Cyanide-insensitive Respiration in Plant Mitochondria

A continuous exposure of intact avocados (Persea americana) to 400 ul/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.The sharp rise in respiration, the climacteric, which attends the ripening of certain fruits, has been attributed alternatively to a decrease in the "organization resistance" of the cell (11,27) and to an increase in protein synthesis, particularly to the synthesis of one or more enzymes putatively involved in the ripening process (1,17,36). Ethylene is considered to be the agent that triggers the climacteric and fruit ripening (34). It is questionable whether protein synthesis constitutes a requisite for the climacteric rise, because the stimulation of glycolysis in preclimacteric fruits by anoxia (11), the acceleration of respiration in preclimacteric slices by uncouplers of oxidative phosphorylation (33), the demonstration of full respiratory capacity of isolated mitochondria from preclimacteric tissue (29), and the development of the climacteric under conditions which may be thought to preclude synthesis of protein (18,31,42) these observations it has been suggested that glycolysis is accelerated in the course of the climacteric rise in respiration (6,15,40). However, activation of glycolysis per se cannot be the cause of ripening or even of the climacteric, because anaerobiosis, while enhancing glycolysis in avocados (see below) and apples (11), prevents ripening, and uncouplers of oxidative phosphorylation, which enhance respiration and presumably glycolysis in banana slices, neither accelerate ripening nor inhibit it when ripening is induced by ethylene or occurs naturally (31,42). Furthermore, the postulated augmentation of glycolysis during the climacteric cannot be attributed to uncoupling of oxidative phosphorylation, for both the rates of 32P esterification and the levels of ATP increase during the climacteric in a variety of fruits (38,43). Because enhancement of glycolysis appears to attend ripening under conditions which normally call for its diminution, i.e. where there is an elevation in energy charge (2, 43), and because the fruit-ripening hormone, ethylene, enhances glycolysis in yeast (41), we undertook to study, in addition to the effect of ethylene on glycolysis, the effect of cyanide on both glycolysis and ripening; because on the o...

Section: Resultsmentioning

confidence: 99%

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

Solomos¹,

Laties²

1974

“…In higher plants, the cyanide-insensitive pathway is not phosphorylating by itself but is branched after the site I of phosphorylation (2 (8), FCCP acts unspecifically on membranes. It may affect not only energyproviding but also energy-consuming reactions at membranes.…”

Section: Discussionmentioning

confidence: 99%

Oxidative Phosphorylation in Germinating Lettuce Seeds (Lactuca sativa) during the First Hours of Imbibition

Hourmant¹,

Pradet²

1981

Experiments with lettuce seeds during the first hours of imbibition showed that oxygen is necessary to sustain high adenine nucleotide ratios and consequently, energy charge values are higher than 0.8 as is usually the case in normally metabolizing tissues. The origin of the energy-rich bonds needed for the biosynthetic work done by germinating seeds during the first min and h after imbibition is a question for debate (7,9,11,15). Some seeds have active fermentative metabolism even in air but 02 is always necessary to permit normal germination. In other seeds, such as lettuce seeds, ATP production from fermentation is very low (17 and unpublished results).In the last 15 years, many improvements in the techniques for extracting mitochondria have permitted researchers to obtain from many plant tissues particles exhibiting very high respiratory control and P/O ratios close to the generally accepted theoretical values. To date, mitochondria extracted from dry or imbibed seeds before the protrusion of the radicle are loosely coupled and do not exhibit respiratory control (27). In germinating seeds, as well as in many plant tissues, a significant percentage of the 02 uptake has been reported to be cyanide-insensitive (11). Many authors have claimed that cyanide is unable to inhibit the germination of seeds (10, 25) and sometimes a stimulatory effect was reported (23).These observations led to the hypothesis that an unknown metabolic pathway was operative until the deficient Cyt oxidase pathway was able to function fully (9,11, 25 Pradet (13), demonstrated that cyanide at low concentration is a potent inhibitor of lettuce seed germination if volatility and chemical instability are controlled. This result was recently confirmed by Yu et al. (26).In vivo studies of adenine nucleotides during lettuce seed germination show that a high ATP level depends upon 02 (14,15, 17). From a comparison between the rate of ATP utilization and 02 uptake, Pradet (15) postulated that oxidative phosphorylation should operate during the beginning of germination. The experiments presented in this paper were done to obtain further evidence on the occurrence of oxidative phosphorylation in vivo during imbibition of lettuce seeds. The rate of energy-rich bond production was estimated from energy charge values (1, 17, 19, 20). Anoxia, cyanide, and FCCP3 were used to limit ATP synthesis. MATERIALS AND METHODSImbibition. Lettuce seeds (Lactuca sativa var. Reine de Mai, pleine terre) were immersed and agitated in water or solutions containing inhibitor for the required time at 20 C.Anoxic Treatments. The presoaked seeds were placed on wet filter papers in stoppered beakers and gassed. The stoppered beakers were fitted as described by Raymond and Pradet (17) to kill the seeds under N2. The O2 content of N2 was controlled at the exit of the beakers as described before (17). The O2 partial pressure in N2 was lower than 10 Pa.Killing, Sampling, Enzyme Inactivation, and Nucleotide Extractions. Metabolic activity was stopped by pouring cold ...

“…Uniformly soaked seeds (100 g) were put between wet filter papers in 4-liter jars. The papers were kept wet by the addition of 4 Md. 20742 through the jars at a rate of 60 ml/min at 20 C. Cherimovas.…”

Section: Methodsmentioning

confidence: 99%

Effects of Cyanide and Ethylene on the Respiration of Cyanide-sensitive and Cyanide-resistant Plant Tissues

Solomos¹,

Laties²

1976

The effects of cyanide and ethylene, respectively, were studied on the respiration of a fully cyanide-sensitive tissue-the fresh pea, a slightly cyanide-sensitive tissue-the germinating pea seedling, and a cyanideinsensitive tissue -the cherimoya fruit. Cyanide inhibition of both fresh pea and pea seedling respiration was attended by a conventional Pasteur effect where fermentation was enhanced with an accumulation of lactate and ethanol and a change in the level of glycolytic intermediates indicative of the activation of phosphofructokinase and pyruvate kinase accompanied by a sharp dedine in ATP level. In these tissues, ethylene had little or no effect on the respiration rate, or on the level of glycolytic intermediates or ATP. By contrast, ethylene as well as cyanide enhanced both respiration and aerobic glycolysis in cherimoya fruits with no buildup of lactate and ethanol and with an increase in the level of ATP. The data support the proposition that for ethylene to stimulate respiration the capacity for cyanide-resistant respiration must be present.We have shown that cyanide as well as ethylene induces a respiratory climacteric and ripening in avocados (18) as vwell as an increase in respiration in potato tubers (19). In both cases, glycolysis is enhanced and the gases produce seemingly identical physiological and biochemical changes. It has been suggested that for ethylene to enhance plant respiration. the cyanideinsensitive, or alternate, path (4) must be present and potentially operative (17). Below we compare the effects of cyanide and ethylene, respectively, on green peas and pea seedlings, where respiration is inhibited by cyanide, and on cherimoyas, where respiration is enhanced by cyanide. MATERIALS AND METHODSFresh green peas were removed from the pods aseptically and samples of 1 0 g were placed in jars through w hich a stream of ethylene-free air was passed at a rate of 60 ml/min at 20 C. Germinating peas were prepared as follows: pea seeds. Pisuni sativum, var. Alaska, were surface-sterilized by soaking for 15 min in 1.5% sodium hypochlorite solution. The seeds were then washed several times, and soaked overnight in distilled H.,O. Uniformly soaked seeds (100 g) were put between wet filter papers in 4-liter jars. The papers were kept wet by the addition of 4-to 10-ml portions of sterile H.,O daily. Md. 20742 through the jars at a rate of 60 ml/min at 20 C. Cherimovas. Annona cherirnola, of unknown variety were collected from a private orchard in San Diego county. Fruits NNere placed singly in respiratory jars. In all cases, the rates of O., uptake and CO., output were measured with a Beckman oxygen and infrared CO., gas analyzer. respectively. Ethylene and cyanide were applied as described previously (18). Sugar phosphates were extracted and estimated according to previously published methods (5, 18). In the case of pea seedlings, the seed coat was removed and the whole seedlings were dropped into liquid nitrogen and poxdered therein. ATP was first isolated by use of an ion exchange ...