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 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.

     

Sulfate Uptake and Respiration of Aging Potato Discs Modified by Malonic Acid and Ultraviolet Radiation

The time course of sulfate uptake in relation to respiration is shown for aging slices of potato, Solanum tuberosum L. cv. Russet. For comparative purposes, a time course for phosphate uptake is also shown. Malonic acid depressed both the respiratory rise and the sulfate accumulating ability of aging discs. A low dose of ultraviolet radiation given at the onset of aging reduced sulfate uptake by one-half without lowering respiration. We hypothesize that sulfate uptake is controlled by at least two mechanisms: one associated with oxidative metabolism and the other mediated by protein synthesis.     

Ribosomal RNA synthesis in newly sliced discs of potato tuber

A burst of ribosomal RNA synthesis is induced in potato tissue by slicing, and continues at a decreasing rate for about 12 hours. Ribosomal RNA synthesis in potato discs is sensitive to puromycin, in contrast to non-ribosomal RNA synthesis. Thus, the influence of puromycin on total RNA synthesis is significant only during the first 12 hours following slicing. The function of RNA made after 12 hours in a puromycin-insensitive manner is unknown. However, it is apparently unrelated to protein synthesis, since it has been shown that total inhibition of RNA synthesis by addition of actinomycin D to potato tissue after 12 hours of aging has no effect upon protein synthesis during the ensuing 12 hours.     

Membrane transformations in aging potato tuber slices

When potato tuber slices (Solanum tuberosum L.) are incubated with radioactive choline, labeled membrane-bound phospholipids are formed. If potato slices are aged for 0 to 24 hours before exposure to radioactive choline, the distribution of the labeled phospholipids undergoes both quantitative and qualitative changes. Quantitatively, there is a marked increase in the total lipoidal radioactivity with aging time. Qualitatively, there is a shift in the kinds of subcellular fractions that are being labeled. Fresh slices incorporate most of the lipoidal radioactivity in the microsomes. Slices aged for 9 hours incorporate most of the label in a fraction consisting of single membrane-bound cisternae, which are presumed to be dictyosomal fragments. Slices aged for 24 hours before incubation with radioactive choline incorporate the greater portion of the label in this same fraction, but a significant portion of the label is found in a heavier, mitochondria-containing fraction.     

Senescense: association of synthesis of Acid phosphatase with banana ripening

During ripening of banana (Musa sapientum L., var. Gros Michel or Valery) acid phosphatase activity increases 13-to 26-fold in the precipitate and 2- to 4-fold in the supernatant fraction of tissue homogenates. These increases are closely correlated with the onset and peak of the climacteric. The precipitate enzyme may be extracted with Triton X-100, CaCl₂ or NaCl; about 80% of it is in a 500g precipitate. Studies on effect of tonicity of the grinding medium indicate that the precipitate enzyme is desorbed from membrane or cell wall surfaces, and is not released as a result of lysis of membranes. The development of acid phosphatase during aging of tissue slices is the same as in intact fruit. Short term studies of tissue slices with cycloheximide and actinomycin D indicate that the increase in activity is owed to new enzyme synthesis, which is dependent upon synthesis of RNA. The possible effects of the increase in acid phosphatase on ripening are discussed.     

内源乙烯对陈化马铃薯切片交替氧化酶表达的诱导作用

抗氰呼吸是植物线粒体区别于动物线粒体的主要功能特征之一,其本质是一条以交替氧化酶（ａｌｔｅｒｎａｔｉｖｅｏｘｉｄａｓｅ,ＡＯＸ）为末端氧化酶,被称为“交替途径”的呼吸电子传递链（ＭｃＩｎｔｏｓｈ１９９４）。该呼吸途径经常发生于产热植物开花、果实成熟、切片陈化、低温胁迫、机械损伤以及病原体侵染等一些较特殊的环境条件或生理过程中,并受乙烯等一些效应剂的诱导（Ｓｏｌｏｍｏｓ和Ｌａｔｉｅｓ１９７６,Ｄａｙ等１９７８,Ｇｕｄｅ和ｖａｎｄｅｒＰｌａｓ１９８５,Ｍａｒｉｓｓｅｎ等１９８６,Ｙｉｐ和Ｈｅｗ１９８…     

Changes in Phospholipid Composition of a Winter Wheat Cultivar during Germination at 2 C and 24 C

Evaluation of various solvent systems for lipid extraction of wheat Triticum aestivum L. cv. Rideau seeds showed that boiling 2-propanol followed by the Bligh-Dyer procedure was the most efficient method, with respect to lipid yield and ability to inactivate lipolytic enzymes. Ten phospholipids were identified in dry seeds; the major components being phosphatidylcholine, lysophosphatidylcholine, N-acyl lysophosphatidyl-ethanolamine, N-acylphosphatidylethanolamine, and phosphatidylethanolamine. After growth for 1 week (2 C) or 31 hours (24 C), the proportions of phosphatidylethanolamine + lysophosphatidic acid and phosphatidic acid increased, lysophosphatidylcholine decreased, and the remaining phospholipids showed little change. At 5 weeks (2 C) or 72 hours (24 C), the seedlings showed 5-fold increases in the proportion of phosphatidic acid largely at the expense of phosphatidylcholine, small decreases in N-acyl lysophosphatidylethanolamine and N-acylphosphatidylethanolamine, and significant increases in lysophosphatidylcholine. The changes in phosphatidic acid and phosphatidylcholine are interpreted as being partially due to increasing phospholipase D activity during germination. In general, the phospholipid composition was similar in morphologically equivalent seedlings grown at 2 C or 24 C. The increased membrane content in seedlings grown at 2 C does not reflect any preferential synthesis of individual phospholipids.     

Oxygen Electrode Measurements of Potato Slice Respiration at 0{degrees} C.

The use of the oxygen electrode in measuring the respirationrate of potato slices between o° C. and 27° C. is described.When slices are transferred from 27° C. to o° C. therespiration rate adjusts to the level characteristic of thelow temperature within 2 or 3 minutes. The slices have a highoxygen affinity and the respiration rate at o° C. is approximately10 per cent. of the rate at 24° C. in both freshly cut slicesand slices aged 24 hours, during which time respiration increasesfourfold.     

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.     

Mobilization of respiratory metabolism in potato tubers by carbon dioxide

Applying high concentrations of CO₂ to whole potato tubers stimulated a rapid and pronounced respiratory gas exchange, which persisted for a prolonged time. The upsurge in respiration was proportional to the applied CO₂ concentrations and was further augmented by high O₂ levels. Tests using whole potatoes, or potato tissue slices from tubers previously treated with CO₂, indicated that the rapid CO₂-induced respiration is sensitive to cyanide during the first 24 hours of CO₂ application. The respiratory rise cannot be attributed to the emergence of a cyanide-resistant alternative electron transport pathway, although prolonged applications of CO₂, up to 72 hours, led to a gradual development of the pathway. CO₂-stimulated respiration was accompanied by a pronounced decline in the content of starch and glucose 6-phosphate, suggesting an active utilization of respiratory substrates. The ATP content in the CO₂-treated potatoes increased markedly, resembling similar increases in tissues undergoing respiratory upsurge.     

Alternative respiration and heat evolution in plants

The alternative respiratory pathway dissipates most of the chemical energy of respiratory substrates as heat. We have shown that this heat can be quantified by microcalorimetry and is a measure of alternative pathway activity in vivo. The alternative pathway is known to increase in aged potato (Solanum tuberosum) slices and in chill-stressed leaves. Aging of potato slices for 24 hours was accompanied by an almost fourfold increase in the rate of heat evolution. This heat increase was resistant to KCN but could be blocked by an alternative pathway inhibitor, salicylhydroxamic acid (SHAM). In cucumber (Cucumis sativus) leaves subjected to chilling stress (between 4 and 16°C), the rate of heat evolution was inversely related to temperature. As in aged potato slices, the increased rate of heat evolution in cucumber leaves was blocked by SHAM, but not by KCN. Nitrogen or the combination of SHAM and KCN blocked most of the heat evolution in both aged potato slices and chill-stressed cucumber leaves. Calorimetric measurements of the alternative pathway corresponded to respiration measurements performed using an oxygen electrode.     

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.

     

Alternative Oxidase of Potato Is an Integral Membrane Protein Synthesized de Novo during Aging of Tuber Slices

The rise in alternative respiratory capacity upon aging of potato (Solanum tuberosum) tuber slices is correlated with changes in mitochondrial membrane protein composition and a requirement for cytoplasmic protein synthesis. However, the lack of an antibody specific to the alternative oxidase has, until recently, prevented examination of the alternative oxidase protein(s) itself. We have employed a monoclonal antibody raised against the Sauromatum guttatum alternative oxidase to investigate developmental changes in the alternative pathway of aging potato slice mitochondria and to characterize the potato alternative oxidase by one- and two-dimensional gel electrophoresis. The relative levels of a 36 kilodalton protein parallel the rise in alternative path capacity. A plausible interpretation is that this alternative oxidase protein is synthesized de novo during aging of potato slices.     

Relationship Between Endogenous Ethylene and the Development and Operation of the Alternative Respiratory Pathway in Aged Potato Tuber Slices

The relationship between endogenous ethylene and the development and the operation of the alternative respiration pathway in aged potato (Solanum tuberosum L. ) tuber slices were investigated. During the 24 h aging period under 30 ℃, along with the great increase of the total respiratory rate (Vt) of the slices, the alternative pathway capacity (Valt) developed continuously, and the Valt/Vt values kept increasing as well. Both the alternative pathway activity (ρValt) and its contribution to Vt(ρValt/Vt) also increased gradually before 12 h of aging, but kept constant from 12 h to 24 h. The time course of the endogenous ethylene production of the aged slices was similar to the changing trends of Valt and Valt/Vt, but different from those of ρValt and ρValt/Vt values. ACC and Cu2 + treatment which stimulated ethylene production of the aged slices enhanced their Valt and Valt/ Vt values, Co2 + and Ag+ treatment resulted in a decreased effect. However, all the above treat ments of ethylene-related agents could not alter the continuously decreasing trend of the ρ value of the alternative pathway of the aged slices. And their effects on ρValt and ρValt/Vt values were only observed before 12 h of aging. These results suggested that the endogenous ethylene was essential to the development of the capacity of alternative pathway, but could only slightly influence the operation of the activity of the alternative pathway in aged potato tuber slices.     

Binding of glycolytic enzymes to a particulate fraction in carrot and sugar beet storage roots : dependence on metabolic state

Numerous studies have demonstrated a rapid increase in the respiration rate during aging of slices of tuber and storage roots. To determine the molecular mechanisms of this phenomenon, the role of enzyme binding to the subcellular particulate fraction has been assessed in carrot (Daucus carota L.) and sugar beet (Beta vulgaris L.). Soluble versus particulate fractions were separated by centrifugation at 16,000g and both fractions assayed for the activities of six glycolytic enzymes. Preparations from sliced and aged tissues showed elevated percentages of five enzymes associated with the particulate fraction as compared with controls. The stimulation of respiration which occurs during aging of underground storage organ slices may result, in part, from an association of enzymes with the particulate fraction of the cell promoting an elevated glycolytic rate.     

用于测定抗氰呼吸途径活性的氧同位素分馏法和氧肟酸抑制法的比较

氧同位素分馏法和氧肟酸抑制法的测定结果都表明,马铃薯切片在１２ ～２４ ｈ 陈化期间抗氰呼吸途径的实际活性基本保持恒定,尽管前者的测定结果是后者的２ 倍。此外,用上述２ 种不同方法进行研究的结果都表明,内源乙烯在诱导陈化切片抗氰呼吸途径容量产生的同时并不调控其实际运行。上述结果表明尽管氧肟酸抑制法不能准确测定抗氰呼吸途径的实际活性,但用于一些比较研究仍是可行的     

Die Trennung von Zellteilung und Suberinsynthese in dereprimiertem pflanzlichem Speichergewebe durch Tris-(hydroxymethyl-)aminomethan

Summary Differential derepression of the genome of potato tuber cells by slicing of the tuber tissue leads to cell divisions. This mitotic activity is totally suppressed by Tris-(hydroxymethyl)-aminomethane (Tris)-buffer widely used in biochemical research. The blockage is reversible if tissue slices are transferred to water in order to wash out the Tris-ions.The actual reason for the inhibition of mitotic activity by Tris is as yet unknown. As a possible mechanism of action an uncoupling of electron transfer from phosphorylation in the mitochondrial respiratory chain is discussed. However, experiments show no difference in O₂-absorption between Tris-treated tissue and control in water. Moreover the application of several inhibitors of respiration causes exactly the same effects in both tissues. Amytal (blockage of flavoproteids) has no influence on the respiratory rate at any time. Antimycin A (blockage of electron flow from cytochrome b to cytochrome c) as well as HCN (inhibition of cytochrome-oxidase) inhibit respiration of both tissues during the first 24 hours after derepression. Later on the respiration becomes resistant to both inhibitors. So the quality of respiration is assumed to be the same in mitotic active potato slices as in the Tris-treated tissue.Recent results of biochemical analyses of events in carbohydrate breakdown of the tissues in question point to a differential effect of Tris on several enzymes as possible reason for its inhibitory action.