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.     

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。     

Aging and the Development of Enhanced Respiration in Potato Tuber Tissue

Disks of potato tissue were aged by two separate methods. The resulting enhaiicetl respirations differed both qualitatively and quantitativety. It is suggested that the diverse results previously reported indicating changes in the terminal oxidative steps and/or in the relative contributions by competing metabolic pathways to the total respiration reflect variations in the methods employed to age the tissues. The nature of the stimulated respiration is discussed in terms of removal or retention of metabolites within the tissue during aging.     

Respiration and Carbohydrate Metabolisms of Potato Tuber

When a potato tuber is cut into slices, the activity of dehydrogenase system linked to DPN decreases but the activities of tyrosinase system and G-6-P dehydrogenase increase in the slices. Accompanying the changes of these enzymatic activities, the metabolic pathway in the slices turns from glycolytic path to pentose phosphate path. Tyrosinase oxidizes phenols which are formed from an intermediate of the pentose phosphate path, sedo-heptulose via shikimate as in microorganisms, resulting in increase in the oxygen uptake of the slices. It seems impossible to play a role as a terminal oxidase linked to diphosphopyridine nucleotide.     

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.     

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.

     

Mechanism of the Increase in Ribonuclease Activity in Potato Tuber Slices

Upon wounding of potato tubers (Solanum tuberosum L. cv. Spunta)RNase activity increases, peaks in about 16 hours, then declines.To see if the increase of the activity is due to de novo synthesisof the enzyme protein, the extracts were compared for theirability to react with a rabbit antibody prepared against thewound activated RNase. The enzyme was purified by polyacrylamidegel electrophoresis of a RNase preparation, which had been partiallypurified from aged potato slices by ammonium sulfate precipitation,carboxymethyl-Sephadex column chromatography and gel filtrationthrough Sephadex G-100. Using rocket immunoelectrophoresis RNase-proteinimmunoprecipitated by the antibody increased in wounded tissue.This observation implies that the activity increase involvesenzyme synthesis. The increase was inhibited by actinomycinD and cordycepin, but not by 5-fluorouracil, suggesting a requirementfor mRNA synthesis. (Received April 9, 1985; Accepted December 16, 1985)     

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.

     

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…     

The Fate of Patatin and Its mRNA in Slices of Potato Tuber

The fate of patatin mRNA was investigated in slices of potatotuber since this mRNA appeared to be a potential example ofa preexisting mRNA that is involved in the rapid formation ofpolysomes which occurs in such slices. Levels of patatin, whichis the major storage protein in mature potato tubers, decreasedslightly during the first 4 h after slicing but remained constantfor the next 44 h. Analysis of products of in vitro translationshowed that patatin mRNA was present and stable in the tubercells even after several months of storage. The translationalactivity of patatin mRNA relative to total translational activityin total cellular RNA fraction increased transiently duringthe first hour and then decreased rapidly to undetectable levelswithin 6 h. By contrast, the activity in polysomal RNA fractiondecreased immediately after slicing. The difference betweenthe relative activities of patatin mRNA in total and polysomalRNA fractions during the first hour suggests that the extentof incorporation of patatin mRNA into polysomes was not in directproportion to its abundance in the cells of the slices. Additionof actinomycin D to the slices did not prevent the transientincrease in the translational activity of patatin mRNA in totalRNA fraction at 1 h, indicating that the transient increasewas not due to synthesis of patatin mRNA de novo after slicing.However, the inhibitor prevented the degradation of patatinmRNA in the slices. This result indicates that the synthesisof new mRNAs is necessary for the degradation of patatin mRNA. ¹Present address: Aburahi Laboratories, Shionogi and Co., Ltd.,Koka-cho, Shiga, 520-34 Japan (Received June 30, 1989; Accepted May 9, 1990)     

Respiration and Carbohydrate Metabolism of Potato Tuber. Part I

The activity of tyrosinase, isolated from potatoes is suppressed in the presence of redox dyes and the composed dehydrogenase systems. The oxygen uptake of potato tuber increases, but after being cut into slices the dehydrogenase activity of tuber decreases in the lapse of time. This increase in oxygen uptake is caused by tyrosinase. Accordingly, tyrosinase does not seem to play the role as a terminal oxidase in the electron transferring systems.     

水杨酸对陈化马铃薯切片乙烯产生的促进作用 (英)

外源水杨酸（talicylicacid,SA）处理可明显促进陈化马铃薯切片的乙烯产生。SA促进乙烯产生的作用强度与SA浓度。pH值、陈化温度以及块茎的生理状态有关：在90μmol－1内,作用强度与SA浓度呈正相关;30℃下的作用强于20℃下的作用;pH6．4的SA溶液作用最强;对休眠块茎切片的作用强于破除休眠块茎切片。     

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.     

The Effect of Natural Growth Inhibitors in Potato Tuber on Respiration

The effects of natural growth inhibitors in potato tuber periderm on respiration were studied. The following results are presented in this paper: 1. The respiratory rate of potato tuber slices increased by application of inhibitors at concentration suppressing the growth of wheat coleoptile sections. This fraction of respiration induced by inhibitors was insensitive to DNP. According to inhibiting ex- periments with iodoacetic acid, malonic acid and KCN, it seems that this induced respiration is mediated by the EMP-TCA pathway and the CN-sensitive electron transfer chain. 2. The effects of various growth regulators on respiration are different depending on whether they are used in single or in combination. When the endogenous inhibitors at Rf 0.4—0.8 plus IAA or GA were studied, the inhibitors induced respiration rise was partially reduced by IAA, but completely depressed by GA. It seems reasonable to consider that the manner and the site of action of various growth zegulators on, respiration are not same.     

Sequential Induction of mRNAs for Phenylalanine Ammonia-lyase in Slices of Potato Tuber

Induction of the mRNA that encodes for phenylalanine ammonia-lyase(PAL, EC 4.3.1.5 [EC] ), which catalyzes the first reaction in thebiosynthesis of a wide variety of phenylpropanoid natural productsfrom phenylalanine, was investigated in. wounded tuber tissuesof the potato (Solanum tuberosum L. cv. Irish Cobbler). Northernblot analysis showed that hybridizable RNA was not present inunwounded tissue, but the amount of hybridizable PAL-specificmRNA increased rapidly in the polysomal RNA fraction with asharp, high peak at the early stage (0 h to 6 h) and two broadlower peaks at the later stage (6 h to 48 h) of the wound response.Addition of actinomycin D to the tissue prevented the appearanceof hybridizable mRNA in the total RNA fraction, confirming thatthe increase resulted from synthesis of PAL mRNA de novo. Levelsof translatable PAL mRNA activity in vitro increased in thepolysomal RNA fraction in parallel with the changes in levelsof hybridizable mRNA, with a subsequent increase in levels ofPAL subunit polypeptides and enzymatic activity in wounded tissues.PAL subunits synthesized both in vivo and in vitro had the samemolecular masses, of about 79 kDa, on SDS-polyacrylamide gelelectrophoresis, but isoelectric focusing revealed the presenceof isoforms of the native tetrameric enzyme with different pIvalues and changes in the relative levels of the isoforms afterwounding. Furthermore, two-dimensional gel electrophoresis ofPAL subunits synthesized in vitro showed that at least eightmRNAs that encoded subunit isoforms with different pI valueswere expressed sequentially after wounding. (Received May 24, 1990; Accepted October 24, 1990)