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.

     

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.     

丙酮酸对陈化马铃薯块茎切片线粒体抗氰呼吸的激活作用

用从陈化马铃薯切片纯化的线粒体进行实验发现：丙酮酸对总呼吸只有微弱的刺激作用,但可明显激活抗氰呼吸,并显著增强抗氰呼吸对总呼吸的贡献;丙酮酸对抗氰呼吸的激活作用可通过洗涤线粒体除去,重新加入丙酮酸又对抗氰呼吸产生激活作用;丙酮酸对抗氰呼吸的半最大激活浓度约为1．0mmol／L。上述结果表明丙酮酸对植物线粒体抗氰呼吸的激活作用可能具有普遍性。     

Cyanide-resistant Respiration of Sweet Potato Mitochondria

The oxidation of malate and succinate by sweet potato mitochondria (Ipomoea batatas [L.] Lam.) was blocked only partly by inhibitors of complexes III (2-heptyl-4-hydroxyquinoline-N-oxide) and IV (cyanide and azide). The respiration insensitive to inhibitors of complexes III and IV was inhibited by salicylhydroxamic acid. Essentially complete inhibition was obtained with inhibitors of complex I (rotenone, amytal, and thenoyltrifluoroacetone) and complex II (thenoyltrifluoroacetone). The observations indicated that electrons were transferred to the cyanide-resistant pathway from ubiquinone or from nonheme iron (iron-sulfur) proteins of complexes I and II before reaching the b cytochromes. In contrast, the oxidation of exogenous NADH did not involve the alternate pathway, as indicated by complete inhibition by inhibitors of complexes III and IV and the absence of an effect of inhibitors of complexes I and II. Hence, electrons from exogenous NADH appear to pass directly to complex III in sweet potato mitochondria.     

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.

     

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.     

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.     

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 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.     

铵态氮和硝态氮营养与大豆幼苗的抗氰呼吸作用

在植物体内,NO云还原成NHI是一个耗能很多的生化过程,每还原一个NO。大约要消耗15个ATP分子（Salsac等1987）。植物直接吸收和利用NHi＋,可以减少能量消耗,所节约的能量可用于植物的生长。因此,从理论上讲,供NHI的植株要比供NOt的植株能够获得更高的生物产量。然而,对于大多数植物,供NOS的植株常具有更大的生长量和产量。对于这种现象,过去人们一直在矿物质和有机物积累的差异（Robin和Salsac1985）、根际的酸化程度（Ruftv等1983,Tolley－Henry和Rapen1986）、NHt有害浓度的积累（Haynes和Goh1978）、对光合作用的…     

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.     

Cyanide-sensitive and Cyanide-resistant Respiration in the Germination of Cocklebur Seeds

Interrelation between the CN-sensitive cytochrome path and the CN-resistant, benzohydroxamic acid (BHM)-sensitive, or n-propylgallate (nPG)-sensitive alternative path in seed respiration during germination was examined using the nondormant upper and lower seeds of Xanthium pensylvanicum Wallr. The operation of both paths was required not only for normal germination of the lower seed but also for KCN- or NaN₃-induced germination of both. From the sensitivity to BHM of the germination response, it became obvious that the alternative path exerts its physiological activity as soon as it develops during the early period of water imbibition. Pretreatments with KCN and NaN₃ for promoting germination, strikingly decreased only the engagement of the cytochrome path in the subsequent respiration without affecting that of the alternative path. Nevertheless, no germination occurred without the operation of the cytochrome path. This suggested that excess operation of the cytochrome path is detrimental to germination, being maximal following the BHM-sensitive phase.     

Effects of Controlled Atmospheres on Production of Sesquiterpenoid Stress Metabolites by White Potato Tuber: Possible Involvement of Cyanide-resistant Respiration

Levels of katahdinone (solavetivone), lubimin, rishitin, and phytuberin, sesquiterpenoid stress metabolites of white potato (Solanum tuberosum), were monitored in tuber slices which were challenged with an extract of Phytophthora infestans and incubated under controlled atmospheres. A mixture of ethylene in air enhanced stress metabolite production. This enhancement was amplified by higher partial pressures of oxygen. Stress metabolite production was inhibited by salicylhydroxamic acid. These results suggest the involvement of cyanide-resistant respiration in the production of potato stress metabolites, compounds which may serve as phytoalexins.     

Suppression by Exogenous Phospholipid of Cyanide-Insensitive Respiration of Submitochondrial Particles from Sweet Potato Root Tissue

Submitochondrial particles from sweet potato root tissue retainedthe respiratory characteristics of the intact mitochondria withrespect to the sensitivity to cyanide and salicylhydroxamicacid. The activities of total, cyanide-insensitive, and salicylhydroxamate-sensitiverespiration of the submitochondrial particles yielded from adefinite weight of tissue slices incubated under aerobic conditions,particularly in ethylenecontaining air, were higher than thosefrom the same weight of intact tissue. The less phospholipidthe submitochondrial particles contained relative to protein,the higher the activities of cyanide-insensitive and salicylhydroxamate-sensitiverespiration tended to be relative to total respiratory activity.When the submitochondrial particles were incubated with phospholipidliposomes, the activities of cyanide-insensitive and salicylhydroxamate-sensitive,but not cyanide-sensitive, respiration became extremely low.All phospholipids showed this effect. Such incubation of thesubmitochondrial particles with phospholipid liposomes yieldedlighter particles, indicating close association of exogenouslyadded phospholipid with the particles. Phospholipid moleculesseemed to enter the membrane of the particles. We propose thatphospholipid deficiency in the mitochondrial inner membranefacilitates operation of the cyanide-insensitive electron transportpath. (Received March 30, 1984; Accepted June 15, 1984)     

Dark Respiration during Photosynthesis in Wheat Leaf Slices

The metabolism of [¹⁴C]succinate and acetate was examined in leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthetically active radiation). In the dark [1,4-¹⁴C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO₂. An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after ¹⁴CO₂ production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of ¹⁴CO₂ over 2 hours indicates that, in the dark, the tricarboxylic acid cycle was the major route for succinate metabolism with CO₂ as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate and large amounts of the label entered the organic and amino acids. While carbon dioxide contained much less radioactivity than in the dark, other products such as sugars, starch, glycerate, glycine, and serine were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in addition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylic acid cycle operated in the light and that CO₂ was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the ¹⁴CO₂ release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark.