Cyanide-resistant Respiration in Fresh and Aged Sweet Potato Slices

The respiration of fresh sweet potato (Ipomoea batatas) slices is resistant to, and often stimulated by, cyanide and antimycin A. m-Chlorobenzhydroxamic acid (CLAM), a selective inhibitor of the alternate path, inhibits respiration in the presence of cyanide and has a limited inhibitory effect in the presence of antimycin A. Thus, a partial bypass of the antimycinsensitive site is indicated. Respiration rises 2-fold at best with slice aging, the increment being cytochrome-mediated. The cyanide-resistant pathway contributes neither to coupled fresh slice respiration nor to the induced respiration in the absence of inhibitors of the cytochrome path. In the presence of uncoupler, however, the alternate path is engaged both in fresh and aged slices. V_cyt, the maximal capacity of the cytochrome path, remains essentially the same with slice aging, whereas V_alt decreases from 20 to 60 per cent. The induced respiration is readily accommodated by the potential cytochrome path capacity of fresh slices, which is realized on aging. Accordingly, there is no need to invoke mitochondrial proliferation in explanation of the development of the induced respiration. The engagement of the alternate path in response to uncoupler reflects substrate mobilization to a degree that substrate oxidation exceeds the electron transport capacity of the cytochrome path.

Fresh slices do not utilize exogenous substrates, whereas aged slices do so readily. Cerulenin, a specific inhibitor of fatty acid synthesis, prevents the development of the induced respiration as well as the capacity to oxidize exogenous substrates. It is suggested that lipid, and ultimately membrane, biosynthesis is central to the development of the induced respiration and the ability to use exogenous substrates, much as in potato.

     

Respiratory Contribution of the Alternate Path during Various Stages of Ripening in Avocado and Banana Fruits

The respiration of fresh slices of preclimacteric avocado (Persea americana Mill. var. Hass) and banana (Musa cavendishii var. Valery) fruits is stimulated by cyanide and antimycin. The respiration is sensitive to m-chlorobenzhydroxamic acid in the presence of cyanide but much less so in the presence of antimycin. In the absence of cyanide the contribution of the cyanide-resistant pathway to the coupled preclimacteric respiration is zero. In uncoupled slices, by contrast, the alternate path is engaged and utilized fully in avocado, and extensively in banana. Midclimacteric and peak climacteric slices are also cyanide-resistant and, in the presence of cyanide, sensitive to m-chlorobenzhydroxamic acid. In the absence of uncoupler there is no contribution by the alternate path in either tissue. In uncoupled midclimacteric avocado slices the alternate path is fully engaged. Midclimacteric banana slices, however, do not respond to uncouplers, and the alternate path is not engaged. Avocado and banana slices at the climacteric peak neither respond to uncouplers nor utilize the alternate path in the presence or absence of uncoupler.

The maximal capacities of the cytochrome and alternate paths, V_cyt and V_alt, respectively, have been estimated in slices from preclimacteric and climacteric avocado fruit and found to remain unchanged. The total respiratory capacity in preclimacteric and climacteric slices exceeds the respiratory rise which attends fruit ripening. In banana V_alt decreases slightly with ripening.

The aging of thin preclimacteric avocado slices in moist air results in ripening with an accompanying climacteric rise. In this case the alternate path is fully engaged at the climacteric peak, and the respiration represents the total potential respiratory capacity present in preclimacteric tissue. The respiratory climacteric in intact avocado and banana fruits is cytochrome path-mediated, whereas the respiratory climacteric of ripened thin avocado slices comprises the alternate as well as the cytochrome path. The ripening of intact fruits is seemingly independent of the nature of the electron transport path.

Uncouplers are thought to stimulate glycolysis to the point where the glycolytic flux exceeds the oxidative capacity of the cytochrome path, with the result that the alternate path is engaged.

     

Antimycin-insensitive Cytochrome-mediated Respiration in Fresh and Aged Potato Slices

The effect of antimycin A on the respiration of fresh potato (Solanum tuberosum var. Russet Burbank) slices has been determined in the presence and absence of m-chlorobenzhydroxamic acid (CLAM). Two antimycin-binding sites are indicated. At low concentrations antimycin alone inhibits respiration only slightly. When CLAM and low antimycin are added together, respiration is sharply inhibited, as in response to cyanide. High antimycin alone is as inhibitory as cyanide. The branch point to the alternate path is intact in fresh slices, as is the hydroxamate-sensitive component. The full alternate path is inoperative, however, as indicated by the sensitivity to cyanide. The data suggest an alternate path loop which bypasses the high affinity antimycin site and returns electrons to the cytochrome path. Antimycin at high concentrations prevents articulation of the loop with the cytochrome path.

The respiration of aged slices is not only markedly resistant to antimycin at high concentrations, but quite insensitive to CLAM in the presence of antimycin. A model is proposed which involves parallel paths within complex III of the cytochrome path, with one path bearing the high affinity, and the other the low affinity antimycin site. With slice aging the antimycin affinity of the latter site is even further reduced, providing a relatively antimycin-insensitive bypass to both the high affinity antimycin-sensitive cytochrome path, and the CLAM-sensitive alternate path. The alternate path loop in fresh slices is presumed to feed into the low affinity antimycin-sensitive arm of the cytochrome path.

     

Alternative path mediated chloride absorption in cyanide-resistant tissues

The ability of two cyanide-resistant tissues—aged potato slices and fresh preclimacteric banana slices—to take up chloride in the presence of cyanide has been established. Extensive inhibition of chloride uptake by cyanide and chlorbenzhydroxamate together indicates that chloride absorption in the presence of cyanide is supported by respiration mediated by the alternative path. The partial inhibition of respiration-dependent chloride uptake by chlorbenzhydroxamate alone is independent of its effect on the alternative oxidase, and points to inhibition of the transport process per se.     

Studies on the Wound-Induced CN-Resistant Respiration in Potato Tuber Slices

The respiration of fresh potato slices was sensitive to CN-, and was not inhibited by m-CLAM in the presence or absence of CN-. By contrast, the wound-induced respiration of slices, incubated in air for 24 hours, was not only relatively resistant to CN-, but also markedly inhibited by m-CLAM in the presence or absence of CN-. When m-CLAM and CN- were added togather, the inhibitory effect was higher than that of them when used separately. The observations indicated that the alternate path is operative in aged potato slices. The data determined by the method of m-CLAM titration showed that the actual contribution of alternate path and cytochrome path in aged potato slices was approximately 28% and 54% of the total respiration respectively in the absence of CN-. When the cytochrome path was inhibited by CN-, the maximal capacity of alternate path (Valt was higher than the actual contribution of them (ρ·Valt). The increased contribution of alternate path in presence of CN- might be thought to indicate that there is a diversion of electron flux from the cytochrome path to the alternate path. When the respiratory flux of aged slices was reduced by treatment with iodoacetate and malonate, the proportions of respiration inhibited by CN- and m-CLAM respectively were not changed.     

Development of cyanide-resistant respiration in mitochondria from potato tubers treated with ethanol,acetaldehyde, and acetic acid

Treatment of intact potato (Solanum tuberosum L.) tubers with acetaldehyde, ethanol or acetic-acid vapors led to a respiratory upsurge which was further increased when the volatiles were applied in 100% O₂. Mitochondria from tubers held in 100% O₂ (O₂ control) displayed a substrate state, state 3, and state 4 in respiration, whereas in mitochondria from the volatile-treated tubers the respiratory rate of the different states was virtually indistinguishable. This respiratory pattern was companied by the development of a cyanide-resistant respiration since these mitochondria exhibited resistance to CN and sensitivity to CN+salicylhydroxamic acid. Acetaldehyde-treated potatoes showed a time-course development (up to 36 h) of cyanide resistance and concomitant sensitivity to salicylhydroxamic acid, indicating the onset of synthetic processes leading to the observed changes in mitochondrial respiration.Abbreviations V total respiration rate - V_cyt velocity of O₂ uptake attributable to cytochrome oxidase - V_alt velocity of O₂ uptake attributable to the alternate oxidase - RCR respiratory control ratio - SHAM salicylhydroxamic acid Paper of the Journal Series, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick, N.J., USA     

Membrane Lipid Breakdown in Relation to the Wound-induced and Cyanide-resistant Respiration in Tissue Slices: A COMPARATIVE STUDY

A study of a variety of bulky storage organs and fruits reveals that fresh slices fall into two categories with respect to their sensitivity to CN. Fresh slices in the first class are CN-sensitive, whereas slices of the second class are resistant to, and often stimulated by, CN. In tissue slices which are initially CN-sensitive, cutting initiates a burst of lipolytic activity. In CN-resistant fresh slices, there is no measurable lipid breakdown.Slicing evokes the wound-respiration which is 5- to 10-fold that of the parent organ. Slice aging, in turn, evokes a further 2- to 3-fold respiratory increase, the wound-induced respiration, whether fresh slice respiration is CN-sensitive or -resistant. Estimation of the contribution by the cytochrome and alternative paths shows that the wound respiration in both groups is mediated by the cytochrome path. On the other hand, the wound-induced respiration in the first class is cytochrome path mediated, whereas, in some members of the second group, both pathways are utilized. Uncouplers of oxidative phosphorylation elicit a CN-sensitive increment in fresh slices as great or greater than the wound-induced respiration. Accordingly, de novo synthesis of mitochondria is ruled out as an explanation of the latter.The integrity of endomembranes, perhaps including mitochondrial membranes, is seemingly a prerequisite for the operation of the alternative path, that is, alternative path activity is lost concomitantly with membrane lipid breakdown. The development of the wound-induced respiration is not co-extensive with the development of the CN-resistant path in all tissue slices. The fundamental process of aging appears to involve activation of pre-existing respiratory capacity.Fresh slices from whatever source fail to utilize exogenous (14)C-labeled glucose, whereas aged slices do so readily. A transport lesion is indicated, the healing of which does not depend on the development of the wound-induced respiration but does depend on fatty acid, and presumably membrane lipid, biosynthesis.     

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.     

Inhibition of the Development of Induced Respiration and Cyanide-insensitive Respiration in Potato Tuber Slices by Cerulenin and Dimethylaminoethanol

The interdependence of the development of wound-induced respiration and membrane-related phospholipid biosynthesis in potato tuber (Solanum tuberosum var. Russet) slices was established by the use of agents which selectively affect lipid and phospholipid synthesis. Cerulenin, a specific inhibitor of de novo fatty acid synthesis, inhibited the ultimate development of wound-induced respiration and of cyanide resistance only when given in the critical first 10 to 12 hours of slice aging. Similarly, when slices were exposed to the choline analogue dimethylaminoethanol within the first 10 hours, the phospholipid composition of the membrane lipids was drastically altered, the wound-induced respiration in a 24-hr period was substantially curtailed, and the development of cyanide insensitivity was sharply inhibited. These observations indicate that time-restricted membrane-related phospholipid synthesis is prerequisite to the development of wound-induced respiration and concurrent cyanide insensitivity.     

Cyanide-resistant Respiration in Freshly Cut Potato Slices

Treating intact white potato (Solanum tuberosum L.) tuber with ethylene in air or O₂ made it possible to obtain freshly cut slices which exhibit cyanide-resistant respiration. The cyanide-resistant path requires induction in whole tubers. The data also indicate that high O₂ concentration is necessary for the full development of cyanide-resistant respiration.     

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.     

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.     

Appearance of an Alternate Pathway Cyanide-resistant during Germination of Seeds of Cicer arietinum

The combined action of the inhibitors antimycin A and cyanide with benzohydroxamic acid indicates the presence of a cyanide-resistant pathway of respiration in chick pea (Cicer arietinum L.) seeds. The appearance of this pathway takes place during germination. During the first 12 hours of germination, the respiration is predominantly cyanide-sensitive, showing after this time a shift to an “alternate” respiration which is sensitive to benzohydroxamic acid, reaching the maximal cyanide resistance between 72 and 96 hours of germination. The appearance of the alternate pathway is initiated by high O₂ concentrations and depends on cytoplasmic protein synthesis, since its appearance is inhibited by cycloheximide but not by chloramphenicol. Actinomycin D has no effect on the appearance of the alternate pathway. Our results indicate, in agreement with other authors, that the branching point is located between the flavoproteins and cytochromes b, probably at the level of ubiquinone, but the possibility of more than one branching point of the electron flow is also considered.     

A comparison of thermo- and skotodormancy in seeds of Lactuca serriola in terms of induction,alleviation, respiration,ethylene and protein synthesis

Lactuca serriola L., which was found to enter skotodormancy at a relatively low temperature, was used to try and ascertain differences in thermo- and skotodormancy.Abbreviations SE standard error - V_T total respiratory rate - V_cyt respiratory rate inhibited by cyanide - V_alt total respiratory capacity inhibited by SHAM and denoted alternate respiration - V_res residual respiration - PMSF phenylmethylsulfonyl fluoride; panacide, 5,5-2,2-dihydroxydiphenylmethane - SHAM salicyl-hydroxamic acid     

Ethylene and wound-induced cyanide-resistant respiration climaxes in potato: The synergistic role of CO2 and the selective role of lycorine

When treated with ethylene in O₂, conditioned potato (Solanum tuberosum L. cv. Russet Burbank) tubers – that is, tubers kept at room temperature for 10 days or more – yield slices that are CN^? resistant. Ten % CO₂ in the gas mixture not only synergizes the effect of ethylene, but replaces the need for conditioning as well. The response to CO₂ is more pronounced with increasing time from harvest. By contrast fresh slices from untreated tubers are CN^? sensitive, as are slices from tubers incubated in O₂ or O₂ plus CO₂. The suggestion is made that CN^? resistance is constitutive, and that treatment with ethylene/CO₂ in O₂ confers on potato tuber tissue a resistance to the extensive degradation of membrane phospholipids that normally attends slicing and leads to the loss of CN^? resistance. In this connection respiration inhibition by imidazole, an inhibitor of fatty acid α-oxidation, is extensive in slices of untreated tubers, and sharply diminished in slices of ethylene-treated tubers in proportion to their CN^? resistance. The coextensive rise of respiration rate and CN^? resistance in aged potato slices has led to the presumption that the CN^?-resistant path mediates the respiration climax. Accordingly the alkaloid, lycorine, has been considered to inhibit the development of CN^? resistance in aging potato slices because it curtails the wound-induced respiration. A comparison was carried out on the effect of lycorine on CN^?-sensitive and CN^?-resistant fresh slices – the latter obtained from ethylene/CO₂-treated tubers. Lycorine suppressed the development of the wound-induced respiration without restricting the development of CN^? resistance.     

Factors Influencing the Development of Cyanide-resistant Respiration in Potato Tissue

Ethylene, cyanide gas, and volatalized ethanol, acetaldehyde, and acetic acid were applied in a continuous flow to whole potato tubers. Freshly cut slices were obtained periodically during the treatment, and showed a progressive development of a cyanide-resistant respiration. The application of the employed volatiles in 100% O₂ accelerated the onset and the magnitude of the cyanide-resistant respiration.     

Cyanide-and rotenone-resistant respiration in mitochondria of sugar beet taproots during plant growth and development

Effects of cyanide and rotenone were examined on respiration (oxygen uptake) in mitochondria isolated from sugar beet (Beta vulgaris L.) taproots at various stages of plant growth and development. In mitochondria from growing and cool-stored taproots, the ability of cyanide-resistant, salicylhydroxamic acid-sensitive alternative oxidase (AO) to oxidize malate, succinate, and other substrates of tricarboxylic acid cycle (TCA) was low and constituted less than 10% compared to predominant activity of the cytochrome oxidase pathway during State 3 respiration. Artificial aging of storage tissue (2-day incubation of tissue sections under high humidity at 20°C) substantially activated AO, but the highest capacity (V _alt) of this pathway of mitochondrial oxidation was only observed in the presence of pyruvate and a reducing agent dithiothreitol. At the same time, mitochondria from growing taproots exhibited high rates of rotenone-resistant respiration, and these rates gradually declined during plant growth and development. The slowest rates of this respiration were observed during oxidation of NAD-dependent TCA substrates in mitochondria from dormant storage organ. The results are discussed in relation to significance of alternative electron transport pathways during growth and storage of sugar beet taproots.     

Similarities between the Actions of Ethylene and Cyanide in Initiating the Climacteric and Ripening of Avocados

A continuous exposure of intact avocados (Persea americana) to 400 μl/l of cyanide results in a rapid increase in the rate of respiration, followed by a rise in ethylene production, and eventual ripening. The pattern of changes in the glycolytic intermediates glucose 6-phosphate, fructose diphosphate, 3-phosphoglyceric acid, and phosphoenolpyruvate during the rapid rise in respiration in both ethylene and cyanide-treated fruits is similar to that found in fruits made anaerobic where a 2.3- to 3-fold increase in the rate of glycolysis is observed. It is suggested that both during the climacteric and in response to cyanide, glycolysis is enhanced. It is proposed that cyanide implements the diversion of electrons to the cyanide-resistant electron path through structural alterations which are independent of the simultaneous inhibition of cytochrome oxidase.     

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.