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.     

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.     

Wound-induced respiration and pyrophosphate:fructose-6-phosphate phosphotransferase in potato tubers

A seven fold increase in the rate of respiratory O2 uptake was observed 24 h after slicing of potato tuber disks. The maximum activity of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was 5-7 times greater than that of ATP-dependent phosphofructokinase (PFK) in fresh or aged potato slices. Thus, PFP may participate in glycolysis which supplies respiratory substrate in potato tubers. The PFP activity of desalted extracts determined in the absence of fructose-2,6-bisphosphate (F2,6BP) increased by 4.5 fold 24 h after slicing. However, maximal PFP activity determined with saturating (1 microM) F2,6BP was not changed. The Ka values of PFP for F2,6BP was lowered from 33 to 7 nM after 24 h of aging treatment. This increased susceptibility of the PFP activity to its allosteric activator, F2,6BP, may be involved in the increased respiration in wounded disks of potato tubers. Immunoblotting experiments indicated that both the alpha (66 kDa) and the beta (60 kDa) subunits of PFP were present in fresh or 24 h aged tuber slices.     

Influx and efflux of inorganic phosphate in sweet potato slices. effects of abscisic acid on bi-phasic kinetics

Influx and efflux of inorganic phosphate was determined in freshly cut and “aged” slices of sweet potato tubers, in the presence or absence of abscisic acid. Slices aged in the phytohormone took up considerably less phosphate than did the control slices. No difference in the rates of uptake of phosphate by the fresh tissue was observed. Kinetics of uptake of the aged untreated slices, proved to be bi-phasic. This was not the case for the treated slices. Neither the treated nor the untreated uptake curves for the fresh slices were bi-phasic. Efflux of phosphate from the slices aged in abscisic acid was greater than from the untreated slices. The results strongly suggest an effect of abscisic acid on the cell membrane.     

Inhibition of o(2) consumption resistant to cyanide and its development by N-propyl gallate and salicylhydroxamic Acid

Kinetics of inhibition of cyanide-insensitive O₂ uptake by n-propyl gallate (PG) and salicylhydroxamic acid (SHAM) were determined in fresh slices from ethylene-treated tubers of Solanum tuberosum `Norchip' and with mitochondria and lipoxygenase (EC 1.13.11.12) isolated from these tubers. PG and SHAM appeared to be inhibiting at identical sites in mitochondria but at disparate sites in slices. The apparent K_I for SHAM was similar in mitochondria and slices. However, the apparent K_I for PG in mitochondria was about 40-fold lower than the K_I for PG inhibition of lipoxygenase activity. The amount of lipoxygenase associated with mitochondria increased when tubers were treated with ethylene. PG, but not SHAM, inhibited aging-induced development of cyanide-insensitive respiration. The latter two phenomena are in accord with the hypothesis that lipid metabolism is required for the development of the alternative pathway.     

H2O2和水杨酸对陈化马铃薯切片交替呼吸途径影响的比较

外源5.0 mmol/L H2O2和0.1 mmol/L 水杨酸(salicylic acid, SA)处理均可明显提高陈化24 h的马铃薯切片的交替呼吸途径容量(Valt)及其与总呼吸的比值(Valt/Vt).应用交替氧化酶的单克隆抗体进行Western杂交的结果表明,H2O2和SA处理均可明显提高陈化马铃薯切片中交替氧化酶的表达水平.用氧同位素分辨法研究,结果表明:H2O2处理对陈化马铃薯切片中交替呼吸途径的实际运行没有影响,而SA处理对交替呼吸途径的实际运行具有明显的促进作用.上述结果表明,H2O2和SA对植物组织交替呼吸途径的影响存在差异,二者均可促进交替氧化酶的表达从而诱导交替呼吸途径容量的发生,但H2O2不影响其实际运行,而SA还可同时诱导其实际运行.     

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…     

Stimulation by Ethylene of Mitochondrial Development in Wounded Sweet Potato Root Tissue

Ethylene (about 100 µl per liter) markedly stimulatedincreases in respiratory, Cyt c oxidase and succinate dehydrogenaseactivities of the crude mitochondrial fraction as well as mitochondrialmembrane protein during aging of sliced sweet potato root tissue,indicating that it stimulated mitochondrial development in woundedtissue. It had such an effect even when slices were pre-agedin its absence for 1 day and thereafter aged in its presence.The mitochondrial inner membrane from slices aged in ethylene-containingair was denser than that from fresh slices, while the membranefrom slices aged in ethylene-free air was lighter. Chloramphenicolcompletely inhibited the increase in Cyt c oxidase activitywhether slices were aged in the presence or absence of ethylene.Cycloheximide did not inhibit the increase in slices aged inethylene-free air, but did by 50% in those aged in ethylene-containingair. ¹ This work was supported in part by a Grant-in-Aid (No. 411308)for Scientific Research from the Ministry of Education, Scienceand Culture, Japan. (Received April 4, 1981; Accepted July 7, 1981)     

Alternative pathway respiration and lipoxygenase activity in aged potato slice mitochondria

Mitochondrial preparations isolated from aged white potato (Solanum tuberosum L.) slices exhibited classical cyanide-insensitive O(2) uptake which was inhibited by salicylhydroxamic acid and tetraethylthiuram disulfide (disulfiram). These mitochondria also possessed lipoxygenase activity, as determined by O(2) uptake in the presence of 4 millimolar linoleic acid. Purification of the mitochondrial preparation on a continuous Percoll gradient resulted in a large decrease in lipoxygenase activity whereas cyanide-insensitive (disulfiram sensitive) O(2) consumption was still observed. These data indicate that cyanide-insensitive O(2) consumption in mitochondrial preparations isolated from aged white potato slices is of mitochondrial origin and not due to lipoxygenase contamination.     

Effect of ethylene and carbon dioxide on potato metabolism: stimulation of tuber and mitochondrial respiration, and inducement of the alternative path

The respiration of potato tubers (Solanum tuberosum var. Russet Burbank) which have been kept at room temperature for 10 days is stimulated upon subsequent treatment with C₂H₄ (10 microliters per liter) and O₂. The respiratory rise reaches a peak in 24 to 30 hours and thereafter declines. Coincident with the rise in tuber respiration is an increase in the respiratory rates of fresh slices and isolated mitochondria. Slices and mitochondria from C₂H₄- and O₂-treated tubers also display substantial resistance to CN, and the resistant respiration is inhibited by hydroxamates.

The longer the tubers are stored after harvest, the less effective is C₂H₄ in causing CN resistance in slices and mitochondria from treated tubers. Addition of 10% CO₂ to the C₂H₄-O₂ mixture, however, causes extensive CN resistance to develop, even in slices and mitochondria from old tubers. The results show that C₂H₄, O₂, and CO₂ act synergistically to induce alternative path development in potatoes.

     

Binding of Butyl Gallate to Plant Mitochondria : II. Relationship to the Presence or Absence of the Alternative Pathway

[¹⁴C]butyl gallate was used in binding studies to investigate the cyanide-resistant respiratory pathway in mitochondria isolated from a variety of sources displaying varying levels of cyanide resistance. Highly cyanide-resistant mitochondria were isolated from aroid spadices, while moderately cyanide-resistant mitochondria were isolated from either mung bean (Vigna radiata L.) hypocotyls or carbon dioxide/oxygen/ethylene-treated tubers. Totally cyanide-sensitive mitochondria were isolated from untreated tubers and rat liver. With one exception, all the plant mitochondria showed a reversible butyl gallate binding site which saturated at a level of 1.0 to 2.0 nanomoles per milligram protein. The exception, freshly harvested white potato tubers (<1 month from harvest), showed little specific butyl gallate binding, and also showed no appreciable induction of the cyanide-resistant pathway following carbon dioxide/oxygen/ethylene treatment. Only a low level, linear binding, well below that seen with plant mitochondria, was observed with rat liver mitochondria. Taken together, these results suggest a model for the interaction of the alternative pathway with the cytochrome pathway. In this model, the butyl gallate binding site (alternative oxidase) is a constitutive component in those mitochondria that are capable of developing the alternative pathway, and the binding sites associated with a second, inducible component that functions to couple the oxidase to the cytochrome pathway.     

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.     

Oxidation of reduced nicotinamide adenine dinucleotide phosphate by plant mitochondria.

Mitochondria isolated from various plant tissues (leaves, etiolated shoots and hypocotyls, and stem tubers) oxidize exogenous NADPH with respiratory control values and ADP:O ratios similar to those obtained with exogenous NADH as substrate. In all the mitochondria investigated, the electron-transfer inhibitors rotenone and amytal each had the same effect on the oxidation of NADPH as they had on the oxidation of NADH. The oxidation of exogenous NADPH by white potato tuber mitochondria was much more sensitive to inhibition by citrate or ethylene glycol bis-(beta-aminoethyl ether)-N,N-tetraacetic acid than was the oxidation of NADH. Mitochondria isolated from aged beetroot slices showed an increased capacity for the oxidation of exogenous NADH (compared with mitochondria from fresh tissue) but no such increase in the capacity to oxidize exogenous NADPH. These results suggest that exogenous NADPH and NADH are oxidized via different flavoproteins in plant mitochondria.     

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.     

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.

     

Changes in properties of the inner mitochondrial membrane during mitochondrial biogenesis in aging sweet potato tissue slices in relation to the development of cyanide-insensitive respiration.

Protein and phospholipid of the inner mitochondrial membrane in sweet potato root tissue increased after a lag phase during aging of the sliced tissue. The protein, but not the phospholipid, from aged slices was more insoluble in a solution containing sodium deoxycholate and sodium cholate than that from fresh tissue. There were differences in polypeptide composition between deoxycholate-cholate-soluble and -insoluble fractions as determined by polyacrylamide-gel electorophoresis of membrane fragments in the presence of sodium dodecyl sulfate. However, no difference was observed between mitochondrial membranes from fresh and aged slices. When disrupted mitochondrial membrane was subjected to equilibrium density centrifugation, two bands were obtained from aged slices but only one band from fresh tissue. The lighter band from aged slices was indentical to the single band from fresh tissue. The denser band was very poor in phospholipid, and the protein was very insoluble in deoxycholate-cholate solution. The denser membrane fragments possessed a cyanide-insensitive respiratory chain whereas the lighter did not. It is proposed that the development of the cyanide-insensitive respiration in aging slices is related to the formation of phospholipid-deficient mitochondrial membrane.     

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.     

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.     

Inhibition of nitrate accumulation and in vivo nitrate reductase activity in potato tuber slices by dimethysuIfoxide

Dimethylsulfoxide at concentrations of 0.5 to 5.0% inhibitednitrate reductase activity and the accumulation of nitrate in"fresh" and "aged" potato tuber slices. Fresh slices were moresensitive to concentrations of 0.5 to 2.5% and suppression ofenzyme activity paralleled a decline in NO₃ accumulation. With aged slices concentrations of 0.5 to 2.5% progressivelysuppressed enzyme activity without affecting NO₃ accumulation.These results are discussed in relation to the known effectsof dimethylsulfoxide on the permeability of biological membranesand protein structure. (Received November 29, 1978; )     

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.