Catalase Degradation in Sunflower Cotyledons during Peroxisome Transition from Glyoxysomal to Leaf Peroxisomal Function

First order rate constants for the degradation (degradation constants) of catalase in the cotyledons of sunflower (Helianthus annuus L.) were determined by measuring the loss of catalase containing ¹⁴C-labeled heme. During greening of the cotyledons, a period when peroxisomes change from glyoxysomal to leaf peroxisomal function, the degradation of glyoxysomal catalase is significantly (P = 0.05) slower than during all other stages of cotyledon development in light or darkness. The degradation constant during the transition stage of peroxisome function amounts to 0.205 day⁻¹ in contrast to the constants ranging from 0.304 day⁻¹ to 0.515 day⁻¹ during the other developmental stages. Density labeling experiments comprising labeling of catalase with ²H₂O and its isopycnic centrifugation on CsCl gradients demonstrated that the determinations of the degradation constants were not substantially affected by reutilization of ¹⁴C-labeled compounds for catalase synthesis. The degradation constants for both glyoxysomal catalase and catalase synthesized during the transition of peroxisome function do not differ. This was shown by labeling the catalases with different isotopes and measuring the isotope ratio during the development of the cotyledons. The results are inconsistent with the concept that an accelerated and selective degradation of glyoxysomes underlies the change in peroxisome function. The data suggest that catalase degradation is at least partially due to an individual turnover of catalase and does not only result from a turnover of the whole peroxisomes.     

Catalase Synthesis and Turnover during Peroxisome Transition in the Cotyledons of Helianthus annuus L

Based on measurements of total catalase hematin and the degradation constants of catalase hematin, zero order rate constants for the synthesis of catalase were determined during the development of sunflower cotyledons (Helianthus annuus L.). Catalase synthesis reached a sharp maximum of about 400 picomoles hematin per day per cotyledon at day 1.5 during the elaboration of glyoxysomes in the dark. During the transition of glyoxysomes to leaf peroxisomes (greening cotyledons, day 2.5 to 5) catalase synthesis was constant at a level of about 30 to 40 picomoles hematin per day per cotyledon. In the cotyledons of seedlings kept in the dark (day 2.5 to 5) catalase synthesis did not exceed 10 picomoles hematin per day per cotyledon. During the peroxisome transition in the light, total catalase hematin was maintained at a high level, whereas total catalase activity rapidly decreased. In continuous darkness, total catalase hematin decreased considerably from a peak at day 2. The results show that both catalase synthesis and catalase degradation are regulated by light. The turnover characteristics of catalase are in accordance with the concept that glyoxysomes are transformed to leaf peroxisomes as described by the one population model and contradict the two population model and the enzyme synthesis changeover model which both postulate de novo formation of the leaf peroxisome population and degradation of the glyoxysome population.     

The in vivo turnover of rat liver microsomal epoxide hydrolase and both the apoprotein and heme moieties of specific cytochrome P-450 isozymes

The in vivo turnover rates of liver microsomal epoxide hydrolase and both the heme and apoprotein moieties of cytochromes P-450a, P-450b + P-450e, and P-450c have been determined by following the decay in specific radioactivity from 2 to 96 h after simultaneous injections of NaH14CO3 and 3H-labeled delta-aminolevulinic acid to Aroclor 1254-treated rats. Total liver microsomal protein was characterized by an apparent biphasic exponential decay in specific radioactivity, with half-lives of 5-9 and 82 h for the fast- and slow-phase components, respectively. Most (approximately 90%) of the rapidly turning over microsomal protein fraction was immunologically distinct from membrane-associated serum protein, and thus appeared to represent integral membrane proteins. The existence of two distinct populations of cytochrome P-450a was suggested by the apparent biphasic turnover of both the heme and apoprotein moieties of the holoenzyme. The half-lives of the apoprotein were estimated to be 12 and 52 h for the fast- and slow-phase components, respectively, and 7 and 34 h for the heme moiety. The turnover of cytochromes P-450b + P-450e was identical to that of cytochrome P-450c, with half-lives of 37 and 28 h for the apoprotein and heme moieties, respectively. In all cases, the shorter half-lives of the heme component compared to the protein component were statistically significant. In contrast to the cytochrome P-450 isozymes, epoxide hydrolase (t1/2 = 132 h) turned over slower than the "average" microsomal protein (t1/2 = 82 h). The differential rates of degradation of these major integral membrane proteins during both the rapid and slow phases of total microsomal protein turnover argue against the concepts of unit membrane degradation and unidirectional membrane flow of liver endoplasmic reticulum.     

Metabolism of arginine by aging and 7 day old pumpkin seedlings

The metabolism of arginine by etiolated pumpkin (Cucurbita moschata) seedlings was studied over various time and age intervals by injecting arginine-U-¹⁴C into the cotyledons. At most, 25% of the ¹⁴C was transported from the cotyledon to the axis tissue and the amount of this transport decreased with increasing age of the seedlings. The cotyledons of 25 day old plants contained 60% of the administered ¹⁴C as unmetabolized arginine. Little ¹⁴C was in sugars and it appeared that arginine was the primary translocation product. Time course studies showed that arginine was extensively metabolized and the labeling patterns suggest that different pathways were in operation in the axis and cotyledons. The amount of arginine incorporated into cotyledonary protein show that synthesis and turnover were occurring at rapid rate. Only 25% of the label incorporated into protein by 1.5 hr remained after 96 hr. The label in protein was stable in the axis tissue. By 96 hr 50% of the administered label occurred as ¹⁴CO₂ and it appeared that arginine was metabolized, through glutamate, by the citrio acid cycle in the cotyledons. The experiments showed that an extensive conversion of arginine carbon into other amino acids did not occur.     

Turnover of phytochrome in pumpkin cotyledons

By using density labeling, it was found that the protein moiety of phytochrome is synthesized de novo in the red-absorbing form in cotyledons of dark-grown pumpkin (Cucurbita pepo L.) seedlings, as well as those irradiated with red light and returned to the dark. The rate of synthesis appears to be unaffected by the light treatment. Turnover of the red-absorbing form was also detected in dark grown seedlings using density labeling, while turnover of the far red-absorbing form is already implied from the well known “destruction” observed in irradiated seedlings. In both cases, true degradation of the protein is involved, but the rate constant of degradation of the far red-absorbing form may be up to two orders of magnitude greater than that of the red-absorbing form. The data indicate that, in pumpkin cotyledons, phytochrome levels are regulated against a background of continuous synthesis through divergent rate constants of degradation of the red and far red-absorbing forms and the relative proportions of the two forms present.     

Cytochrome P-450 heme and the regulation of hepatic heme oxygenase activity

The degradation of cytochrome P-450 heme in the liver has been studied by a new approach. In rats, hepatic heme was labeled by administration of a tracer pulse of [5-¹⁴C]δ-aminolevulinic acid (ALA), and its degradation was analyzed in terms of labeled carbon monoxide (¹⁴CO) excretion, which is a specific degradation product of the labeled heme. Within minutes after administration of [5-¹⁴C]ALA, 14CO was detectable and increased after 2 h to an “early peak,” reflecting the elimination of labeled heme from a rapidly turning over pool in the liver. Beyond the early peak, the rate of ¹⁴CO production decreased in a log-linear manner, consistent with the degradation of heme in stable hepatic hemoproteins. From the rate at which ¹⁴CO production declined during this phase, from the predominant labeling of cytochrome P-450 heme by the administered [5-¹⁴C]ALA and from the known turnover characteristics of this hemoprotein in the liver, it could be inferred that production of ¹⁴CO—between 16 and 30 h after administration of labeled ALA—largely reflected degradation of cytochrome P-450 heme. This approach, which permits serial measurements in a single animal, was used to study the effect on cytochrome P-450 heme of administered heme or endotoxin, both of which are potent stimulators of hepatic heme oxygenase activity. Both of these substances caused marked acceleration of the degradation of cytochrome P-450 heme, the effect occurring over the same dose range as that for stimulation of hepatic heme oxygenase. The findings suggest that stimulation of this enzyme activity in the liver is closely related to the rate of degradation of cytochrome P-450 heme.     

Fate of the porphyrin cofactors during the light-dependent turnover of catalase and of the photosystem II reaction-center protein D1 in mature rye leaves

The apoprotein of the enzyme catalase (EC 1.11.1.6) was shown to exhibit a light-dependent turnover in leaves. Present results indicate that photoinactivation of the enzyme was not accompanied by a synchronous destruction and new synthesis of its heme moiety. In rye (Secale cereale L.) leaves the catalase content was not depleted in light when porphyrin synthesis was inhibited by gabaculine. Photoinactivation of purified bovine liver or rye leaf catalase in vitro was not accompanied by concomitant damage to the heme groups. Both the incorporation of -[³H]aminolevulinic acid ([³H]ALA) into catalase-heme and its apparent turnover increased with irradiance. However, the apparent half-life of the catalase-heme was much longer than that of its apoprotein. It is probable that not only degradation but also an exchange with the free heme pool contributed to the apparent turnover of radioactivity of the catalase-heme. Part of the chlorophyll (Chl) associated with photosystem II (PS II) had a preferential light-induced turnover, and repair of PS II appeared to require new Chl synthesis also in mature green rye leaves. The activity of PS II, indicated by the ratio of variable to maximal fluorescence (F_v/F_m), rapidly declined in the presence of gabaculine in light and the reaction-center proteins D1 and D2 were depleted. When segments of mature green rye leaves were labeled with [³H]ALA and incorporation into Chl-protein complexes analysed after electrophoretic separation in the presence of Deriphat, the highest radioactivity was observed in the core complex of PS II, while PS I and the light-harvesting complex of PS II (LHC II) were unlabeled. In greening etiolated leaves highest incorporation was observed in LHC II. Both the incorporation of [³H]ALA into the PS II core complex of green rye leaves and its turnover increased with irradiance. However, the apparent half-life of the PS II-bound labeled porphyrin compounds (mainly Chl) was considerably longer than that of the reaction-center protein D1 under identical conditions.Abbreviations ALA -aminolevulinic acid - CII Core complex of PS II - Chl chlorophyll - DMSO dimethyl sulfoxide - F_v/F_m ratio of variable to maximal chlorophyll fluorescence - LHC light-harvesting complex - PAR photosynthetically active radiation We thank the Deutsche Forschungsgemeinschaft for financial support. Technical assistence by B. Kramer and Ch. van Oijen is greatly appreciated. We are grateful to Dr. Johanningmeier and Dr. Godde (Lehrstuhl für Biochemie der Pflanzen, Universität Bochum, Germany) for providing antisera against the D1 and D2 proteins and Dr. M. Schmidt (Botanisches Institut, Universität Frankfurt am Main, Germany) for valuable advice. Deriphat 160 was kindly supplied by Henkel Corp., Hoboken, N.J., USA.     

Role of ethylene in chlorophyll degradation in radish cotyledons

The effect of age of radish seedlings on changes in chlorophyll concentration caused by ethylene was examined. Ethylene was produced at 2–4 nl g^?1 h^?1 following excision of cotyledons from 5-to 20-day-old seedlings. The youngest cotyledons maintained this rate, whereas ethylene synthesis declined by as much as 80% during a 24-h period in older cotyledons. The youngest cotyledons continued to accumulate chlorophyll in the dark, but after 7 days cotyledons lost chlorophyll and the proportion of chlorophyll lost increased with age. Ethylene promoted, and norbornadiene inhibited, this loss of chlorophyll; in combined treatments the effects of ethylene and norbornadiene were competitive. The maximal rate of chlorophyll loss occurred in 1μl L^?1 ethylene; extrapolation of the response to concentration indicated that half-maximum loss would occur at 0.005–0.01 μl L^?1 ethylene. In cotyledons from 20-day-old seedlings, chlorophyll degradation occurred mainly after 24 h from excision and transfer to the dark. Chlorophyll degradation during 48 h in the dark was affected by norbornadiene or ethylene applied from 0–24 h or from 24–48 h.     

Purification of glyoxysomal catalase and immunochemical comparison of glyoxysomal and leaf peroxisomal catalase in germinating pumpkin cotyledons

As a step to study the mechanism of the microbody transition (glyoxysomes to leaf peroxisomes) in pumpkin (Cucurbita sp. Amakuri Nankin) cotyledons, catalase was purified from glyoxysomes. The molecular weight of the purified catalase was determined to be 230,000 to 250,000 daltons. The enzyme was judged to consist of four identical pieces of the monomeric subunit with molecular weight of 55,000 daltons. Absorption spectrum of the catalase molecule gave two major peaks at 280 and 405 nanometers, showing that the pumpkin enzyme contains heme. The ratio of absorption at 405 and 280 nanometers was 1.0, the value being lower than that obtained for catalase from other plant sources. These results indicate that the pumpkin glyoxysomal catalase contains the higher content of heme in comparison with other plant catalase.

The immunochemical resemblance between glyoxysomal and leaf peroxisomal catalase was examined by using the antiserum specific against the purified enzyme preparation from pumpkin glyoxysomes. Ouchterlony double diffusion and immunoelectrophoretic analysis demonstrated that catalase from both types of microbodies cross-reacted completely whereas the immunotitration analysis showed that the specific activity of the glyoxysomal catalase was 2.5-fold higher than that of leaf peroxisomal catalase. Single radial immunodiffusion analysis showed that the specific activity of catalase decreased during the greening of pumpkin cotyledons.

     

Untersuchungen zur Funktionsänderung der Microbodies in den Keimblättern von Helianthus annuus L.

Summary The enzyme patterns in sunflower cotyledons indicate that the glyoxysomal function of microbodies is replaced by the peroxisomal function of these organelles during the transition from fat degradation to photosynthesis. The separation of the microbody population into glyoxysomes and peroxisomes during this transition period is reported. The mean difference in density between the activity peaks of glyoxysomal and peroxisomal marker enzymes on a sucrose gradient was calculated to be 0.007±0.004 g/cm³ and turned out to be significant (t=7.8>4.04=t _5;0.01). The activity peak of catalase coincides with that of isocitrate lyase in early stages of development, but shifts to the activity peak of peroxisomal marker enzymes during the transition period. No isozymes of the catalase could be detected by gel electrophoresis in the microbodies with the two different functions.During the rise of the peroxisomal marker enzymes no synthesis of the common microbody marker, catalase, could be demonstrated using the inhibitor allylisopropylacetamide. Using D₂) for density labeling of newly-formed catalase, no difference is observed between the density of catalase from cotyledons grown on 99.8% D₂O during the transition period and the density of enzyme from cotyledons grown on H₂O. The activity of particulate glycolate oxidase is reduced 30–50% by allylisopropylacetamide, but is not affected by D₂O. The chlorophyll formation in the cotyledons is strongly inhibited by both substances.     

In vivo biosynthesis and turnover of 35S-labeled glomerular basement membrane

Renal glomerular basement membrane was labeled in vivo by the injection of tracer amounts of radioactive sulfate into normal adult rats. The biosynthesis and turnover of [³⁵S]glycosaminoglycans in purified basement membrane was determined from the specific activity of ³⁵S in pronase digests of basement membranes isolated 1–7 days after injection. Peak radioactive labeling occurred 24 h after injection following which the specific activity of basement membrane sulfate, expressed as cpm/μg uronic acid, progressively declined over the ensuing period of study. The biologic half-life of radioactive sulfate in basement membrane was estimated at about 7 days, which is within the range previously reported for [³⁵S]glycosaminoglycans in whole renal cortex. The findings indicate that ³⁵S-labeled components of glomerular basement membrane have a relatively rapid turnover.     

On the synthesis and degradation of the multiple forms of catalase in mouse liver

The incorporation of ⁵⁵Fe-labeled ferrous sulfate and ³H-labeled γ-aminolaevulinic acid into the catalase of mouse liver was measured at intervals up to 96 hr after intraperitoneal injection, and the intracellular location of radioactive catalase followed, as well as the distribution of radiolabel between the multiple forms of this enzyme. At 10 min, catalase radioactivity was present in all the cellular fractions studied, but after this time, label began to disappear from the microsomal fraction and from the peroxisomal detergent extract. By comparison, catalase incorporation reached a peak at about 6 hr in the peroxisomal aqueous extract, and rose to a broad peak after about 30 hr in the cytosol fraction. On resolving the multiple forms of catalase in the supernatant fraction by electrophoresis, it was found that label first appeared in the fastest moving heteromorph, and appeared sequentially in the other multiple forms over a period of 96 hr.The sequence of degradation of catalase was also studied by examination of residual catalase activity subsequent to the injection of allyl-isopropyl acetamide, a heme synthesis antagonist which blocks catalase synthesis. Blood catalase levels did not seem to be significantly affected by this treatment, but in the liver, the decay rates of catalase activity were appreciable, and varied significantly between the intracellular pools. The rate of decrease was greatest in the peroxisomal detergent extract, and least in the supernatant fraction.These findings have been discussed in relation to current understanding of the subcellular disposition, multiplicity, and turnover of hepatic catalase.     

Tracking animal-mediated seedling establishment from dispersed acorns with the aid of the attached cotyledons

Various methods have been used to track seed dispersal of large-seeded species; however, the influence of different seed tracking methods on ecological outcomes of seed dispersal by animals is not well evaluated. Acorn removal by food hoarding animals and the following seedling establishment of Mongolian oak (Quercus mongolica) were investigated in Xiaoxing’anling Mountain, Heilongjiang, northeastern China, by using four different marking methods: plastic tagging, nail insertion, hole drilling, and isotope labeling. The acorn removal speed differed among marking methods, with plastic-tagged acorns being removed more slowly than those marked with nails, holes, and isotope. By checking the attached cotyledons and performing isotope analyses, more seedlings were found to establish from nailed acorns and isotope-soaked acorns than from drilled acorns and plastic-tagged acorns. Plastic-tagged acorns were transported closer than those marked with nails, holes, and isotope. Moreover, seedlings were often found clustered in caches containing acorns marked with plastic tags. Low level of cotyledon predation by animals makes it possible to directly identify focal seedlings of white oaks based on the attached cotyledons. Considering cotyledon predation by animals, coupling minor modification of cotyledons with isotope labeling appears to be an easy way to explore the actual pattern of seed dispersal of large-seeded trees, e.g., oaks.     

Role of ethylene in chlorophyll degradation in radish cotyledons

The effect of age of radish seedlings on changes in chlorophyll concentration caused by ethylene was examined. Ethylene was produced at 2–4 nl g^–1 h^–1 following excision of cotyledons from 5-to 20-day-old seedlings. The youngest cotyledons maintained this rate, whereas ethylene synthesis declined by as much as 80% during a 24-h period in older cotyledons. The youngest cotyledons continued to accumulate chlorophyll in the dark, but after 7 days cotyledons lost chlorophyll and the proportion of chlorophyll lost increased with age. Ethylene promoted, and norbornadiene inhibited, this loss of chlorophyll; in combined treatments the effects of ethylene and norbornadiene were competitive. The maximal rate of chlorophyll loss occurred in 1l L^–1 ethylene; extrapolation of the response to concentration indicated that half-maximum loss would occur at 0.005–0.01 l L^–1 ethylene. In cotyledons from 20-day-old seedlings, chlorophyll degradation occurred mainly after 24 h from excision and transfer to the dark. Chlorophyll degradation during 48 h in the dark was affected by norbornadiene or ethylene applied from 0–24 h or from 24–48 h.     

Immunogold labelling indicates high catalase concentrations in amorphous and crystalline inclusions of sunflower (Helianthus annuus L.) peroxisomes

Summary Immunogold labelling and electron microscopy were used to investigate whether catalase was present in peroxisomal inclusions, the composition of which has not yet been determined in plant cells. In the mesophyll cells of sunflower (Helianthus annuus L.) cotyledons, the catalase gold label was confined to peroxisomes. At day 2 of postgerminative growth in darkness, peroxisomes were free of inclusions, and the matrix was homogeneously labelled with gold particles. Thereafter, amorphous inclusions appeared, but by day 5 of growth, conspicuous crystalline inclusions (cores) were the predominant type. This developmental change, first observed in cotyledons grown in continuous light between day 2.5 and 5, also took place in cotyledons kept in permanent darkness. Both amorphous and crystalline inclusions showed a much higher immunogold label than did the peroxisomal matrix, indicating that catalase was a component of both types of peroxisomal inclusions. In contrast to catalase, the immunogold label of glycolate oxidase was almost completely absent from cores and was confined to the peroxisomal matrix. Together with reports on the absence of other enzymes from peroxisomal inclusions in sunflower and other species (Vaughn, 1989) our results suggest that catalase is a major constituent of amorphous and crystalline peroxisomal inclusions in plants.     

THE SYNTHESIS AND TURNOVER OF RAT LIVER PEROXISOMES : IV. Biochemical Pathway of Catalase Synthesis

Early events in the biosynthesis of liver catalase were studied on female rats receiving [³H]leucine or [³H]δ-aminolevulinic acid or a mixture of [³H]leucine with [¹⁴C]δ-aminolevulinic acid by intraportal injection. Catalase antigen was selectively separated from homogenates by immunoprecipitation, both without and after partial purification of the enzyme. Label from both precursors appeared first in immunoprecipitable material which was lost upon purification of catalase; the label subsequently became associated with material indistinguishable from catalase. Kinetic analysis of the results indicates that the nonpurifiable material identified by early labeling consists of two distinct biosynthetic intermediates, the first lacking heme and representing about 1.6% of the total catalase content or 13 µg/g liver, the second containing heme and representing about 0.5% of the total catalase content or 4 µg/g liver. The first intermediate migrates at the same rate as catalase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and therefore has a monomeric molecular weight of about 60,000.     

Apparent Catalase Synthesis in Sunflower Cotyledons during the Change in Microbody Function: A Mathematical Approach for the Quantitative Evaluation of Density-labeling Data

Density-labeling with 10 mm K¹⁵NO₃/70% ²H₂O has been used to investigate catalase synthesis in different developmental stages of sunflower (Helianthus annuus L.) cotyledons. A mathematical approach is introduced for the quantitative evaluation of the density-labeling data. The method allows, in the presence of preexisting enzyme activity, calculation of this synthesized activity (apparent enzyme synthesis) which results from the balance between actual enzyme synthesis and the degradation of newly synthesized enzyme at a given time. During greening of the cotyledons, when the catalase activity declines and the population of leaf peroxisomes is formed, the apparent catalase synthesis is lower than, or at best equal to, that occurring during a developmental stage when the leaf peroxisome population is established and catalase synthesis and degradation of total catalase are in equilibrium. This result suggests a formation, in fatty cotyledons, of the leaf peroxisomes by transformation of the glyoxysomes rather than by de novo synthesis.     

Specific Effects of Kinetin on the Uptake of Monovalent Cations by Sunflower Cotyledons

Kinetin promoted the uptake of K⁺ and Rb⁺ into detached sunflower cotyledons. This action was concomitant with an acceleration of growth. A slighter promotion of Li⁺ uptake was also noted, but there was no consistent influence on that of Na⁺. A small inhibitory effect on NH₄⁺₄ uptake was apparent when the latter was computed per average weight of sample during the course of incubation. Light also promoted the growth of the cotyledons, but depressed their capacity to absorb potassium. The action of kinetin on cotyledons removed from 5–7 day old seedlings was weaker than on those removed from 2–4 day old seedlings with regard to growth but stronger with regards to K⁺ uptake. When K⁺ uptake by cotyledons taken from 7-day old seedlings was followed with time the kinetin effect was already detectable within a few hours, but it became more pronounced after 10 hours' incubation. Kinetin did not accelerate growth or K⁺ uptake in hypo-cotyl segments. IAA, which was previously shown to promote these processes in hypocotyl segments, inhibited them in cotyledons. A working hypothesis is suggested according to which endogenous auxins and cytokinins regulate the absorption of K⁺ in shoot cells of the intact plant in a manner similar to that in which they act in excised tissues and in this way affect the distribution and redistribution of K⁺ in the shoot; and that they are among the factors which determine the selectivity of ion uptake in the intact plant.     

Activities of Ribulose Bisphosphate Carboxylase and Phosphoenolpyruvate Carboxylase and C-Bicarbonate Fixation during in Vitro Culture of Pinus radiata Cotyledons

The activities of ribulose bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxylase (PEPC), as indicators of autotrophic and nonautotrophic CO₂ fixation, were measured in excised cotyledons of Pinus radiata D. Don cultured for 21 days under shoot-forming (SF) and nonshoot-forming (NSF) conditions. The activity of RuBPC was found to increase in both SF and NSF cultures during the initial 5 days of culture. However, it leveled off from day 5 to day 10 and subsequently began to decrease until the end of the culture period under the SF conditions. In contrast, in the NSF cultures, RuBPC activity increased until day 15, when it was twofold higher than the maximum activity found in the SF cultures. An increase in PEPC activity of about 2.5 times the level of activity in freshly excised cotyledons was observed during the initial 5 days of culture under the SF conditions. PEPC activity began to decline after day 5 until it reached the level of activity seen in NSF cotyledons by day 15. In contrast, the activity of PEPC did not show any significant increase during the initial 10 days of culture under the NSF conditions. The K_m (phosphoenolpyruvate) of PEPC from SF cotyledons was about 35% higher than that of NSF cotyledons. Cotyledons from two culture periods (days 5 and 15) were incubated for 15 seconds with NaH¹⁴CO₃. The label in the malate and asparatate fractions as a percentage of total ¹⁴C incorporation was 3 times higher in the SF cotyledons than in the NSF cotyledons. A higher incorporation of ¹⁴C into products of photosynthesis under the NSF conditions was also observed.     

Regulation of ascorbate peroxidase activity in dark-grown radish cotyledons by a catalase inhibitor, 3-Amino-1,2,4-Triazole

The present study was performed to see the physiological role of cytosolic ascorbate peroxidase (APX) and its relationship to other enzymes involved in the H₂O₂ scavenging metabolism, and also to elucidate the regulation of APX expression in dark-grown radish (Raphanus sativus L. cv Taiwang) cotyledons. To do so, 3-amino-l,2,4-triazole (aminotriazole), a known specific inhibitor of catalase, was used to simulate a catalase-deficient phenomenon in cotyledons. Aminotriazole, in very low concetration (10^-4 M), inhibited remarkably the development of catalase activity in cotyledons during dark germination. This inhibition of catalase by aminotriazole, however, did not result in any significant changes in the growth response and the H₂O₂ level of developing cotyledons. In addition, the development of guaiacol peroxidase (GPX) activity was also not significantly affected. Unlike GPX, cytosolic APX activity was induced rapidly and reached a 1.7-fold increase in aminotriazole treated cotyledons at day 7 after germination. However,in vitro incubation of cytosolic APX preparation from cotyledons with aminotriazole did not result in any significant change in activity. One cytosolic APX isozyme (APXa) band involved in this APX activation was predominantly intensified in a native polyacrylamide gel by activity staining assay. This means that this APXa isozyme seems to play a key role in the expression of cytosolic APX activity. On the other hand, 2-day-old control seedlings treated with exogenous 1 mM H₂O₂ for 1 h showed a significant increase of cytosolic APX acitivity even in the absence of aminotriazole. Also, 2 μM cycloheximide treatment substantially inhibited the increase of APX activity due to aminotriazole. Based on these results, we suggest that a radish cytosolic APX could probably be substituted for catalase in H₂O₂ removal and that the expression of APX seems to be regulated by a change of endogenous H₂O₂ level which couples to APX protein synthesis in a translation stage in cotyledons.