophyllum spicatum leaves was not stimulated by sucrose, malate or glycine in the absence of the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP), but was greatly increased by CCCP, either in the presence or in the absence of substrates. These results suggest that respiration was limited by the adenylate system, and not by substrate availability. The increase in the respiratory rate by CCCP was due to a large increase in the activities of both the cytochrome and alternative pathways. The respiration rate of M. spicatum leaves in the presence of substrates was little inhibited by SHAM alone, but the SHAM-resistant rate (that is, the cytochrome path) was greatly stimulated by the further addition of CCCP. Similarly, the cyanide-resistant rate of O₂ uptake was also increased by the uncoupler.     

Energy metabolism of Plantago lanceolata, as affected by change in root temperature

The long and short term metabolic effects of a shift in root temperature was investigated in Plantago lanceolata L. with special reference to the role of the cyanide resistant alternative pathway in root respiration. After a 10-day period of growth at a 13°C root temperature, a decrease in root as well as shoot growth was observed, compared to control plants grown continuously at 21°C. Apart from an increase in shoot soluble and insoluble sugar level, no changes in metabolism were found, neither in root respiration, shoot photosynthesis, nor in root sugar and plant protein level.
Decreasing the root temperature from 21 to 13°C gave several clear short term changes in metabolism. Within one hour a decrease in cytochrome chain activity of the roots was found together with an increase in activity of the alternative chain. After 24 h a recovery to the initial level of both chains was observed. An increase in root temperature from 13 to 21°C gave an immediate increase in activity of both respiratory chains that was still present 24 h after the switch.
It is concluded that the activity of the alternative respiratory pathway in the root is strongly affected by a sudden temperature change in the root environment. This pathway acts in a way which is described by 'the energy overflow model'. The presence of the alternative electron transport pathway should be taken into account in determinations of the respiratory Q₁₀. Moreover, the length of time between the temperature change and respiration measurements is an important factor.     

Effects of cyanide, salicylhydroxamic acid, and temperature on respiration and germination of spores of the fern Sphaeropteris cooperi

During the first 96 h of culture, germinating spores of the fern Sphaeropteris cooperi (F. v. Muell.) Tryon showed a gradual rise in respiratory activity to a maximum of about 6.5 μl 0₂ h⁻¹ mg⁻¹ dry wt. This was followed by a transitory decline in rate, concluded by a second respiratory rise preceding the emergence of the rhizoid after 192 h of culture. Oxygen uptake during the first 120 h of germination was insensitive to 1 m M potassium cyanide (KCN) but was inhibited by 1 m M salicylhydroxamic acid (SHAM); however, beyond this time cyanide showed increasing inhibitory effectiveness whereas SHAM became less effective. Regardless of time of application, KCN had no effect on germination. Maximum inhibition of germination by SHAM was achieved if applied up to 120 h after culture initiation, after which spores became insensitive to SHAM. Heat treatment (50°C for 90 min) during the cyanide-resistant phase of respiration (0 h–120 h) induced cyanide-sensitive respiration and completely inhibited spore germination. Elevated temperatures had little effect if applied during the cyanide-sensitive phase (beyond 120 h). Temperature inhibited spores regained their ability to germinate if maintained in culture until the cyanide-resistant pathway was restored and then subjected to a second photoinductive light treatment. These results suggest the presence and possible involvement of the cyanide-resistant, alternative respiratory pathway during germination of Sphaeropteris cooperi spores.     

Cyanide-resistant respiration in roots and leaves. Measurements with intact tissues and isolated mitochondria

A comparison was made between the oxygen uptake of roots and leaves and of mitochondria isolated from the same tissues. Ten species were included in this study: three legumes, one C3-monocotyledon, one C4-monocotyledon, the rest non-leguminous C3-dicotyledons. Root and leaf respiration in all species examined displayed substantial resistance to KCN (0.1–1.0 mM) and the cyanide-resistant respiration was completely inhibited by salicylhydroxamic acid (SHAM; 10–20 mM). SHAM alone inhibited oxygen uptake to varying degrees, depending on the species. Mitochondria were isolated from roots and leaves of many of the species examined and also displayed cyanide-resistant oxygen uptake, which was sensitive to both SHAM and tetraethylthiuram disulfide (disulfiram). Concentrations of SHAM greater than 2 mM caused inhibition of the cytochrome path as well as of the alternative path in isolated mitochondria. Respiration rates of intact roots and leaves in the presence of varying concentrations of SHAM alone were plotted against those obtained in the presence of both SHAM and KCN. This plot showed that in vivo the cytochrome pathway was not affected by 10 or 20 mM SHAM in the external solution. We conclude that the activity of the alternative pathway in intact roots and leaves can be reliably estimated by comparing SHAM-sensitivity and cyanide-resistance of respiration.     

The effect of phosphate deficiency on mitochondrial activity and adenylate levels in bean roots

Bean plants ( Phaseolus vulgaris ) were grown for 16–20 days with or without phosphate in Knop nutrient medium. It was found in previous experiments that for roots grown on a P_i-deficient medium respiration is mainly carried out by the cyanide-insensitive pathway. Mitochondria isolated from—P_i, roots had poor respiratory control and their respiration exhibited 62% inhibition by cyanide and was inhibited (30%) by salicylhydroxamic acid (SHAM). In contrast, mitochondria obtained with control (+P_i) roots had respiratory control and ADP/O ratios typical for succinate as the substrate; their respiration was inhibited to 95% by cyanide and insensitive to SHAM. The integrity of mitochondrial membranes was similar in both types of mitochondria. Cytochrome oxidase activity, however, was about 20% lower in -P_i mitochondria, but the cytochrome composition was the same in both types of mitochondria. The cytochrorae pathway was not operating at full capacity in mitochondria isolated from—P_i roots but the alternative oxidation pathway participated in a great part in mitochondrial respiration, similar to in vivo whole roots. The participation of the non-phosphorylating., alternative pathway decreased the respiratory control ratio in mitochondria and had an effect on the total adenine nucleotide pool and energy charge values which were lower (16 and 13% respectively) in -P_i roots. About 50% lower ADP and 20% lower ATP levels were observed whereas AMP levels were several times higher.     

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.     

Sulfide-Resistant Respiration in Leaves of Elodea canadensis Michx: Comparison with Cyanide-Resistant Respiration

The rate of dark O₂ uptake of Elodea canadensis leaves was titrated with either cyanide or sulfide in the presence and in the absence of 5 millimolar salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase. The inhibition of O₂ uptake by SHAM alone was very small (3-6%), suggesting that actual respiration mainly occurred through the cytochrome pathway. O₂ uptake was slightly stimulated by cyanide at concentrations of 50 micromolar or higher, but in the presence of SHAM respiration was strongly suppressed. The effects of sulfide on O₂ uptake were similar to those of cyanide, except that the percent stimulation of O₂ uptake by sulfide alone was somewhat higher than that of cyanide. However, the estimates of the capacity of the alternative pathway were similar with both inhibitors. Another difference is that maximal inhibition of respiration in the presence of SHAM was observed with lower concentrations of sulfide (50 micromolar) than cyanide (250 micromolar). The results suggest that sulfide can be used as a suitable inhibitor of cytochrome c oxidase in studies with intact plant tissues, and that sulfide does not apparently inhibit the alternative oxidase.     

Effect of water deficit on respiration of conducting bundles in leaf petioles of sugar beet

Isolated fibrovascular bundles from source leaf petioles of sugar beet (Beta vulgaris L.) and hog-weed (Heracleum sosnovskyi L.) were used to study the influence of long-term drought on the oxygen uptake rate and activities of mitochondrial oxidases, i.e., cytochrome oxidase and salicylhydroxamic acid-sensitive alternative oxidase (AO). Under normal soil moisture content (70% of full water-retaining capacity, WRC), the oxygen uptake by sugar beet conducting bundles was characterized by a high rate (> 700 μl O₂/(g fr wt h)) and by distinct cytochrome oxidase-dependent manner of terminal oxidation (up to 80% inhibition of respiration in the presence of 0.5 mM KCN). After long-term water deficit (40% of WRC), the bundle respiration proceeded at nearly the same rate but featured an elevated resistance to cyanide. At early drought stage (10 days), a decrease in the activity of cytochrome-mediated oxidation pathway was largely counterbalanced by activation of mitochondrial AO, whereas long-term dehydration of plants was accompanied by activation of additional oxidative systems insensitive to both KCN and SHAM. Similar but even more pronounced changes in activities of terminal oxidases were discovered in conducting bundles of wild-grown hogweed plants exposed to long-term natural drought. It is supposed that the suppression of cytochrome-mediated oxidation coupled with ATP synthesis in the cells of sugar beet source leaves impedes the translocation of assimilates and their accumulation in the taproot, which represents an important factor of drastic decrease in the yield of this agricultural crop under conditions of water deficit.     

Action of Respiratory Inhibitors on Seed Germination and Oxygen Uptake in Avena fatua L.

The effects of sodium azide, potassium cyanide (cytochrome oxidaseinhibitors), and salicylhydroxamic acid (SHAM; an alternativerespiration inhibitor) on germination and respiration of Avenafatua L. seeds were studied. Azide and cyanide released seeddormancy at similar concentrations and treatment durations.Cyanide, however, stimulated germination of seeds with littleafter-ripening, whereas azide had no effect under similar conditionsunless the seeds were after-ripened for several months; theduration of after-ripening required for seeds to respond toazide varied with seed batch. There was also a greater lag priorto germination in the case of azide, compared to cyanide treatedseeds. SHAM inhibited the stimulation of germination and respirationby azide, but not by cyanide. Furthermore, respiration induced by azide or cyanide could notbe inhibited by the subsequent application of SHAM. These findingssuggest that the respiration stimulated by azide and cyanideis not alternative (SHAM-sensitive) and, therefore, this respiratorypathway cannot be involved in the stimulation of germinationby cytochrome oxidase inhibitors. While embryos excised fromcontrol, azide or cyanide pretreated seeds had the capacityto perform alternative respiration, the actual contributionof this pathway was negligible. A large proportion of respirationof embryos excised from azide or cyanide pretreated seeds wasresidual, i.e. insensitive to both SHAM and cyanide. Alternative respiration, azide, cyanide, dormancy, salicylhydroxamic acid, wild oats     

Cyanide-resistant respiration in light- and dark-grown soybean cotyledons

Measurements of respiration were made on intact tissue and mitochondria isolated from soybean (Glycine max [L.] Merr. cv `Corsoy') cotyledons from seedlings of different ages grown in light and darkness. Effects of cyanide (KCN) and salicylhydroxamic acid (SHAM) on O₂ uptake rates were determined. O₂ uptake was faster in light-grown tissue and was inhibited by both KCN and SHAM in all except light-grown tissue older than 9 days. Both inhibitors stimulated O₂ uptake in tissues more than 9 days old. Mitochondria in which O₂ uptake was coupled to ATP synthesis were isolated from all tissues. O₂ uptake by mitochondrial preparations from light- and dark-grown cotyledons was equally sensitive to KCN. Similarly, age did not affect KCN sensitivity, but sensitivity to SHAM declined with age both in the presence and absence of KCN. Estimated capacities of the cytochrome and alternative pathways of the mitochondrial preparations indicated considerably larger cytochrome than alternative pathway capacities. The cytochrome pathway capacities paralleled the state 3 mitochondrial respiration rates, which increased from day 5 to day 7 then declined thereafter. The alternative pathway capacities were not affected by light. The uncoupler, p-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), increased the flow of electrons through the cytochrome pathway at the expense of flow through the alternative pathway in isolated mitochondria. However, the combined capacities did not exceed the rate in the presence of FCCP. The results are interpreted to indicate that the stimulation of respiration by KCN and SHAM observed in the 12-day-old green cotyledons and previously observed in older soybean leaves is not explained by characteristics of the mitochondria.     

Changes in Properties of Barley Leaf Mitochondria Isolated from NaCl-Treated Plants

Treatment of barley (Hordeum vulgare) seedlings with 400 millimolar NaCl for 3 days resulted in a reduction in plant growth and an increase in the leaf content in ions (K⁺ + Na⁺) and proline. Purified mitochondria were successfully isolated from barley leaves. Good oxidative and phosphorylative properties were observed with malate as substrate. Malate-dependent electron transport was found to be only partly inhibited by cyanide, the remaining oxygen uptake being SHAM sensitive. The properties of mitochondria from NaCl-treated barley were modified. The efficiency of phosphorylation was diminished with only a slight decrease in the oxidation rates. In both isolated mitochondria and whole leaf tissue of treated plants, the lower respiration rate was due to a lower cytochrome pathway activity. In mitochondria, the activity of the alternative pathway was not modified by salt treatment, whereas this activity was increased in whole leaf tissue. The possible participation of the alternative pathway in response to salt stress will be discussed.     

Salicylic Acid induces cyanide-resistant respiration in tobacco cell-suspension cultures

Cyanide-resistant, alternative respiration in Nicotiana tabacum L. cv Xanthi-nc was analyzed in liquid suspension cultures using O₂ uptake and calorimetric measurements. In young cultures (4-8 d after transfer), cyanide inhibited O₂ uptake by up to 40% as compared to controls. Application of 20 μm salicylic acid (SA) to young cells increased cyanide-resistant O₂ uptake within 2 h. Development of KCN resistance did not affect total O₂ uptake, but was accompanied by a 60% increase in the rate of heat evolution from cells as measured by calorimetry. This stimulation of heat evolution by SA was not significantly affected by 1 mm cyanide, but was reduced by 10 mm salicylhydroxamic acid (SHAM), an inhibitor of cyanide-resistant respiration. Treatment of SA-induced or uninduced cells with a combination of cyanide and SHAM blocked most of the O₂ consumption and heat evolution. Fifty percent of the applied SA was taken up within 10 min, with most of the intracellular SA metabolized in 2 h. 2,6-Dihydroxybenzoic and 4-hydroxybenzoic acids also induced cyanide-resistant respiration. These data indicate that in tobacco cell-suspension culture, SA induces the activity and the capacity of cyanide-resistant respiration without affecting the capacity of the cytochrome c respiration pathway.     

Cold hardening of raspberry plants in vitro is enhanced by increasing sucrose in the culture medium

The influence of exogenously applied sucrose on cold hardening of raspberry ( Rubus idaeus L.) in vitro was examined. Raspberry plants (cv. Preussen) were cultured on Murashige-Skoog (MS) media with different levels (1, 3, 5 and 7%) of sucrose and subjected to low-temperature acclimation (3/−3°C day/night temperature, 8-h photoperiod) for 14 days. Cold hardiness (LT₅₀ in controlled freezing), shoot moisture content, osmolality and the amounts of sucrose, glucose and fructose were determined. Exogenously applied sucrose was taken up by plants, but the uptake corresponded to less than 10% of total sugar reserves in the culture. Cold hardiness was primarily affected by acclimation treatment, but sucrose increased cold hardiness of nonacclimated plants and significantly enhanced the effect of acclimation treatment, 5% sucrose in the culture medium being optimal for cold hardening. LT₅₀ values ranged between −4.1 and −7.1°C for nonacclimated, and between −14.2 and −20.7°C for cold-acclimated shoots. Shoot moisture content was inversely related to medium sucrose level and declined only slightly during cold acclimation. After cold acclimation, plant osmolality predicted hardiness better than shoot moisture content. Plant osmolality and sugar content were increased by increasing the medium sucrose level and, to a greater extent, by cold acclimation. Sucrose, glucose and fructose accumulated during hardening. Sucrose was the predominant sugar, and the rate of sucrose accumulation during cold acclimation was independent of the medium sucrose level or the initial plant sucrose content. A close correlation between cold hardiness and total sugars, sucrose, glucose and fructose was established. These results suggest that sugars have more than a purely osmotic effect in protecting acclimated raspberry plants from cold.     

Respiration during seed maturation

The contribution of cytochrome and alternative pathways to respiration during soybean [ Glycine max (L.) Merr. cv. Chippewa 64] seed formation and maturation was measured by oxygen consumption of embryonic axis and cotyledon tissues and of mitochondria isolated from immature seeds. Respiration rates (dry weight basis) declined duing the last month of seed maturation. Cyanide sensitive respiration (cytochrome pathway) accounted for 80 to 90% of total oxygen consumption throughout seed formation and maturation. The proportion of the total respiration resistant to 1 m M cyanide and inhibited by 1 m M salicylhydroxamic acid (alternative pathway) did not exceed 6%. A residual oxygen consumption, accounting for 5 to 15% of total respiration, persisted in the presence of both inhibitors. Likewise, virtually no alternative respiration was found in mitochondria prepared from immature seeds. The alternative respiratory pathway seems absent during soybean seed formation and maturation regardless of whether grown in the greenhouse or field.     

Evidence for a significant contribution by peroxidase-mediated O2 uptake to root respiration of Brachypodium pinnatum

This study describes the O₂ uptake characteristics of intact roots of Brachypodium pinnatum. In the presence of 25 mM salicylhydroxamic acid (SHAM), concentrations of KCN below 3.5 νM had no effect on the rate of root respiration, whereas in the absence of 25 mM SHAM a significant inhibition of approx. 18% was observed. This indicates that an O₂-consuming reaction, not associated with the cytochrome pathway, the alternative pathway or the “residual component”, operates in the absence of any inhibitors in roots of B. pinnatum. We demonstrate here that this fourth O₂-consuming reaction is mediated by a peroxidase. A peroxidase which catalyzed O₂ reduction in the presence of NADH was readily washed from the roots of B. pinnatum. This peroxidase was stimulated by 5 mM SHAM, whereas ascorbic acid, catalase, catechol, gentisic acid, low concentrations potassium cyanide (3.5 μM), sodium azide, sodium sulfide, superoxide dismutase and high concentrations SHAM (25 mM) inhibited this reaction. Except for high concentrations of SHAM and concentrations of KCN higher than approx. 3.5 μM, these effectors could not be used to inhibit the peroxidase-mediated O₂ uptake in intact roots of B. pinnatum. Concentrations of SHAM below 10 mM stimulated O₂ uptake up to 15% of the control rate, depending on concentration, whereas 25 mM SHAM inhibited O₂ uptake by 35%. The stimulation at low concentrations resulted from a SHAM-stimulated peroxidase activity, whereas 25 mM SHAM completely inhibited both the peroxidase-mediated O₂ uptake and the activity of the alternative pathway. A method is presented for determining the relative contributions of each of the four O₂-consuming reactions, i.e. the cytochrome pathway, the alternative pathway, the “residual component” and the peroxidase-mediated O₂ uptake. The peroxidase-mediated O₂ uptake contributed 21% to the total rate of oxygen uptake in roots of B. pinnatum, the cytochrome pathway contributed 41%, the alternative pathway 14% and the “residual component” 24%.     

Respiration of barley roots: Assessment of activity of the alternative path using SHAM

The activity of the alternative path of O₂ consumption in detached and intact roots of barley [ Hordeum distichum (L.) Lam. cv. Maris Mink] was determined by titration with salicylhydroxamic acid (SHAM) in the presence and absence of cyanide. In the absence of cyanide, only high concentrations were inhibititory (> 5 m M ). whilst in its presence low SHAM concentrations (2.5–5.0 m M ) gave maximum inhibition: the resulting ϱ V_alt plots were non-linear. A SHAM-stimulated peroxidase could readily be washed from these roots, but non-linearity cannot be explained in terms of SHAM-stimulation of this peroxidase as it is not active in the absence of an exogenous supply of NADH. In detached roots the degree of inhibition of respiration with 25 m M SHAM was nearly double the capacity of the alternative path (measured as the degree of inhibition by SHAM in the presence of cyanide), suggesting non-specific inhibition. Effects of SHAM on cytochrome path activity in intact roots were examined by reverse titration with cyanide in the presence and absence of SHAM. At 5 m M SHAM had no effect on the cytochrome path, but at 25 m M it inhibited. We conclude that the only factor causing non-linearity of ϱV_alt plots in barley roots is non-specific inhibition of the cytochrome path by high concentrations of SHAM; consequently only low concentrations of SHAM (2.5–5.0 m M ) are suitable for estimating alternative path activity in barley roots.     

Effects of KCN and Salicylhydroxamic Acid on Respiration of Soybean Leaves at Different Ages

Measurements of respiration were made on leaf discs from glasshouse-grown soybean (Glycine max [L.] Merr. cv `Corsoy') plants in the presence and absence of cyanide (KCN) and salicylhydroxamic acid (SHAM). O₂ uptake by mature leaves measured at 25°C was stimulated by 1 millimolar KCN (63%) and also by 5 millimolar azide (79%). SHAM, an inhibitor of the alternative oxidase and a selection of other enzymes, also stimulated O₂ uptake by itself at concentration of 10 millimolar. However, in combination, KCN and SHAM were inhibitory. The rate of O₂ uptake declined consistently with leaf age. The stimulation of O₂ uptake by KCN and by SHAM occurred only after a certain stage of leaf development had been reached and was more pronounced in fully expanded leaves. In young leaves, O₂ uptake was inhibited by both KCN and SHAM individually. The uncoupler, p-trifluoromethoxy carbonylcyanide phenylhydrazone, stimulated leaf respiration at all ages studied, the stimulation being more pronounced in fully expanded leaves. The uncoupled rate was inhibited by KCN and SHAM individually. The capacity of the cytochrome path declined with leaf age, paralleling the decline in total respiration. However, the capacity of the alternative path peaked at about full leaf expansion, exceeding the cytochrome capacity and remaining relatively constant. These results are consistent with the presence in soybean leaves of an alternative path capacity that seems to increase with age, and they suggest that the stimulation of O₂ uptake by KCN and NaN₃ in mature leaves was mainly by the SHAM-sensitive alternative path. The stimulation of O₂ uptake by SHAM was not expected, and the reason for it is not clear.     

The acclimation of photosynthesis and respiration to temperature in the C3–C4 intermediate Salsola divaricata: induction of high respiratory CO2 release under low temperature

Photosynthesis in C₃–C₄ intermediates reduces carbon loss by photorespiration through refixing photorespired CO₂ within bundle sheath cells. This is beneficial under warm temperatures where rates of photorespiration are high; however, it is unknown how photosynthesis in C₃–C₄ plants acclimates to growth under cold conditions. Therefore, the cold tolerance of the C₃–C₄ Salsola divaricata was tested to determine whether it reverts to C₃ photosynthesis when grown under low temperatures. Plants were grown under cold (15/10 °C), moderate (25/18 °C) or hot (35/25 °C) day/night temperatures and analysed to determine how photosynthesis, respiration and C₃–C₄ features acclimate to these growth conditions. The CO₂ compensation point and net rates of CO₂ assimilation in cold‐grown plants changed dramatically when measured in response to temperature. However, this was not due to the loss of C₃–C₄ intermediacy, but rather to a large increase in mitochondrial respiration supported primarily by the non‐phosphorylating alternative oxidative pathway (AOP) and, to a lesser degree, the cytochrome oxidative pathway (COP). The increase in respiration and AOP capacity in cold‐grown plants likely protects against reactive oxygen species (ROS) in mitochondria and photodamage in chloroplasts by consuming excess reductant via the alternative mitochondrial respiratory electron transport chain.     

Respiration, cyanide-insensitive oxygen uptake and oxidative phosphorylation in cyanobacteria

Cyanide-insensitive oxygen uptake in the dark of 9 species of cyanobacteria was 6–20% of the total oxygen uptake of intact cells. In Phormidium , no cyanideinsensitive oxygen uptake was observed. In intact cells, the I₅₀ value for cyanide was significantly lower in cyanobacteria of the taxonomic sections I to III (1–9 μ M ) than in those from section IV and V (10–60 μ M ). Cyanide-insensitive oxygen uptake in the cell-free system of Anabaena variabilis was not affected by typical inhibitors of the alternative pathway of plants. Cell-free oxidation of cytochrome c was completely inhibited by cyanide with an I₅₀ value of 0.5–1 μ M . Electron transport of intact cells without cyanide present yielded P/O ratios of 0.7–3.0. The data on oxidative phosphorylation using intact cells and the cell-free system, indicate that cyanide-insensitive oxygen uptake is not coupled to ATP formation.     

The respiratory behavior of Lewis carcinoma cells--existence of a cyanide-resistant respiration

The respiratory O2 consumption of vegetative, non-necrotic, Lewis lung carcinoma cells was found to be very low compared with that of non-tumor tissues and was highly resistant to cyanide. However, the resistant rate was inhibited by salicylhydroxamic acid (SHAM) in either isolated cells or tissue fragments. In addition, this compound did not affect their cytochrome-c oxidase activity. The results support the existence of an alternative oxidase system that significantly contributes to oxygen uptake in Lewis carcinoma cells. To our knowledge, this is the first report showing significant SHAM-sensitivity of tumor respiration and perhaps of higher animal cell respiration.