Collapse of Peroxide-Scavenging Systems in Apple Flower-Buds Associated with Freezing Injury

Changes in the metabolic activities of peroxide-producing systemsand peroxide-scavenging systems after freezing and thawing inflower buds of the apple, Malus pumila Mill., were studied withspecial reference to freezing injury. In flower buds of the‘McIntosh’ apple that were frozen below lethal temperatures,the activity of NADH-Cyt c reductase (EC 1.6.99.3 [EC] ), one of theenzymes in the electron-transport chains that are related tothe peroxide-producing systems, decreased slightly, while thatof Cyt c oxidase (EC 1.9.3.1 [EC] ) hardly changed. By contrast, theactivities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49 [EC] ),dehydroascorbate reductase (EC 1.8.5.1 [EC] ) and ascorbate peroxidase(EC 1.11.1.11 [EC] ), which are involved in the peroxide-scavengingsystems, decreased to very low levels. The activity of glyceraldehyde-3-phosphatedehydrogenase (EC 1.2.1.12 [EC] ) also decreased markedly. However,little change was observed in the activities of hexokinase (EC2.7.1.1 [EC] ), glucosephosphate isomerase (EC 5.3.1.9 [EC] ), glutathionereductase (EC 1.6.4.2 [EC] ) and glutathione peroxidase (EC 1.11.1.9 [EC] ).Examination of substrates involved in the peroxide-scavengingsystems revealed that the levels of glucose-6-phosphate andfructoses-phosphate decreased to approximately 10^–4 to10^–5 M and 10^–5 M, respectively, and the levelsof GSH decreased to about 10^–5 M or became barely detectable.A decrease in the levels of GSSG also occurred while levelsof ascorbate rose slightly. Similar results were observed withflower buds from ‘Starking Delicious’ and ‘Jonathan’apple trees. These results suggest that the freezing injury to apple flower-budsis closely related to the collapse of the peroxide-scavengingsystems that are coupled with the pentose phosphate cycle. Theresults also suggest that the dysfunction of these peroxide-scavengingsystems is caused by H₂O₂, which may be produced during freezingand thawing. (Received March 14, 1992; Accepted June 5, 1992)     

Metabolic Response to Treatment with Cold,Paraquat, or 3-Amino-1,2,4-triazole in Leaves of Winter Wheat

We treated leaves of winter wheat (Triticum aestivum L.) with cold, paraquat, or 3-amino-1,2,4-triazole and compared the responses. We assayed the activities of glucose-6-phosphate dehydrogenase, catalase, dehydroascorbate reductase and ascorbate free radical reductase and levels of hydrogen peroxide, glucose-6-phosphate, fructose-6-phosphate, ascorbate, dehydroascorbate, reduced and oxidized glutathione. With any of the three treatments, contents of cellular peroxides and hexose phosphates were raised. The content of ascorbate was lowered markedly by paraquat treatment, which produces active oxygen species, whereas such a decrease did not occur in other two treatments. When the plants were treated with 3-amino-1,2,4-triazole, which is a specific inhibitor of catalase, the content of oxidized glutathione increased severalfold. The glucose-6-phosphate dehydrogenase activity increased with all three treatments, but it decreased after glyphosate treatment, which does not stimulate the formation of peroxides. The activities of catalase and dehydroascorbate reductase were increased by the treatment of cold and paraquat, while 3-amino-1,2,4-triazole did not affect the dehydroascorbate reductase activity. The activity of ascorbate free radical reductase increased after treatment by paraquat only.     

Antioxidant enzyme responses to chilling stress in differentially sensitive inbred maize lines

Antioxidant enzyme activities were determined at the first,third and fifth leaf stages of four inbred lines of maize (Zeamays L.) exhibiting differential sensitivity to chilling. Plantswere exposed to a photoperiod of 16:8 L:D for one of three treatments:(a) control (25C), (b) control treatment plus an exposure toa short-term chilling shock (11C 1 d prior to harvesting),and (c) long-term (11 C constant) chilling exposure. Catalase(CAT; EC 1.11.1.6 [EC] ), ascorbate peroxidase (ASPX; EC 1.11.1.11 [EC] ),superoxide dismutase (SOD; EC 1.15.1.1 [EC] ), glutathione reductase(GR; EC 1.6.4.2 [EC] ), and mono-dehydroascorbate reductase (MDHAR;EC 1.6.5.4 [EC] ) activities were assessed. Reducing and non-reducingsugars and starch concentrations were determined as generalmetabolic indicators of stress. Reduced activities of CAT, ASPX,and MDHAR may contribute to limiting chilling tolerance at theearly stages of development in maize. Changes in levels of sugarand starch indicated a more rapid disruption of carbohydrateutilization in comparison to photosynthetic rates in the chilling-sensitiveline under short-term chilling shocks and suggested a greaterdegree of acclimation in the tolerant lines over longer periodsof chilling. Key words: Antioxidant enzymes, differential chilling sensitivity, maize, soluble carbohydrates, Zea mays     

Scavenging of Hydrogen Peroxide in the Endosperm of Ricinus communis by Ascorbate Peroxidase

The fat-storing endosperm of Ricinus communis L. was found tocontain an ascorbate peroxidase (EC 1.11.1.11 [EC] ), which is nearlyas active as catalase (EC 1.11.1.6 [EC] ) in degradation of hydrogenperoxide (H₂O₂) at its physiological concentrations. This ascorbateperoxidase probably functions together with monodehydroascorbatereductase (EC 1.6.5.4 [EC] ) or dehydroascorbate reductase (EC 1.8.5.1 [EC] )and glutathione reductase (EC 1.6.4.2 [EC] ) to remove the H₂O₂ producedduring the transformation of fat to carbohydrate in the glyoxysomes.The activities of these enzymes as well as the content of ascorbateand glutathione increase parallel to the activities of glyoxysomalmarker enzymes during the course of germination. Inhibitionof catalase by aminotriazole results in increases of the ascorbateperoxidase activity and of the glutathione content. All fourenzymes are predominantly localized in the cytosol of the Ricinusendosperm with low activities found in the plastids and themitochondria. The results suggest, that the ascorbate-dependentH₂O₂ scavenging pathway, which has been shown to be responsiblefor the reduction of photosynthetically derived H₂O₂ in thechloroplasts, operates also in the Ricinus endosperm. (Received June 5, 1990; Accepted July 31, 1990)     

CHANGES IN ACTIVITY OF DGLUCOSE-6-PHOSPHATE: NADP AND 6-PHOSPHO-D-GLUCONATE: NADP OXIDOREDUCTASES IN RELATION TO LIGNIFICATION OF BAMBOO

D-Glucose-6-phosphate: NADP oxidoreductase (glucose-6-phosphatedehydrogenase; EC 1.1.1.49 [EC] ) and 6-phospho-D-gluconate: NADPoxidoreductase (6-phosphogluconate dehydrogenase; EC 1.1.1.44 [EC] )were found to be present in immature bamboo. Optimal pHs ofthe glucose-6-phosphate- and 6-phosphogluconate dehydrogenaseswere found to be 8.0 and 8.5, respectively. Both enzymes were demonstrated to be NADP-specific and NADPcould not be replaced by NAD. Fructose-6-phosphate was indirectlyutilized after convrsion to glucose-6-phosphate by glucose-6-phosphateisomerase coexisting in the enzyme preparation. Pattern of enzyme activity and of respiratory breakdown of glucose-1-¹⁴Cand glucose-6-¹⁴C were investigated in connection with lignificationof bamboo and discussed in comparison with sugar metabolismof fungi-infected plant tissues. As for the changes in the enzymeactivity with growth of bamboo, it was recognized that therewas a tendency that the activity of both enzymes increased andwas maintained at a certain level even in the aged tissues.In addition there was a drop of the C₆/C₁ ratio toward the tissuesof lower parts containing considerable amount of lignin andthis phenomenon was the same as that observed in pentose phosphatemetabolism of fungi-infected plant tissues. (Received September 5, 1966; )     

Monodehydroascorbate Reductase in Spinach Chloroplasts and Its Participation in Regeneration of Ascorbate for Scavenging Hydrogen Peroxide

The primary reaction product of chloroplast ascorbate peroxidaseactivity was shown to be monodehydroascorbate radical (MDA).MDA reductase (EC 1.6.5.4 [EC] ) was localized in spinach chloroplaststroma. The MDA reductase activity of spinach chloroplasts,using NAD(P)H as electron donor, could account for the regenerationof ascorbate from MDA produced by ascorbate peroxidase activity.In the absence of MDA reductase, MDA disproportionated to ascorbate(AsA) and dehydroascorbate (DHA). The DHA was reduced to AsAby DHA reductase (EC 1.8.5.1 [EC] ) in chloroplasts. Both NADH andNADPH served as the electron donor of partially purified MDAreductase from spinach leaves. (Received September 24, 1983; Accepted January 23, 1984)     

Metabolic response to treatment with cold, paraquat, or 3-amino-1,2,4-triazole in leaves of winter wheat.

We treated leaves of winter wheat (Triticum aestivum L.) with cold, paraquat, or 3-amino-1,2,4-triazole and compared the responses. We assayed the activities of glucose-6-phosphate dehydrogenase, catalase, dehydroascorbate reductase and ascorbate free radical reductase and levels of hydrogen peroxide, glucose-6-phosphate, fructose-6-phosphate, ascorbate, dehydroascorbate, reduced and oxidized glutathione. With any of the three treatments, contents of cellular peroxides and hexose phosphates were raised. The content of ascorbate was lowered markedly by paraquat treatment, which produces active oxygen species, whereas such a decrease did not occur in other two treatments. When the plants were treated with 3-amino-1,2,4-triazole, which is a specific inhibitor of catalase, the content of oxidized glutathione increased severalfold. The glucose-6-phosphate dehydrogenase activity increased with all three treatments, but it decreased after glyphosate treatment, which does not stimulate the formation of peroxides. The activities of catalase and dehydroascorbate reductase were increased by the treatment of cold and paraquat, while 3-amino-1,2,4-triazole did not affect the dehydroascorbate reductase activity. The activity of ascorbate free radical reductase increased after treatment by paraquat only.     

Changes in some Calvin Cycle Enzymes of the Tomato during Acclimation to Irradiance

The activities of three Calvin cycle enzymes, RuBPc (E.C. 4.1.1.39 [EC] ),3PGA phosphokinase (E.C. 2.7.2.3 [EC] ) and NADP-G3P dehydrogenase(E.C. 1.2.1.13 [EC] ), and the cytoplasmic enzyme PEPc (E.C. 4.1.1.31 [EC] )together with soluble protein and chlorophyll were measuredin extracts from young tomato leaves during acclimation to achange in irradiance. Leaf area and fresh weight were also measuredto show changes due to growth during treatments. Soluble proteinhad doubled on a unit leaf area basis 7 d after transfer from100 µmol quanta m^–2s^–1 PAR (low light) to400 µmol quanta m^–2s^–1 PAR (high light). Duringthis period the protein/chlorophyll ratio rose from 4•6to 10, RuBPc activity almost doubled and PEPc almost trebled.Following the reverse transfer from high to low light, solubleprotein decreased by 30% after 7 d and the protein/chlorophyllratio fell from 12 to 5•6. There was no change in RuBPcactivity 3 d after transfer from high to low light while PEPcactivity decreased by over 30%. There was no decrease in theactivity of 3PGA phosphokinase or NADP-G3P dehydrogenase 1 dafter transfer to low light, but decreases were apparent after3 d. The extracted kinase and dehydrogenase when fully activatedwere able to phosphorylate and reduce 3PGA at more than 2•5-foldits calculated rate of synthesis in the leaf. The data are discussedin relation to changes in the CO₂ exchange of the leaf. Key words: Photosynthetic acclimation, irradiance, tomato leaf, RuBP carboxylase     

Contribution of the Pentose Phosphate Pathway and Glycolytic Pathway to Dormancy Breakage and Germination of Peanut (Arachis hypogaea L.) Seeds

Levels of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADPH oxidoreductase, E.C. 1.1.1.49 [EC] ) 6-phosphogluconate dehydrogenase(6-phospho-D-gluconate : NADP⁺ oxidoreduc tase, E.C. 1.1.1.44 [EC] )and aldolase (fructose 1, 6-diphosphate, D-glyceraldehyde, 3-phosphatelyase, E.C. 4.1.2.13 [EC] ) were assayed in the seeds of geneticallydormant and non-dormant pure lines of groundnut. In dormantlines cotyledons showed increased levels of activity of G-6-PDHand 6-PGDH during dry storage after-ripening. While the embryonicaxis did not exhibit detectable levels of enzyme activitiesimmediately after harvest, the activity started after a lapseof time during dry storage. When seeds of dormant lines wereincubated with kinetin (6-furfurylaminopurine) a distinct increasein the levels of both the enzymes was observed. The levels ofaldolase activity gradually decreased in the cotyledons andincreased in the embryonic axis of both control and kinetintreated seeds during the period of after-ripening. Comparedto control, kinetin treatment increased the aldolase activityin the embryonic axis and decreased it in the cotyledons. In non-dormant lines the activity of both the enzymes of PPpathway increased sharply both in the cotyledons and embryonicaxis while aldolase activity decreased in the cotyledons andincreased in the embryonic axis during germination i.e., from24 h to 96 h of germination. Abscisic acid caused inhibitionof enzyme activities to a large extent. Key words: PP pathway, dormancy breakage, germination, peanut     

The Development of Activities of Some Enzymes Concerned with Glycollate Metabolism in Greening Bean Leaves

The activities of phosphoglycollate phosphatase (EC 3.1.3.18 [EC] ),glycollate oxidase (EC 1.1.3.1 [EC] .). catalase (EC 1.11.1.6 [EC] ), theperoxisomal NADH-glyoxylate reductase (EC 1.1.1.26 [EC] ) which isconsidered to function as a hydroxypyruvate reductase in theperoxisomes, and the chloro-plastic NADPH-dependent glyoxylatereductaae, have been measured in extracts prepared from 14-d-olddark-grown bean leaves during the course of their greening inresponse to exposure to continuous illumination. All of theenzymes were found in the dark-grown leaves and on a per-leafbasis the activities increased from 6- to 12-fold with the exceptionof a 2–3-fold increase of NADPH-dependent glyoxylate reductaseduring 96-h greening, while the activities either remained constantor declined during similar periods in darkness. Initial lagperiods were evident before the illumination-induced increasesin enzyme activities. As D-threo-chloramphenicol did not affectthe increase in activity of any of these enzymes it would appearthat the increases were in no way dependent on protein synthesisby 70S ribosomes, or on the development of photosynthetic activity.     

Glyoxylate Cycle Enzymes in Peroxisomes Isolated from Petals of Pumpkin (Cucurbita sp.) during Senescence

The activities of the two unique enzymes of the glyoxylate cycle,isocitrate lyase (EC 4.1.3.1 [EC] ) and malate synthase (EC 4.1.3.2 [EC] ),were undetectable in petals of pumpkin (Cucurbita sp. AmakuriNankin) until the end of blooming, but they appeared duringsenescence. The activity of catalase (EC 1.11.1.6 [EC] ) increased,glycolate oxidase (EC 1.1.3.1 [EC] ) activity did not change, whilehydroxypyruvate reductase (EC 1.1.1.81 [EC] ) activity peaked at fullblooming stage and declined thereafter. After fractionationof cellular organelles on a sucrose density gradient, we detectedisocitrate lyase and malate synthase activities in peroxisomalfractions only from petals at the senescing stage. Northernblot analysis revealed that malate synthase mRNA increased duringpetal senescence. Citrate synthase (EC 4.1.3.7 [EC] ) and malate dehydrogenase(EC 1.1.1.37 [EC] ) activities were also present, while aconitase(EC 4.2.1.3 [EC] ) was not detectable in peroxisomal fractions. Moreoverthe presence of 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35 [EC] )and urate oxidase (EC 1.7.3.3 [EC] ) in the peroxisomal fractionsfrom senescing petals indicates that peroxisomes could be involvedboth in the ß-oxidation pathway and in the purinecatabolism during petal senescence. (Received May 25, 1991; Accepted September 25, 1991)     

Accumulation of Hydrogen Peroxide in Chloroplasts of SO2-Fumigated Spinach Leaves

Illuminated chloroplasts isolated from SO₂-fumigated spinachleaves accumulated more H₂O₂ than those from non-fumigated ones.This H₂O₂ formation was dependent on light and was inhibitedby DCMU. It also was depressed by cytochrome c and superoxidedismutase (EC 1.15.1.1 [EC] ). The addition of sulfite to rupturedchloroplasts isolated from non-fumigated leaves caused an H₂O₂accumulation that accompanied O₂ uptake. Spinach leaves losttheir catalase (EC 1.11.1.6 [EC] ), ascorbate peroxidase and glutathionereductase (EC 1.6.4.2 [EC] ) activities at the beginning of SO₂ fumigation,when H₂O₂ was accumulated. These results suggest that the accumulationof H₂O₂ in SO₂-fumigated spinach leaves is caused by the increasein O₂^–production, the precursor for H₂O₂, with a sulfite-mediatedchain reaction at the reducing site of photosystem I, and byinactivation of the H₂O₂ scavenging system. (Received October 7, 1981; Accepted June 16, 1982)     

Senescence- and drought-related changes in peroxidase and superoxide dismutase isoforms in leaves of Ramonda serbica

Ramonda sp. (Gesneriaceae) is an endemic and relic plant ina very small group of poikilohydric angiosperms that are ableto survive in an almost completely dehydrated state. Senescence-and drought-related changes in the activity of peroxidase (POD;EC 1.11.1.7 [EC] ), ascorbate peroxidase (EC 1.11.1.11 [EC] ), and superoxidedismutase (SOD; EC 1.15.1.1 [EC] ) were determined in leaves of differentage and relative water content. The results indicate that differentPOD isoforms were stimulated during senescence and dehydration.Two of the soluble POD isoforms were anionic with pI 4.5, andtwo were cationic with pI 9.3 and 9.0. The activity of ascorbateperoxidase remained unchanged either by drought or senescence.For the first time, SOD isoforms have now been determined inthis resurrection plant. Several SOD isoforms, all of the Mntype, were found to be anionic with pI 4 and a few others hadpI from 5 to 6, while one band of FeSOD with a lower molecularweight was neutral. Rehydration brought about a remarkable decreaseover the first hour in the activity of all the antioxidant enzymesexamined but activity recovered 1 d after rehydration. The resultsconfirmed that dehydration and senescence caused disturbancein the redox homeostasis of Ramonda leaves, while inducing differentPOD isoforms. A physiological role of peroxidase reaction withhydroxycinnamic acids in conservation and protection of cellularconstituents of desiccated Ramonda leaves is suggested. Key words: Desiccation, peroxidase, Ramonda, senescence, superoxide dismutase     

Suppression of Inhibitory Effects of Copper on Enzymatic Activities by Copper-Binding Substances from Athyrium yokoscense Assayed in vitro

A metal-tolerant fern, Athyrium yokoscense, is capable of growingin highly copper-contaminated soil, but cupric chloride inhibitedthe activities of some enzymes extracted from the fern. Thefunction in the detoxification of copper of two copper-bindingsubstances was investigated by examination of their effectson various enzymes assayed in vitro, i.e. acid phosphatase (orthophosphoric-monoesterphosphohydrolase [acid optimum], EC 3.1.3.2 [EC] ), glucose-6-phosphatedehydrogenase (D-glucose-6-phosphate: NADP⁺ 1-oxidoreductase,EC 1.1.1.49 [EC] ) and isocitrate dehydrogenase (threo-Ds-isocitrate:NADP⁺ oxidoreductase [decarboxylating], EC 1.1.1.42 [EC] ). The twocopper-binding substances, whose apparent molecular weightsare 9.5 kDa and 2 kDa, were previously obtained from the solublecytoplasmic fraction of the fern root. The 9.5-kDa substance,which is a cysteine-rich peptide induced as a result of exposureof the fern to copper, was found to suppress almost entirelythe inhibitory effects of the metal on the enzymes. The suppressoractivity of the peptide was nearly as effective as that of ethylenediaminetetraaceticacid. The 2-kDa substance, which is also found in fern thathas not been exposed to copper, had a more modest suppressoractivity. These results indicate that the 9.5-kDa substancemay contribute to the copper-tolerance of the fern growing incopper-contaminated soil. (Received August 26, 1988; Accepted March 17, 1989)     

Activation Energies of Reactions Catalyzed by the Nitrate Reductase from Chlorella vulgaris

The thermal dependence of two of the reactions catalyzed bythe nitrate reductase from Chlorella vulgaris was determined.The activation energies for NADH:nitrate oxidoreductase (EC1.6.6.1 [EC] ) and NADH:Cytochrome c oxidoreductase (EC 1.6.99.3 [EC] )are 42.1 kJ?mol^–1 and 21.5 kJ?mol^–1, respectively.Since the thermal dependency of the two enzymes is different,ratios of the activities will vary with temperature. The importanceof both rigorous thermal control during nitrate reductase assaysas well as the need to specify the temperature at which theratio of activities for the enzyme are clearly established. ¹Present Address: Cropping Systems Research Laboratory, USDA-ARS,Route 3, Box 215, Lubbock, TX 79401, U.S.A. (Received November 25, 1987; Accepted March 2, 1988)     

The Regulation of the Starch-Malate Balances during Volume Changes of Guard Cell Protoplasts

The enzymatic activities of phosphoenolpyruvate carboxylase(EC 4.1.1.31 [EC] ), ‘malic enzyme’ (EC 1.1.1.40 [EC] ), phosphofmctokinase(EC 2.7.1.11 [EC] ) and fructosebisphosphatase (EC 3.1.3.11 [EC] ) weremeasured during the swelling and shrinking of isolated and purifiedguard cell protoplasts (Vicia faba) in darkness. The volumeincrease was accompanied by the activation of phosphofructokinaseand a short stimulation of phosphoenolpyruvate carboxylase,at the same time the ‘malic enzyme’ and fructosebisphosphatasewere inhibited. However, during the shrinkage of guard cellprotoplasts these two enzymes were activated in contrast tophosphoenolpyruvate carboxylase and phospho-fructokinase. Becauseof the dramatic increase of phosphoenolpyruvate carboxylaseactivity during the swelling, this enzyme was assumed to actas a trigger for the swelling phase.     

Purification of NADP-Malic Enzyme and Phosphoenolpyruvate Carboxylase from Sugar Cane Leaves

A procedure is described for the purification of phosphoenolpyruvatecarboxylase (EC 4.1.1.31 [EC] ) and NADP-dependent malic enzyme (EC1.1.1.40 [EC] ) from sugar cane leaves. Each enzyme was purified tohomogeneity as judged by sodium dodecyl sulfate-polyacrylamidegel electro-phoresis, with about 30% yield. Phosphoenolpyruvatecarboxylase was purified 54-fold. A molecular weight of 400,000and a homotetrameric structure were determined for the nativeenzyme. The purified carboxylase had a specific activity of20.0 {diaeresis}mol (mg protein)_–1 min_–1, and wasactivated by glucose-6-phosphate and inhibited by L-malate.K_m values at pH 8.0 for phosphoenolpyruvate and bicarbonatewere 0.25 and O.l0 mM, respectively. NADP-malic enzyme, 356-foldpurified, exhibited a specific activity of 71.2 {diaeresis}mol(mg protein)_–1 min_–1 and was characterized as ahomotetramer with native molecular weight of 250,000. Purifiedmalic enzyme showed an absolute specificity for NADP⁺ and requireda divalent metal ion for activity. K_m values of 0.33 and 0.008mM for L-malate and NADP⁺, respectively, were determined. Thisenzyme was inhibited by several organic acids, including ketoand amino acids; while succinate and citrate increased the enzymeactivity when assayed with 10{diaeresis}M L-malate. The effectsshown by amino acids and by citrate were dependent on pH, beinghigher at pH 8.0 than at pH 7.0. (Received October 26, 1988; Accepted February 3, 1989)     

Kinetic Properties of Four Enzymes Involved in Proline Metabolism in Cold-acclimated Wheat

Two varieties of wheat (Triticum aestivum L.) a winter (Kharkov)and a spring (Glenlea), were acclimated under controlled conditionsat 5 °C and 25 °C (12 h photoperiod). Kinetic properties(K_m1 V_max/Km ratio and Q₁₀ as a function of reduction of substrateconcentration) were investigated for enzymatic systems involvedin two pathways of proline metabolism: the glutamic acid andthe ornithine pathways. Four enzymes were studied, namely prolinedehydrogenase (PDH, EC 1.5.1.2 [EC] ), glutamate dehydrogenase (GDH,EC 1.4.1.2 [EC] -4), glutamine synthetase (GS, EC 6.3.1.2 [EC] ) and ornithinetransaminase (OT, EC 2.6.1.13 [EC] ). Kinetic properties of thesefour enzymes proved to be modulated by cold acclimation, especiallyin Kharkov, the winter cultivar, which accumulates proline.Firstly, the synthesis of precursors of proline may be augmentedand the degradation of proline lessened by either decreasingthe K_m values of OT or increasing the K_m values of PDH. Secondly,the catalytic efficiency (V_max ratio) of GDH, GS, and OT isincreased. Thirdly, the lower values of Q₁₀ indicate a highcapacity of reaction of GS and OT.     

Development of Enzymes of the Glyoxylate Cycle during Senescence of Pumpkin Cotyledons

The presence and activities of isocitrate lyase (EC 4.1.3.1 [EC] )and malate synthase (EC 4.1.3.2 [EC] ) were studied during senescenceof pumpkin cotyledons (Cucurbita sp. Amakuri Nankin). Afterincubation of detached cotyledons in permanent darkness, theactivities appeared and increased up to the eighth day and thendeclined, while the activities of catalase (EC 1.11.1.6 [EC] ), glycolateox-idase (EC 1.1.3.1 [EC] ), and hydroxypyruvate reductase (EC 1.1.1.81 [EC] )decreased dramatically. After fractionation of cell organellesby sucrose density gradient, we detected isocitrate lyase andmalate synthase activities in peroxisomal fractions. The activityof the two key enzymes of the glyoxylate cycle also increasedduring senescence in vivo and we confirmed the presence of thetwo enzymes in the peroxisomal fractions after sucrose gradientcentrifugation. At every point examined, the level of malatesynthase was demonstrated by immunoblotting. It is concludedthat the development of isocitrate lyase and malate synthaseactivities represents the transition from leaf peroxisomes toglyoxysomes and that such a phenomenon is associated with senescence. (Received January 25, 1991; Accepted March 22, 1991)     

Participation of Hydrogen Peroxide in the Inactivation of Calvin-Cycle SH Enzymes in SO2-Fumigated Spinach Leaves

In SO₂-fumigated spinach leaves under light, chloroplast SHenzymes, glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPD)(EC 1.2.1.13 [EC] ), ribulose-5-phosphate kinase (Ru5PK) (EC 2.7.1.19 [EC] )and fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11 [EC] ) weremore remarkably inactivated than other chloroplast enzymes.Their activities recovered after removal of SO₂. The inactivationparalleled light-dependent CO₂-fixation in spinach leaves. Inilluminated chloroplasts isolated from SO₂-fumigated spinachleaves, NADP-GAPD and Ru5PK were more specifically in activatedthan other chloroplast enzymes. These two enzymes could be protectedfrom the inactivation by adding catalase. The NADP-GAPD inactivationwas suppressed by DCMU, cytochrome c or anaerobic conditions.By adding thiol compounds, the NADP-GAPD inactivation was dischargedand the activity increased. In chloroplasts or crude extractsfrom non-fumigated spinach leaves, NADP-GAPD and Ru5PK weremore strongly inhibited by externally added H₂O₂ than otherchloroplast enzymes. All results supported the idea that thesuppression of photosynthesis at the beginning of SO₂ fumigationwas caused by the reversible inhibition of chloroplast SH enzymewith H₂O₂. (Received October 7, 1981; Accepted June 16, 1982)