Dependence of Wound-induced Respiration in Potato Slices on the Time-restricted Actinomycin-sensitive Biosynthesis of Phospholipid

Actinomycin D prevents the full development in a 24-hour period of both wound respiration and cyanide resistance only when given in the first 10 to 12 hours following the cutting of potato tuber (Solanum tuberosum var. Russet) slices. The capacity for choline incorporation into phosphatidylcholine increases with slice aging and is inhibited by actinomycin D in the same time-restricted way. The time-restricted effectiveness of actinomycin D applies to the cutting-elicited enhanced synthesis of three critical enzymes of phosphatidylcholine synthesis, namely phosphorylcholine-glyceride transferase, phosphorylcholine-cytidyl transferase, and phosphatidylphosphatase. By contrast, actinomycim D given at any time is without effect on the measurable levels after 24 hours of a selection of glycolytic and mitochondrial respiratory enzymes. Neither succinic dehydrogenase nor cytochrome oxidase activity increases with time in aging potato slices in the presence or absence of chloramphenicol. The foregoing observations emphasize the central role of phospholipid, and ultimately membrane biosynthesis, in the development of wound-induced respiration.     

Effect of Ethylene and Oxygen on the Development of Cyanide-resistant Respiration in Whole Plant Mitochondria

Mitochondria from whole potatoes (Solanum tuberosum) ordinarily fail to oxidize respiratory substrates and to consume molecular O₂ in the presence of cyanide. Mitochondrial preparations obtained from tubers previously held for 24 hours in ethylene (10 microliters per liter) in air are only partially inhibited by cyanide. Application of ethylene in 100% O₂ led to an additional increase in the resistance of the mitochondrial respiration to cyanide. The resistance to cyanide was accompanied by a decrease in the respiratory control but no change in oxidative phosphorylation as shown by the measurement of ATP synthesis.     

Relative Contribution of Cytochrome-mediated and Cyanide-resistant Electron Transport in Fresh and Aged Potato Slices

The respiration of fresh potato (Solanum tuberosum, var. Russet Burbank) slices is predominantly cyanide-sensitive whether in the presence or absence of uncoupler. By contrast, the wound-induced respiration which develops in thin slices with aging is cyanide-resistant, and in the presence of cyanide, sensitive to chlorobenzhydroxamic acid, a selective inhibitor of the cyanide-resistant respiration. Titration of the alternate path in coupled slices with chlorobenzhydroxamic acid, in the presence and absence of cyanide, shows that the contribution of the cyanide-resistant pathway to the wound-induced respiration is zero. Similar titrations with uncoupled slices reveal that the alternate path is engaged and utilized extensively.

The maximal capacity of the cytochrome path (V_cyt) has been estimated in fresh and aged slices in the presence of the uncoupler carbonyl-cyanide m-chlorophenyl hydrazone. It has been found that V_cyt of aged slices is but 30 to 40% higher than that of fresh slices. The results suggest that the bulk of the wound-induced respiration is mediated through the cytochrome pathway which exists in fresh slices in suppressed form, and which is fully expressed by slice aging. The engagement of the alternate path by uncouplers in aged slices is attributed to an increase in substrate mobilization, with the result that the electron transport capacity of the cytochrome chain is exceeded.

     

Appearance and Disappearance of Cyanide-Resistant Respiration in Vigna mungo Cotyledons during and following Germination of the Axis

Mitochondrial preparations isolated from black gram (Vigna mungo L.) cotyledons exhibited cyanide-resistant respiration which was of mitochondrial origin. The appearance and the disappearance of this alternative respiration took place during and following imbibition. During the first 6 hours of imbibition, the respiration was completely inhibited by cyanide, but after this time the alternative respiration markedly developed, reaching a maximal cyanide-resistance 12 to 16 hours after the start of imbibition. Subsequently, the alternative respiration gradually disappeared. The actions of cycloheximide and chloramphenicol indicated that the appearance was dependent on cytoplasmic protein synthesis and that the disappearance depended on both cytoplasmic and mitochondrial protein synthesis. The alternative pathway contributed to state 4 respiration, but not to state 3 respiration, in mitochondria from 1-day-old cotyledons. On day 3, it contributed to neither state 3 nor state 4.     

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.

     

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.     

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.

     

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.     

Cyanide-insensitive Respiration in Pea Cotyledons

Mitochondria isolated by a zonal procedure from the cotyledons of germinating peas possessed a cyanide-resistant respiration. This respiration was virtually absent in mitochondria isolated during the first 24 hours of germination but thereafter increased gradually until the 6th or 7th day of seedling development. At this time between 15 and 20% of the succinate oxidation was not inhibited by cyanide. The activity of the cyanide-resistant respiration was also determined in the absence of cyanide. Relationships among mitochondrial structure, cyanide-resistant respiration, and seedling development are discussed.     

Some Specific Features of the Respiration of Hydrocarbon-utilizable Candida Yeasts

The respiration of both glucose-grown and hydrocarbon-grown cells of Candida tropicalis pK 233 harvested in the stationary phases was not inhibited by cyanide when glucose was used as oxidation substrate, but the former was rather stimulated in the presence of cyanide. When n-alkanes were used as oxidation substrate, cyanide lowered the respiratory activities of both cells to about 50%. With respect to the susceptibility to cyanide, the younger cells growing on n-alkanes were less sensitive in hydrocarbon oxidizing ability than the older cells, whereas the older cells growing on glucose or n-alkanes were more resistant in glucose oxidizing ability than the younger cells. Acetate was oxidized by both glucose-grown and hydrocarbon-grown cells of the yeast. Laurate was oxidized by hydrocarbon-grown cells, but not by glucose-grown cells. The respiration on laurate was inhibited completely by 3.3 mM of cyanide. In general, hydrocarbon-grown cells of Candida tropicalis pK 233 were more sensitive to various respiratory inhibitors than glucose-grown cells, although the oxidation substrates had a significant effect.

The respiration of both glucose-grown and hydrocarbon-grown cells of C. albicans, C. guilliermondii and C. lipolytica harvested in the stationary phases was also resistant to cyanide when glucose was used as oxidation substrate. But the respiration on n-alkanes of these cells was inhibited significantly by 3.3 mM of cyanide except for C. albicans.     

The Relation between Photosynthesis, Respiration, and Crassulacean Acid Metabolism in Leaf Slices of Aloe arborescens Mill

Leaves and leaf slices from Aloe arborescens Mill. were used to study the interrelations between Crassulacean acid metabolism, photosynthesis, and respiration. Oxygen exchange of leaf slices was measured polarographically. It was found that the photosynthetic utilization of stored malic acid resulted in a net evolution of oxygen. This oxygen production, and the decrease in acid content of the leaf tissue, were completely inhibited by amytal, although the rate of respiratory oxygen uptake was hardly affected by the presence of this inhibitor of mitochondrial electron transport. Other poisons of respiration (cyanide) and of the tricarboxylic acid cycle (trifluoroacetate, 2-diethyl malonate) also were effective in preventing acid-dependent oxygen evolution. It is concluded that the mobilization of stored acids during light-dependent deacidification of the leaves depends on the operation of the tricarboxylic acid cycle and of the electron transport of the mitochondria.     

Metabolism of Red Beet Slices II. Effects of Aging

The rate of respiration of red beet slices increased 3- to 4-fold when the slices were aged in a moist atmosphere for 18 to 24 hours. The respiration of fresh slices was severely inhibited by 8 × 10⁻⁵ m HCN but as slices aged the sensitivity of respiration to HCN fell rapidly. The presence of HCN stimulated the respiration of slices after 8 to 12 hours of aging.     

Respiration of Gametangia of the Aquatic Phycomycete Allomyces macrogynus: Inhibition by Cyanide or Antimycin and Salicylhydroxamic Acid or Propyl Gallate

Gametangia of the aquatic phycomycete Allomyces macrogynus have a cyanide- and antimycin A-insensitive respiration, which is sensitive to salicylhydroxamic acid (alternative respiration). Propyl gallate is also an inhibitor of this alternative pathway, and propyl gallate is more efficient than hydroxamic acid. Gametangial respiration is insensitive to propyl gallate, but propyl gallate sensitivity is gradually established when the gametangia are titrated with cyanide. Carbonyl cyanide m-chlorophenyl hydrazone stimulates the cyanide-sensitive respiration and engages the alternative sensitive respiration. Sodium azide inhibits both the alternative and the cyanide-sensitive respiration, but the cyanide-sensitive respiration is inhibited 10 times more efficiently than the alternative respiration. Rotenone inhibits the total respiration and the propyl gallate-insensitive respiration by 33% and the cyanide-insensitive respiration by 43%.

The kinetic results reported here are discussed with respect to the models of de Troostembergh and Nyns (1977 Arch Int Physiol Biochem 85:404-406; 1978 Eur J Biochem 53:423-432) and of Bahr and Bonner (1973 J Biol Chem 248:3446-3450) for the partitioning of electrons between cyanide-insensitive and propyl gallate-insensitive respiration. The results reported here do not agree with the model of de Troostembergh and Nyns.

     

Inhibition of Respiration in Prototheca zopfii by Light

Irradiation of cells of Prototheca zopfii with blue light inhibited the respiratory capacity of the cells. The inhibition of respiration was correlated with a photodestruction of cytochrome c(551), cytochrome b(559), and cytochrome a₃. Cytochrome c(549), cytochrome b(555), and cytochrome b(564) were unaffected by the irradiation treatment. The α-band of reduced cytochrome a was shifted from 599 to 603 nm by irradiation, an effect similar to that observed when methanol was added to nonirradiated cells. The presence of oxygen was required during irradiation for both photoinhibition of respiration and photodestruction of the cytochromes. Cytochrome a₃ was protected against photodestruction by cyanide. Photodestruction of these same cytochromes also occurred when washed mitochondria of P. zopfii were irradiated.     

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.     

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.     

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.     

Respiration and cyanogenesis inPseudomonas aeruginosa

The respiratory ability of batch cultures ofPseudomonas aeruginosa strain 9-D2 peaks during midlog phase at 3.8 nmol O₂/min/10⁸ cells. This ability declines in late log phase, just prior to the time the culture begins to produce cyanide. The respiration of this organism is particularly sensitive to cyanide inhibition during midlog-phase growth, but is extremely resistant to this compound in stationary phase. These inhibition patterns are biphasic for each of these situations and indicate several respiratory responses to HCN. Addition of cyanide to midlog-phase cells resulted in the production of a stationary-phase type of cyanide respiration pattern in 2 h. A non-cyanideproducing mutant of this organism produced significantly less of the cyanide-resistant respiration components.     

The effect of tonicity and metabolic inhibitors on respiration and ripening of avocado fruit slices

The ripening of avocado (Persea americana Mill.) fruit slices was inhibited whether they were floated in water or in buffered aqueous 0.3 m mannitol, 0.25 m KCl, and sucrose. There was no evidence to support the contention that ripening occurred when the tonicity of the bathing medium was increased. Decreased gaseous exchange is considered to be a major cause of this inhibition because by utilizing a technique that afforded better aeration, slices could be water infiltrated and still ripen normally. Metabolic studies on the ripening of slices using this method indicated that several metabolic inhibitors, malonate, cyanide, acetaldehyde, dinitrophenol, and fluoride did not prevent ripening, but that their effect on the respiration pattern was marked. This technique provides a suitable way to study control of ripening at the tissue level.     

Studies on the effect of certain enzymic poisons on the metabolism of storage organs IV. Respiration and nitrogen assimilation by radish root slices in relation to feeding with KCN

At low and high concentrations, respectively, KCN stimulatedand inhibited the CO₂ output of radish root slices. Analysisof tissue slices showed that CO₂ production is nearly accountedfor by the loss in carbohydrates. After 48 hr, the content ofprotein-N was slightly changed in all the cyanide-treated tissues;whereas, that of soluble-N decreased markedly at high cyanideconcentrations and hardly changed at low concentrations. Leakageof soluble-N occurred at high concentrations of cyanide. The nitrogen compounds taken up were incorporated into the nitrogenpool of radish slices. KCN retarded uptake of nitrogen compounds,inhibited their utilization and led to excessive leakage ofsoluble-N from the tissues into the media. Increased CO₂ outputof slices in nitrogen media was largely accounted for by theloss in carbohydrates from the tissues. High concentrations of cyanide are thought to inhibit CO₂ outputthrough inhibition of the oxidase system and accompanying phosphorylations.The present results with low concentrations further substantiatethe uncoupling theory as a main factor involved in respiration. ¹Permanent address: Department of Botany, Faculty of Science,University of A'in Shams, Abbassia, Cairo, Egypt, U.A.R. (Received October 3, 1969; )