The mechanism of chilling injury in sweet potato IX. The relation of chilling to changes in mitochondrial respiratory activities

To examine the mitochondrial activity of chilling-stored sweetpotatoes a method of isolating mitochondria with a good respiratorycontrol (RC) ratio from healthy sweet potato tissue was established.Mitochondria were isolated from two varieties of sweet potatoes(Norin No. 1, moderately sensitive to chilling, and OkinawaNo. 100, very sensitive) kept at about 0°C for about 15to 40 days. Respiratory activity was measured with an oxygenelectrode apparatus. Mitochondrial activities of chilling-storedNorin No. 1, i.e. the RC ratio, respiratory rate at state 3and ADP/O ratio decreased about 2 to 3 weeks after the beginningof incubation. The decline in the RC ratio was most sensitive.Diminution of the activities when malate was used was seen earlierthan when succinate was used. When activities were measuredusing succinate at low concentration (0.2 M) of mannitol, thedecrease in activities was more conspicuous than at a high concentration(0.7 M). Similar experiments with Okinawa No. 100 also showedthe decline in these activities. However, the three kinds ofactivities simultaneously decreased, and the decline appearedfaster than in the case of Norin No. 1. ¹ This paper constitutes part 99 of the phytopathological chemistryof sweet potato with black rot and injury. ² Current address: Nomura Research Institute, Kamakura, Kanagawa247, Japan. (Received June 6, 1972; )     

The mechanism of chilling injury in sweet potato VI. Changes of lipid components in the mitochondrial membrane during chilling storage

Phospholipids from the mitochondrial membrane of the sweet potato,a tropical plant, were composed mainly of phosphatidylethanolamine,phosphatidylcholine, lysophosphatidylcholine and an unknownphospholipid (S4) and their approximate contents were 39, 33,7 and 21 molar percent, respectively. Phosphatidylethanolaminebegan to decrease during the 4 day-chilling storage and thedecrease continued up to 40% during the 14 day-chilling storage.Decreases in phosphatidyicholine, lysophosphatidylcholine andS4 were indicated for the 8 day-chilling storage and their componentswere lost during the 14 day-chilling storage, by 20, 10 and20%, respectively. The amounts of these phospholipids did notseem to be changed further by prolonged chilling sotrage. Phospholipids of the mitochondrial membrane from the white potato,a temperate-zone plant, were composed mainly of phosphatidylethanolamine,phosphatidylcholine, lysophosphatidylethanolamine and an unknownphospholipid (W4), whose approximate contents were 39, 43, 3and 15 molar percent, respectively. Amounts of these four phospholipidswere not altered during the 14 day-chilling storage. ¹ This paper constitutes part 93 of the phytopathological chemistryof the sweet potato with black rot and injury. (Received August 21, 1971; )     

Mechanism of Cold Injury in Sweet Potatoes

The biochemical mechanism of cold injury occurring in sweet potatoes stored at 0°C was studied. Oxygen uptake and RC ratio of mitochondria from sweet potatoes kept at 0°C for about 15 days declined when succinate or malate was used as substrate. As sweet potatoes suffered slight cold injury, a decrease in the respiratory rate of state 3 of mitochondria was observed. This decrease could be restored approximately to the level of that of healthy sweet potato mitochondria by the addition of cytochrome c when succinate was used as substrate. When sweet potatoes suffered severe damage, only partial recovery was observed with cytochrome c. While it was found that the respiratory rate in state 3 of mitochondria from chilled sweet potatoes was less inhibited by cyanide than that of healthy sweet potato mitochondria, the inhibition could be restored to that of healthy sweet potato mitochondria by the addition of cytochrome c. When malate was used as substrate, no effect of cytochrome c and NADH₂ was observed. There was no difference between chilled and healthy sweet potato mitochondria in enzyme activities of the electron transport system except for malate dehydrogenase.     

Localization and Characterization of Peroxidases in the Mitochondria of Chilling-Acclimated Maize Seedlings

We present evidence of two peroxidases in maize (Zea mays L.) mitochondria. One of these uses guaiacol and the other uses cytochrome c as the electron donor. Treatments of fresh mitochondria with protease(s) indicate that ascorbate and glutathione peroxidases are likely bound to the mitochondria as cytosolic contaminants, whereas guaiacol and cytochrome peroxidases are localized within the mitochondria. These two mitochondrial peroxidases are distinct from contaminant peroxidases and mitochondrial electron transport enzymes. Cytochrome peroxidase is present within the mitochondrial membranes, whereas guaiacol peroxidase is loosely bound to the mitochondrial envelope. Unlike other cellular guaiacol peroxidases, mitochondrial guaiacol peroxidase is not glycosylated. Digestion of lysed mitochondria with trypsin activated mitochondrial guaiacol peroxidase but inhibited cytochrome peroxidase. Isoelectric focusing gel analysis indicated guaiacol peroxidase as a major isozyme (isoelectric point 6.8) that is also activated by trypsin. No change in the mobility of guaiacol peroxidase due to trypsin treatment on native polyacrylamide gel electrophoresis was observed. Although both peroxidases are induced by chilling acclimation treatments (14[deg]C), only cytochrome peroxidase is also induced by chilling (4[deg]C). Because chilling induces oxidative stress in the maize seedlings and the mitochondria are a target for oxidative stress injury, we suggest that mitochondrial peroxidases play a role similar to catalase in protecting mitochondria from oxidative damage.     

Mechanism of Chilling Injury in Sweet Potatoes

1. 1. Both amounts of lipid phosphorus and acid-insoluble nitrogen in the mitochondrial fraction from chilling-injured sweet potatoes (var. Okinawa 100) were larger than in the fraction from healthy sweet potatoes. The N-amount appeared to be increased more by chilling-injury than the P-amount.

2. 2. Sweet potato, a tropical plant, showed lower value of the degree of unsaturation of fatty acids in mitochondrial fraction than white potato, a temperate-zone plant.

3. 3. The amount of unsaturated fatty acids of C₁₆, C₁₈ and C₂₀ as percentage of the total fatty acids was higher in mitochondrial fractions from chilling-injured sweet potatoes (var. Okinawa 100 and var. Norin 1) than in mitochondrial fractions from healthy sweet potatoes. However, in the case of white potato mitochondrial fraction no detectable difference was observed between storage at 0~1°C and at 10~14°C.

     

Mechanism of chilling injury in sweet potato Part XII. Temperature dependency of succinoxidase activity and lipid-protein interaction in mitochondria from healthy or chilling-stored tissue

The Arrhenius plot of succinoxidase activity of intact mitochondriafrom healthy sweet potato (Ipomoea batatas Lam.) roots showedtwo transition temperatures of the activation energy at 8–10?Cand 16–18?C. Two transition temperatures were also observedin the case of sweet potatoes stored for 21 days at chillingtemperature, but the activation energy was lower than that ofhealthy tissue. The results on lipiddepleted mitochondria andlipid-rebound mitochondria indicated that the membrane lipidplayed an important role in the transition temperature of succinoxidaseactivity. On the other hand, in the binding of phospholipidto lipid-depleted mitochondria from healthy tissue, the numberof binding sites (n) and the dissociation constant (K) conspicuouslychanged at a temperature between 10 and 15?C. That is, n andK values above the temperature were considerably higher thanthose below the temperature, in spite of addition of phospholipidmicelles whose phase transition temperatures were differentfrom each other. In similar binding experiments with 14-daychilling-stored tissue, n values did not remarkably change,between higher and lower ranges of the above-mentioned temperatureand were lower at higher than at lower temperature. The resultssuggest that the conformation of the protein moiety in the mitochondrialmembrane undergoes reversible change in the early stage of chillingtreatment, which becomes irreversible with prolonged chillingtreatment. ¹This paper constitutes Part 113 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. ²Present address: Division of Breeding, Fruit Tree ResearchStation (Minist. Agric. Forest)., Hiratsuka, Kanagawa 254, Japan. (Received March 11, 1974; )     

Studies on chilling injury in plant cells I. Ultrastructural changes associated with chilling injury in callus tissues of Cornus stolonifera

This study was designed to elucidate the primary ultrastructuralchanges associated with chilling injury of cultured cells ofCornus stolonifera (TK-1). The cultured cells suffered seriousinjury after exposure to 0?C for longer than 24 hr, while noinjury was observed with less than 12 hr of treatment. Earlyultrastructural responses to chilling treatment were detectedin proplastids and the rough endoplasmic reticulum within 12hr of treatment. A remarkable ultrastructural change in thetonoplast, i.e., invagination, infolding and partial disruption,became apparent in the 24-hr stage of chilling treatment. Novisible change, however, was observed in mitochondria, nucleiand plasma membranes within 24 hr. Upon exposure to 0?C for48 hr, an abrupt degradation proceeded in the cytoplasm. Thesequential ultrastructural changes observed in cell organdies,especially proplastids, rough endoplasmic reticulum and thetonoplast, were closely related to the degradation of the cellscaused by chilling treatment. ¹ Contribution No. 1840 from the Institute of Low TemperatureScience, Hokkaido University. ² This work was supported in part by Grant 248004 from the Ministryof Education. (Received September 8, 1977; )     

Evidence that a Proliferation of the Endoplasmic Reticulum Precedes the Formation of Glyoxysomes and Mitochondria in Germinating Castor Bean Endosperm

Excised castor bean endosperm halves incubated with CDP-[Me-¹⁴C]cholineactively incorporated this compound into membrane phosphatidylcholine.The capacity of the tissue to synthesize phosphatidyl-[¹⁴C]cholineincreased during the first 3 d of germination and subsequentlydeclined. At the onset of germination phosphatidyl-[^l4C]cholinewas exclusively recovered in the ER membrane fraction. The rateof incorporation into the ER membranes increased strikinglyduring the first 24 h of germination while that into mitochondriaand glyoxysomes remained low. At later developmental stagesan increasing proportion of the newly synthesized phosphatidyl-[¹⁴C]cholinewas present in mitochondria and glyoxysomes; the rate of incorporationinto the membranes of these organelles increased while thatinto the ER membrane began to level off. The kinetics of CDP-[¹⁴C]cholineincorporation into membrane phosphatidylcholine of the majororganelle fractions of 3-d-old endosperm tissue showed thatthe ER was immediately labelled, whereas a lag period precededthe labelling of mitochondria and glyoxysomes. Assuming that the incorporation of CDP-[¹⁴C]choline into phosphatidylcholineserves as a reliable indicator of membrane synthesis, the resultsobtained suggest that a proliferation of ER membranes precedesthe formation of glyoxysomes and mitochondria in germinatingcastor bean endosperm. A comparison of developmental changesin (a) total ER and glyoxysomal phospholipid content and (b)ER and mitochondrial NADH cytochrome c reductase activity providedadditional evidence supporting this conclusion.     

Acclimation,Hydrogen Peroxide,and Abscisic Acid Protect Mitochondria against Irreversible Chilling Injury in Maize Seedlings

Our previous results indicated that 3-d-old dark-grown chilling-sensitive maize (Zea mays L.) seedlings did not survive 7 d of 4[deg]C chilling stress, but 69% of them survived similar stress when the seedlings were either preexposed to 14[deg]C for 3 d or pretreated with 0.1 mM H2O2 for 4 h at 27[deg]C (T.K. Prasad, M.D. Anderson, B.A. Martin, C.R. Stewart [1994] Plant Cell 6: 65-74) or 1 mM abscisic acid (ABA) for 24 h at 27[deg]C (M.D. Anderson, T.K. Prasad, B.A. Martin, C.R. Stewart [1994] Plant Physiol 105: 331-339). We discovered that chilling imposed oxidative stress on the seedlings. Since H2O2 accumulated during the periods of both acclimation and nonacclimation, we concluded that H2O2 had dual effects at low temperature: (a) During acclimation, its early transient accumulation signals the induction of antioxidant enzymes such as catalase 3 and peroxidase to scavenge H2O2; and (b) at 4[deg]C in nonacclimated seedlings, it accumulates to damaging levels in the tissues because of low levels of these and perhaps other antioxidant enzymes. Three-day-old seedlings pretreated with H2O2 (a mild oxidative stress) or ABA showed induced chilling tolerance. In the present study, we investigated whether mitochondria are a target for chilling-induced oxidative stress and, if so, what differences do acclimation, H2O2, or ABA make to protect mitochondria from irreversible chilling injury. The results indicated that chilling, in general, impairs respiratory activity, the cytochrome pathway of electron transport, and ATPase activity regardless of the treatment. In pretreated seedlings, the activities of catalase 3 and peroxidase in the mitochondria increased severalfold compared with control and nonacclimated seedlings. The increases in these antioxidant enzymes imply that mitochondria are under oxidative stress and such increases could initiate a protective mechanism in the mitochondria. Mitochondrial respiration is partially cyanide resistant during chilling stress and also after the 1st d of recovery. Upon further recovery over 3 d, in contrast to nonacclimated seedlings, the mitochondria of acclimation-, H2O2-, and ABA-treated seedlings showed the following recovery features. (a) The mitochondrial respiration changed from a cyanide-resistant to a cyanide-sensitive cytochrome pathway, (b) cytochrome oxidase activity recovered to control levels, (c) the ability of mitochondria to generate ATP was regained, and (d) the antioxidant enzyme activities remained at or above control levels. Based on these results, we conclude that chilling impairs mitochondrial function and that chilling-induced oxidative stress seems to be a factor, at least in part, for causing possible irreversible damage to the mitochondrial membrance components. Acclimation, H2O2, and ABA provide a protective mechanism by inducing antioxidant enzymes to protect mitochondria from irreversible oxidative damage that is absent in nonacclimated seedlings. Therefore, we conclude that the ability of the seedlings to recover from chilling injury is, at least in part, due to the ability of the mitochondria to resume normal function.     

Incidence of ripening and chilling injury on the oxidative activities and Fatty Acid compositions of the mitochondria from mango fruits

The succinate oxidation capacities of mitochondria isolated from mango fruits (Mangifera indica L.) stored at 4, 8, 12, and 20 C were investigated during storage. In normally ripening fruits (at 12 and 20 C) the oxidative capacities increased during the first 10 days and then decreased slowly. At lower temperatures (4 and 8 C), the fruits showed chilling injury symptoms, after about 10 days of storage and the succinate oxidation capacities of mitochondria decreased progressively. Plots of succinate oxidation capacities as against storage temperature showed a marked discontinuity between 12 and 8 C, only when chilling injury was observed on fruits stored at low temperature.

The variations of mitochondrial fatty acid composition during the storage of fruits at different temperatures were also investigated. A marked decrease of the molar ratio palmitoleic acid/palmitic acid, the predominant fatty acids in mitochondrial lipids, was observed to accompany both the succinate oxidation decrease and the induction of chilling injury.

     

Transfer of phospholipids from microsomes to mitochondria in germinating castor bean endosperm

¹⁴C-labeled microsomes were prepared by feeding [1-¹⁴ C]acetateto endosperm tissues from 4-day-old seedlings of castor beanseeds and incubated with unlabeled mitochondria from the sametissues. The loss of ¹⁴C-lipids from the microsomes was accompaniedby an increase of ¹⁴C-lipids in the mitochondria. The additionof 105,000?g supernatant and also pH 5.1-treated supernatant,both of which had been prepared from castor bean endospermsat the same stage, markedly enhanced the lipid transfer frommicrosomes to mitochondria. The activity in this fraction wasprecipitated by ammonium sulfate and lost with trypsin or heattreatment. The transfer of lipids was limited to phospholipids.Thus, it is concluded that in castor bean endosperms, phospholipidsare transferred from the endoplasmic reticulum to the mitochondriaby a phospholipid-exchange protein contained in the cytosol. (Received August 8, 1977; )     

Subcellular localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase and other membrane-bound enzymes in sweet potato roots

The subcellular localization of 3-hydroxy-3-methylglutaryl coenzymeA reductase and other membrane-bound enzymes in fresh, cut anddiseased sweet potato root tissues was resolved by differentialcentrifugation and sucrose density gradient centrifugation.In fresh, cut and diseased tissues, cytochrome c oxidase wasalmost localized in mitochondria, and NADH cytochrome c reductasewas in mitochondria in fresh and cut tissues, but in both mitochondriaand microsomes in diseased tissue. NADPH cytochrome c reductaseand antimycin A insensitive NADH cytochrome c reductase weremainly associated with microsomes. Catalase was dominantly foundin the mitochondrial fraction. 3-Hydroxy-3-methylglutaryl coenzymeA reductase was localized only in mitochondria and not in microsomaland supernatant fractions in both fresh and cut tissues. Indiseased tissue (infected with Ceratocystis fimbriata), in additionto being present in mitochondria, the enzyme was also localizedin microsomes. These results indicate that microsomal 3-hydroxy-3-methylglutarylcoenzyme A reductase whose activity rapidly increased in responseto the infection, predominandy participates in the formationof terpenes such as ipomeamarone. ¹ This paper constitutes Part 122 in the Series "The PhytopadiologicalChemistry of Sweet Potato with Black Rot and Injury." (Received March 1, 1976; )     

Studies on chilling injury in plant cells II. Ultrastructural changes in cells rewarmed at 26?C after chilling treatment

Both the restoration and deterioration of ultrastructures wereobserved during therewarming of cultured cells of Cornus stoloniferain which chilling at 0?C had caused an apparent change in themorphology of the organelles. Complete restoration of the ultrastructures,moderately altered by the 12-hr chilling, took place within12 hr of wanning at 26?C. Even in cells chilled for 24 hr, severelyaltered ultrastructures were partially or completely repairedin more than fifty percent of the treated cells. Some cellschilled for 24 hr, however, displayed further deteriorationof their ultrastructures during rewarming. Restoration of therough endoplasmic reticulum and the development of polysomesin recovering cells were characteristic of the early stage ofrewarming. Rupture of the tonoplast was sometimes observed duringrewarming of cells chilled for 24 hr. A possible role for therough endoplasmic reticulum and for the integrity of the tonoplastin cell recovery during the chill-warm sequence is discussed. ¹Contribution No. 2026 from the Institute of Low TemperatureScience, Hokkaido University. ²This work was supported in part by Grant 248004 from the Ministryof Education. (Received November 6, 1978; )     

Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland. II. Membrane marker enzymes and membrane ultrastructure

The activity of cytochrome oxidase (an inner mitochondrial membrane marker) in mouse mammary gland homogenates was found to increase five- to sixfold from late pregnancy to day 8 of lactation, while that of monoamine oxidase (an outer membrane marker) increased only about 25%. The specific activity of cytochrome oxidase in the isolated mitochondria decreased slightly over the same period while the specific activity of monoamine oxidase decreased fivefold. This reflects the fact that both cytochrome oxidase and mitochondrial protein are increasing at a much greater rate than is monoamine oxidase activity. Mixing experiments preclude the possibility that the release or removal of an inhibitor or stimulator produces the changes in enzymatic activity. The cytochrome oxidase to monoamine oxidase ratio was followed throughout the pregnancy-lactation cycle in total mammary homogenates, isolated mammary parenchymal cells, and isolated mammary mitochondria. In each preparation the pattern was the same with little change in the ratio until late pregnancy; and then a three- to fourfold increase occurred and the values reached a maximum by day 8 of lactation. These experiments were interpreted as demonstrating that the observed enzymatic changes are reflective of alterations in the mitochondria of the mammary parenchymal cell population. Electron micrographs of mid-pregnant and mid-lactating mammary parenchymal cells in situ were prepared, and distinct changes in the mitochondrial morphology noted. The most significant and obvious change is the large increase in the number of inner membrane cristae and an increase in matrix density in the lactating gland cell. Therefore, both enzymatic and morphological studies support the concept of an expansion of the mitochondrial inner membrane during presecretory differentiation in the mouse mammary parenchymal cell.     

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)     

CHANGE OF B-TYPE HAEM CONTENT IN RELATION TO PEROXIDASE BIOSYNTHESIS IN INJURED SWEET POTATO ROOTS

The methods of quantitative analysis of b-type haem in plantswere investigated. With an improved method developed was determinedthe haem content in the supernatant, mitochondrial, and microsomalfractions of sweet potato tissue. The activities of peroxidase,catalase, and cytochrome oxidase, as well as the contents ofb-type haem and acid-insoluble nitrogen in the cellular fractionswere determined at different incubation times after cuttingof sweet potato tissue. Peroxidase and catalase increased withtime in each celluler fraction, following a short lag phase.In the mitochondrial fraction, b-type haem, cytochrome oxidase,and acid insoluble nitrogen increased linearly with time. Inthe microsomal and supernatant fraction, b-type haem increasedwith time following a short lag phase. The increase in haemcontent of the supernatant fraction appeared to be associatedwith peroxidase formation. Time course analysis showed that ⁵⁹Fe-incorporation into b-typehaem of the supernatant fraction increased with time and thatincorporation was markedly inhibited by blasticidin S. The incorporationof ⁵⁹Fe into mitochondrial haem did not increase with time andwas not inhibited by blasticidin S. Blasticidin S inhibited⁵⁹Fe-incorporation into microsomal haem. Time course analysisof b-type haem content, ⁵⁹Fe-incorporation into b-type haem,and peroxidase activity suggest that in the injured tissue haemis synthesized from low molecular weight compounds and is incorporatedinto peroxidase as the haem moiety. ¹ This paper constitutes Part 57 of the Phytopathological Chemistryof Sweet Potato with Black Rot. ² Present address: Institute for Plant Virus Research, Chiba.     

Effect of antibiotics on biogenesis of mitochondria during aging of sliced sweet potato root tissue

The number of mitochondrial particles, and the activities ofperoxidase and cytochrome oxidase increased during aging ofsliced sweet potato root tissue. Blasticidin S inhibited allthese processes, indicating the occurrence of de now synthesisof protein. Chloramphenicol suppressed the synthesis of cytochromeoxidase and the reproduction of mitochondria but did not suppressperoxidase synthesis. Cylcoheximide suppressed peroxidase synthesisleaving the activities of cytochrome oxidase synthesis and mitochondrialreproduction unimpaired. A differentiation of ribosomal andmitochondrial protein syntheses is suggested. Mitomycin G and5-iododeoxyuridine inhibited the reproduction of mitochondriabut not the synthesis of enzymes. ¹This paper constitutes Part 76 of the Phytopathological Chemistryof Sweet Potato with Black Rot. (Received January 4, 1969; )     

Energy conservation capacity and morphological integrity of mitochondria in hypotonically treated rabbit epididymal spermatozoa

Hypotonic treatment of rabbit epididymal spermatozoa in 10 mM phosphate buffer disrupts the plasma membrane and removes the cytoplasmic droplet from those cells to which it is still attached. There is, however, no effect on the mitochondria, which retain their helical configuration around the axial filament complex, have intact inner and outer membranes, and show the same cristal morphology as do the mitochondria in untreated cells. Hypotonically treated spermatozoa respire with added malate-pyruvate, succinate, and ascorbate plus N,N′-tetramethyl-p-phenylenediamine, but not with added fructose or NADH. Respiration is inhibited by oligomycin and stimulated by uncoupler, showing that the mitochondria have retained their capacity for energy conservation. The uncoupled respiration rate is not further stimulated by added cytochrome c. Reduced-minus-oxidized difference spectra obtained at −196 °C of the hypotonically treated cells show a full complement of cytochromes, including cytochrome c. This result implies that the cytochrome c lost from mammalian spermatozoa on storage or chilling [Mann, T. (1951) Biochem. J. 48, 386–388] is cytoplasmic cytochrome c not yet incorporated into the mitochondria. The mitochondrial cytochrome c remains firmly held inside the intact outer membrane.     

Chloramphenicol Induced Changes in Some Respiratory Characteristics of Potato Tuber Callus

Chloramphenicol (CAP), an inhibitor of the mitochondrial proteinsynthesis inhibits callus induction and subsequent growth ofpotato tuber tissue discs. Tissue respiration increase did notoccur in the presence of CAP. Both with and without CAP theinitially CN^–-sensitive tissue becomes totally CN^–-resistantin 1–2 weeks. CAP blocks the development of mitcohondrial cytochrome oxidase.A gradual decrease in the activities of cytochrome oxidase andof cytochrome pathway-mediated mitochondrial respiration isfound in CAP-tissue. The mitochondrial alternative pathway whichis absent in mitochondria from freshly sliced tissue developsduring incubation both in the absence and presence of CAP. Thealternative pathway is only operative in uninhibited state IIIrespiration in mitochondria from CAP-tissue. Cycloheximide, an inhibitor of the cytoplasmic protein synthesisinhibits the developments of the alternative pathway and ofthe cytochrome pathway. Alcohol dehydrogenase activity increasestenfold in the tissue during two weeks of incubation on mediawith and without CAP. Alcohol production in the tissue did nottake place in the controls nor in the CAP-treated tissue. (Received April 18, 1981; Accepted July 17, 1981)     

Respiratory changes with chilling injury of soybeans

The leakage of solutes from cotyledons of soybeans (cv. Chippewa 64) was markedly stimulated by a chilling treatment (1 to 4 C) during the 1st minute of imbibition, but chilling after even 1 minute of water uptake resulted in little or no leakage increase. The respiratory rate of soybean particles was reduced more than 60% if a chilling treatment (15 minutes at 1 to 4 C) was given during the first minutes of imbibition, and little or no reduction was obtained if the chilling treatment was begun at 5 to 15 minutes after the start of imbibition. Using KCN as an inhibitor of cytochrome oxidase pathway of respiration and salicylhydroxamic acid as an inhibitor of the alternative pathway, it was found that the chilling injury involved a major reduction in the cytochrome pathway in whole axes and cotyledons and an engagement of the alternative pathway of respiration in cotyledon tissue. The suggestion is made that the chilling injury involves lesions resulting from temperature stress during the reorganization of membranes with water entry, and that both the leakage and the respiratory effects are consequences of these membrane lesions.