The Development of Glyoxysomes in Peanut Cotyledons and Maize Scutella

The development of glyoxysomes during germination has been studied in isolated peanut (Arachis hypogaea L.) cotyledons and in maize (Zea mays L.) scutella. In peanut cotyledons isocitratase, malate synthetase, and protein associated with the glyoxysomal fraction increase simultaneously from the 3rd to the 8th day of incubation. In scutella of germinating maize seeds the specific activities of isocitratase, malate synthetase, and catalase associated with the glyoxysomes rise until the 4th day of germination and then decline while the total amount of protein present in the fraction stays constant during the first 5 days. If the peanut cotyledons are cultured in 2% glucose, the development of isocitratase and malate synthetase is severely inhibited, but the level of the glyoxysomal protein is not measurably affected.     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt     

In vivo and in vitro studies on asparagine biosynthesis in soybean seedlings

The biosynthesis of asparagine in plants was investigated by feeding radioactive metabolites to soybean cotyledons and by extracting an asparagine synthetase from the same tissue. Soybean cotyledon slices were supplied with radioactive succinate, malate, or aspartate in the presence or absence of various unlabeled metabolites for periods of up to 80 min. Neither aspartate nor malate was rapidly converted to asparagine; labeled aspartate was converted largely to malate. Labeled succinate was rapidly converted to asparagine, and several lines of evidence suggested that fumarate, malate, and aspartate are intermediates. The results suggest that asparagine biosynthesis in plant cells is compartmentalized beginning with succinate. Although results were also consistent with asparagine formation via aspartate, metabolism of a mixture of [¹⁴C] plus [³H]succinate resulted in a lower ${}^{14}\text{C}{}^3\text{H}$ ratio in asparagine than aspartate, suggesting that some asparagine may be formed via another pathway. Demonstration of a glutamine-linked asparagine synthetase in soybean cotyledons supports the idea that asparagine is formed via aspartate. The enzyme requires aspartate (K_m = 2.2 mm), glutamine (K_m = 0.12 mm), ATP (K_m = 0.066 mm), magnesium ion, and sulfhydryl protection. It has a pH optimum of 7.7 and is not located in mitochrondria. A small amount of asparagine was formed when ammonium ion was substituted for glutamine, but the K_m of the enzyme for ammonium ion was about 25-fold greater than the K_m for glutamine suggesting that glutamine is the physiologically important substrate. Soybean cotyledons actively convert [¹⁴C]-cyanide to asparagine, apparently via β-cyanoalanine. However, malate was also rapidly labeled from [¹⁴C]cyanide and this result cannot be explained by known metabolic pathways.     

Solubilization of enzymes from glyoxysomes of maize scutellum

Glyoxysomes isolated from maize scutella (Zea mays L.) were subjected to several disruptive treatments (osmotic shock, resuspension in an alkaline medium, addition of detergent). The damaged glyoxysomes were centrifuged at 89,500g for 40 minutes and several enzymic activities (isocitratase, malate synthetase, catalase, citrate synthetase, malate dehydrogenase) were measured in the supernatant fraction and in the pellet. Isocitratase is the most easily released of all glyoxysomal enzymes closely followed by malate synthetase. Citrate synthetase is in all instances the most insoluble enzyme. All of the enzymes had higher specific activities in the supernatant than in the pellet. These findings suggest that in corn scutellum glyoxysomes none of these enzymes is truly membrane-bound.     

Evidence that Blastocladiella emersonii zoospore chitin synthetase is located at the plasma membrane

Blastocladiella emersonii zoospores are not encased by a cell wall and do not detectably synthesize or contain chitin; accompanying de novo cell wall formation during zoospore encystment, chitin rapidly accumulates and is incorporated into the cell wall. Essential for understanding this abrupt change in chitin synthesis is the location of zoospore chitin synthetase. The enzyme has previously been reported to the sequestered with distinctive cytoplasmic organelles (gamma particles) characteristic for the zoospore cell type. Using similar differential and equilibrium density centrifugation procedures to those reported previously, we have observed the vast majority of zoospore homogenate chitin synthetase activity in fractions distinct from the gamma particle-enriched fractions. Over 90% of the homogenate enzyme activity could be recovered in a sucrose buoyant density region (1.14–1.18 g/ml) containing membranous elements and well separated from the region enriched for gamma particles (1.30–1.34 g/ml). When zoospores were surface-labelled with [³H]concanavalin A prior to homogenization, the buoyant density regions of radioactivity and of chitin synthetase activity exhibited nearly complete coincidence. At least the bulk of zoospore chitin synthetase appears to be located at the plasma membrane, rather than in gamma particles.     

De Novo Synthesis of Isocitritase in Peanut (Arachis hypogaea L.) Cotyledons

Germination of peanut seed is accompanied by a rapid increase in isocitritase (isocitrate lyase, EC 4.1.3.1) during the first 4 days. The presence of cycloheximide (50 μg/ml) during water imbibition inhibited the increase in isocitritase activity. Actinomycin D conversely did not inhibit isocitritase activity until the second day of imbibition while RNA synthesis was inhibited. Germination of peanut seed in ¹⁴C-reconstituted amino acids followed by fractionation of a 20 to 35% ammonium sulfate preparation on a Sephadex G-200 column (57-fold purification) showed that the active enzymic fraction coincided with a large peak of radioactivity. Germination of peanut seed in 45% D₂O followed by enzyme purification and CsCl equilibrium centrifugation revealed that all the enzyme from D₂O seed had a higher density than normal isocitritase. These data indicate that isocitritase in peanut seed is synthesized de novo.     

Mitochondrial malate dehydrogenase of watermelon cotyledons: Time course and mode of enzyme activity changes during germination

Summary Specific antibodies were prepared against the purified mitochondrial malate dehydrogenase (EC 1.1.1.37) from cotyledons of watermelon seedlings (Citrullus vulgaris Schrad.). The isoenzyme was assayed by means of quantitative radial immunodiffusion. Cotyledons of ungerminated seeds were found to contain mitochondrial MDH. During the first 4 days of germination the enzyme activity increased threefold finally contributing 16% to the total MDH activity extracted from cotyledon tissue. Isopycnic CsCl density centrifugation was used to investigate the mode of activity increase. After a four-day period of labelling with deuterium oxide and purification of the mitochondrial isoenzyme, a density shift of 0.021kgx1^-1, accompanied by considerable band broadening of the enzyme profile was observed. These findings are evidence for the de novo synthesis of mitochondrial MDH and its relatively slow turnover in germinating seeds.Abbreviations mMDH mitochondrial malate dehydrogenase - D₂O deuterium oxide     

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.     

Asparagine synthesis in pea leaves, and the occurrence of an asparagine synthetase inhibitor

Asparagine is present in the mature leaves of young pea (Pisum sativum cv Little Marvel) seedlings, and is synthesized in detached shoots. This accumulation and synthesis is greatly enhanced by darkening. In detached control shoots, [¹⁴C]aspartate was metabolized predominantly to organic acids and, as other workers have shown, there was little labeling of asparagine (after 5 hours, 3.1% of metabolized label). Addition of the aminotransferase inhibitor aminooxyacetate decreased the flow of aspartate carbon to organic acids and enhanced (about 3-fold) the labeling of asparagine. The same treatment applied to darkened shoots resulted in a substantial conversion of [¹⁴C]aspartate to asparagine, over 10-fold greater than in control shoots (66% of metabolized label), suggesting that aspartate is the normal precursor of asparagine.

Only traces of glutamine-dependent asparagine synthetase activity could be detected in pea leaf or root extracts; activity was not enhanced by sulfhydryl reagents, oxidizing conditions, or protease inhibitors. Asparagine synthetase is readily extracted from lupin cotyledons, but yield was greatly reduced by extraction in the presence of pea leaf tissue; pea leaf homogenates contained an inhibitor which produced over 95% inhibition of an asparagine synthetase preparation from lupin cotyledons. The inhibitor was heat stable, with a low molecular weight. Presence of an inhibitor may prevent detection of asparagine synthetase in pea extracts and in Asparagus, where a cyanide-dependent pathway has been proposed to account for asparagine synthesis: an inhibitor with similar properties was present in Asparagus shoot tissue.

     

Fermentative products and dark CO2 fixation during germination of seeds of Cicer arietinum

During the germination of Cicer arietinum L. the amounts of ethanol, lactate and malate reached their highest values at 24 hr, the concentration of ethanol being about 4 times that of lactate and twice that of malate. The activities of phosphoenolpyruvate carboxylase and malic enzyme seem to be correlated with the ability of cotyledons to fix CO₂ from NaH¹⁴CO₃ into malate and with the further decrease in this metabolise from 36 hr onwards.     

N and C NMR determination of allantoin metabolism in developing soybean cotyledons

The metabolism of allantoin by immature cotyledons of soybean (Glycine max L. cv Elf) grown in culture was investigated using solid state ¹³C and ¹⁵N nuclear magnetic resonance. All of the nitrogens of allantoin were incorporated into protein in a manner similar to that of each other and to the amide nitrogen of glutamine. The C-2 of allantoin was not incorporated into cellular material; presumably it was lost as CO₂. About 50% of the C-5 of allantoin was incorporated into cellular material as a methylene carbon; the other 50% was presumably also lost as CO₂. The ¹³C-¹⁵N bonds of [5-¹³C;1-¹⁵N] and [2-¹³C;1,3-¹⁵N]allantoin were broken prior to the incorporation of the nitrogens into protein. These data are consistent with allantoin's degradation to two molecules of urea and one two-carbon fragment. Cotyledons grown on allantoin as a source of nitrogen accumulated 21% of the nitrogen of cotyledons grown on glutamine. Only 50% of the nitrogen of the degraded allantoin was incorporated into the cotyledon as organic nitrogen; the other 50% was recovered as NH₄⁺ in the media in which the cotyledons had been grown. The latter results suggests that the lower accumulation of nitrogen by cotyledons grown on allantoin was in part due to failure to assimilate NH₄⁺ produced from allantoin. The seed coats had a higher activity of glutamine synthetase and a higher rate of allantoin degradation than cotyledons indicating that seed coats play an important role in the assimilation and degradation of allantoin.     

Plant Disease and the Regulation of Enzymes Involved in Lignification: Increased Rate of De Novo Synthesis of the Three Tobacco O-Methyltransferases during the Hypersensitive Response to Infection by Tobacco Mosaic Virus

The mechanism underlying the increase of activity of the three O-methyltransferases of tobacco (Nicotiana tabacum) after infection by tobacco mosaic virus (TMV) has been investigated with a density-labeling method. The three O-methyltransferases from healthy or TMV-infected leaves fed with H₂O or ²H₂O were purified by ion-exchange chromatography and their mean buoyant densities were calculated from their respective distribution profiles after centrifugation to equilibrium on RbCl gradients. Densities were corrected with respect to the mean buoyant density of a radioactive density marker prepared from tobacco leaves floated on a solution containing l-[³H]leucine and selected on a preparative gradient for its density close to those of the O-methyltransferases. The introduction of ²H into the pool of amino acids from which the enzymic proteins were synthesized was monitored. By measurement of the labeling of β-galactosidase synthesized by bacteria from the plant amino acids, it was shown that infection did not alter the rate of labeling of the pool of amino acids. The buoyant-density values of the three O-methyltransferases were determined, and density-labeled enzymes from healthy and infected materials were compared. The largest density shifts from the native enzyme were measured for O-methyltransferases from infected leaves. These results show that the increase in activity of the three enzymes after infection is due to the stimulation of the rate of de novo synthesis of enzyme proteins.     

N-ammonia assimilation, 2-oxoglutarate transport, and glutamate export in spinach chloroplasts in the presence of dicarboxylates in the light

The direct incorporation of ¹⁵NH₄Cl into amino acids in illuminated spinach (Spinacia oleracea L.) chloroplasts in the presence of 2-oxoglutarate plus malate was determined. The amido-N of glutamine was the most highly labeled N-atom during ¹⁵NH₄ assimilation in the presence of malate. In 4 minutes the ¹⁵N-label of the amido-N of glutamine was 37% enriched. In contrast, values obtained for both the N-atom of glutamate and the amino-N of glutamine were only about 20% while that of the N-atom of aspartate was only 3%. The addition of malate during the assimilation of ¹⁵NH₄Cl and Na¹⁵NO₂ greatly increased the ¹⁵N-label into glutamine but did not qualitatively change the order of the incorporation of ¹⁵N-label into all the amino acids examined. This evidence indicates the direct involvement of the glutamine synthetase/glutamate synthase pathway for ammonia and nitrite assimilation in isolated chloroplasts. The addition of malate or succinate during ammonia assimilation also led to more than 3-fold increase in [¹⁴C]2-oxoglutarate transport into the chloroplast as well as an increase in the export of [¹⁴C]glutamate out of the chloroplast. Little [¹⁴C]glutamine was detected in the medium of the chloroplast preparations. The stimulation of ¹⁵N-incorporation and [¹⁴C]glutamate export by malate could be directly attributed to the increase in 2-oxoglutarate transport activity (via the 2-oxoglutarate translocator) observed in the presence of exogenous malate.     

Synthesis of Isocitrate Lyase in Sunflower Cotyledons during the Transition in Cotyledonary Microbody Function

Density-labeling with 10 millimolar K¹⁵NO₃/70% ²H₂O has been used to investigate isocitrate lyase synthesis during greening of sunflower (Helianthus annuus L.) cotyledons when the glyoxysomal enzyme activities sharply decline and the transition in cotyledonary microbody function occurs. A density shift of 0.0054 (kilograms per liter) was obtained for the profile of isocitrate lyase activity in the CsCl gradient with respect to the ¹H₂O control. Quantitative evaluation of the density-labeling data indicates that about 50% of the isocitrate lyase activity present towards the end of the transition stage in microbody function is due to enzyme molecules newly synthesized during this stage.     

Isolation and preliminary characterization of cryptic satellite DNA in pea

Optimum conditions have been established for isolation of ‘cryptic’ satellite DNA from the genome of pea (Pisum sativum), using gradients of CS₂SO₄ containing silver ions. At an Ag⁺ :DNA-P ratio (R) of 0.1, and at alkaline pH, four fractions are obtained: mainband (buoyant density 1.437 g cm³; 67% of total DNA), satellite I (buoyant density 1.582 g/cm³; 7% of total DNA), satellite II (buoyant density 1.520 g/cm³, 11% of total) and satellite III (buoyant density variable between 1.45 and 1.51 g/cm³; 15% of total). The reiterated DNA content of these four fractions has been investigated by reassociation experiments conducted over a Cot range of 1 × 10^?5 to 2.0. All four fractions contain at least two kinetic components; each fraction, including the mainband, consists at least partly of highly reiterated DNA. Ribosomal RNA hybridizes only to the mainband.     

Malate synthesis by dark carbon dioxide fixation in leaves

The rates of dark CO₂ fixation and the label distribution in malate following dark ¹⁴CO₂ fixation in a C-4 plant (maize), a C-3 plant (sunflower), and two Crassulacean acid metabolism plants (Bryophyllum calycinum and Kalanchoë diagremontianum leaves and plantlets) are compared. Within the first 30 minutes of dark ¹⁴CO₂ fixation, leaves of maize, B. calycinum, and sunflower, and K. diagremontianum plantlets fix CO₂ at rates of 1.4, 3.4, 0.23, and 1.0 μmoles of CO₂/mg of chlorophyll· hour, respectively. Net CO₂ fixation stops within 3 hours in maize and sunflower, but Crassulaceans continue fixing CO₂ for the duration of the 23-hour experiment.

A bacterial procedure using Lactobacillus plantarum ATCC No. 8014 and one using malic enzyme to remove the β-carboxyl (C₄) from malate are compared. It is reported that highly purified malic enzyme and the bacterial method provide equivalent results. Less purified malic enzyme may overestimate the label in C₄ as much as 15 to 20%.

The contribution of carbon atom 1 of malate is between 18 and 21% of the total carboxyl label after 1 minute of dark CO₂ fixation. Isotopic labeling in the two carboxyls approached unity with time. The rate of increase is greatest in sunflower leaves and Kalanchoë plantlets. In addition, Kalanchoë leaves fix ¹⁴CO₂ more rapidly than Kalanchoë plantlets and the equilibration of the malate carboxyls occurs more slowly. The rates of fixation and the randomization are tissue-specific. The rate of fixation does not correlate with the rate of randomization of isotope in the malate carboxyls.

     

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.     

Chitin synthetase activity is bound to chitosomes anf to the plasma membrane in protoplasts of Saccharomyces cerevisiae

The sub-cellular distribution of chitin synthetase was studied in homogenates of Saccharomyces cerevisiae protoplasts. Use of a mild disruption method minimized rupture of vacuoles and ensuing contamination of subcellular fractions by vacoular proteinases. After fractionation of whole or partially purified homogenates through an isopycnic sucrose gradient chitin synthetase activity was found to be distributed between two distinct particulate fractions with different buoyant density and particle diameter. When whole homogenates were used, about 52% of the chitin synthetase loaded was localized in a microvesicular population identified as chitosomes (diameter 40–110 nm; bouyant density (d) = 1.146 g/cm³). Another vesicular population containing 26% of the activity was identified as plasma membrane vesicles because of its large mean diameter (260 nm), its high buoyant density (d = 1.203 g/cm³) and by the presence of the vanadate-sensitive ATPase activity. Moreover, after surface labeling of protoplasts with ³H-concanavalin A, the label cosedimented with the presumed plasma membrane vesicles. There was a negligible cross-contamination of the chitosome fraction by yeast plasma membrane markers. In both the plasma membrane and the chitosome fractions, the chitin synthetase was stable and essentially zymogenic. Activation of the chitosome fraction produces microfibrils 100–250 nm in length. Our results support the idea that chitosomes do not originate by plasma membrane vesiculation but are defined sub-cellular organelles containing most of the chitin synthetase in protoplasts of Saccharomyces cerevisiae.     

Leucine specific transfer ribonucleic acids and synthetases in the cotyledons of mature and germinating pea seeds

Changes in isoaccepting species of tRNA^Leu were determined in germinating pea seedlings and in developing pods. Leucine specific transfer ribonucleic acids of pea cotyledons can be fractionated into four isoaccepting species by reversed-phase chromatography (RPC-5) on a Plaskon column. In contrast, only two species of tRNA^Leu were observed in developing seed pods. Leucyl-tRNA synthetase purified by ammonium sulfate precipitation and DEAE cellulose column chromatography retained the full range of specificity towards all four tRNA^Leu species of pea cotyledons. This partially purified pea cotyledon enzyme could be further separated on a hydroxylapatite (HA) column into two peaks of leucyl-tRNA synthetase activity. Enzyme 1 is dominant in seed pods while 2 is predominant in cotyledons. Enzymes 1 and 2 from cotyledons were examined for the amino acid acceptor activity of twelve different amino acids. Both these fractions showed less than 3% acceptor activity for eleven other amino acids as compared to leucine-tRNA synthetase activity. Preliminary characterization of enzyme 2 from cotyledon, by isoelectric focusing and polyacrylamide gel electrophoresis indicates at least three subspecies.