Arginase activity in the cotyledons of soybean seedlings

The influence of naphthylacetic acid, abscisic acid, gibberellic acid and kinetin on the formation of aerenchyma in seedling roots of Zea mays L. cv. Capella has been studied in relation to reported changes of their concentration in poorly aerated roots, which readily form aerenchyma, and to the effects of these hormones on the production of ethylene, a major factor promoting aerenchyma formation. Because the absence of nitrate accelerates aerenchyma formation in aerated roots, their influence on these roots was compared. The growth regulators were added to roots growing in non-aerated and aerated nutrient solutions, and aerenchyma formation and the production and endogenous concentration of ethylene were measured. Naphthylacetic acid prevented aerenchyma formation in both aerated roots without nitrate and in non-aerated roots although it enhanced the ethylene concentration of the roots. Abscisic acid also prevented aerenchyma formation, but without affecting the ethylene concentration. Gibberellic acid promoted aerenchyma formation in aerated roots only, but ethylene production in both aerated and non-aerated roots. Kinetin promoted aerenchyma formation in both aerated and non-aerated roots. It stimulated ethylene production in aerated roots, but slightly inhibited it in non-aerated roots. Co²⁺ and Ag⁺, which suppress ethylene production and action, respectively, reduced the promoting effects of gibberellic acid, but not those of kinetin. It is concluded that the effects of the plant growth regulators on aerenchyma formation in maize roots were, with a possible exception for gibberellic acid, not the result of altered ethylene concentrations in the roots. Their influence on aerenchyma formation is discussed in relation to their reported actions on cell membranes.     

Immunolocalization of glyoxysomal malate synthase from soybean cotyledons (Glycine max L)

Summary— Malate synthase (MS; EC 4.1.3.2), an enzyme specific to the glyoxylate cycle, was studied in cotyledons of dark-grown soybean (Glycine max L) seedlings with light and electron microscopy techniques. Immunogold localization confirmed biochemical evidence that MS from soybean is a glyoxysomal matrix enzyme.     

Gluconeogenesis from storage wax in the cotyledons of jojoba seedlings

The cotyledons of jojoba (Simmondsia chinensis) seeds contained 50 to 60% of their weight as intracellular wax esters. During germination there was a gradual decrease in the wax content with a concomitant rise in soluble carbohydrates, suggesting that the wax played the role of a food reserve. Thin layer chromatography revealed that both the fatty alcohol and fatty acid were metabolized. The disappearance of wax was matched with an increase of catalase, a marker enzyme of the gluconeogenic process in other fatty seedlings. Subcellular organelles were isolated by sucrose gradient centrifugation from the cotyledons at the peak stage of germination. The enzymes of the β oxidation of fatty acid and of the glyoxylate cycle were localized in the glyoxysomes but not in the mitochondria. The glyoxysomes had specific activities of individual enzymes similar to those of the castor bean glyoxysomes. An active alkaline lipase was detected in the wax bodies at the peak stage of germination but not in the ungerminated seeds. No lipase was detected in glyoxysomes or mitochondria. After the wax in the wax bodies had been extracted with diethyl ether, the organelle membrane was isolated and it still retained the alkaline lipase. The gluconeogenesis from wax in the jojoba seedling appears to be similar, but with modification, to that from triglyceride in other fatty seedlings.     

Involvement of phospholipids in triglyceride biosynthesis by developing soybean cotyledons

The incorporation of phospholipids specifically labeled with glycerol-2³H and acyl-¹⁴C by whole cell tissues of developing soybean cotyledons (Glycine max L.) reveals that phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, N-acylphosphatidylethanolamine, and phosphatidic acid can be metabolized to diglyceride. The diglyceride formed may be recylced into phospholipid or acylated to triglyceride. Diglyceride from phosphatidic acid and phosphatidylethanolamine is used readily in triglyceride biosynthesis compared to the other phospholipids. Incorporation of N-acylphosphatidylethanolamine having [9-10-³H(N)]oleic acid esterified at sn-3 in cotyledons shows rapid acyltransfer of ³H into triglyceride and therefore N-acylphosphatidylethanolamine appears to participate in triglyceride biosynthesis as an acyl donor. These studies emphasize phospholipid metabolism in developing soybean cotyledons is a dynamic process which plays a key role in triglyceride formation.     

Effect of light on the development of glyoxysomal functions in the cotyledons of mustard (Sinapis alba L.) seedlings

The degradation of storage fat in the cotyledons of mustard seedlings is unaffected by phytochrome and photosynthesis (irradiation with continuous red or far-red light from sowing of the seeds) although light imposes a strong constraint on the translocation of organic matter from the cotyledons into the seedling axis. Likewise, the development and disappearance of glyoxysomal enzyme activities (isocitrate lyase, malate synthase, citrate synthase) takes place independently of light. It is concluded that the mobilization of storage fat (fatcarbohydrate transformation) is independent of photomorphogenesis. The surplus of carbohydrate produced from fat in the light seems to be converted to starch grains in the plastids, which function as a secondary storage pool in the cotyledons.Abbreviations CS citrate synthase - ICL isocitrate lyase - MS malate synthase     

Involvement of phospholipids in polyunsaturated Fatty Acid synthesis in developing soybean cotyledons

Developing soybean (cv. Dare) cotyledons harvested at 30 days after flowering were pulse-labeled with [1-(14)C]oleoyl-CoA. The metabolic interrelation of radiolabeled unsaturated fatty acids between the major glycerolipid classes was determined at various time intervals. At chase time zero, [(14)C]oleic acid accounted for 99.2% of the total glycerolipid radioactivity, and phospholipids contained 92% of the total incorporated radioactivity. With time, phospholipids were metabolized in triacylglycerol biosynthesis and radioactivity was detected in polyunsaturated fatty acids. The hypothesis that phospholipids were metabolic intermediates in polyunsaturated fatty acid biosynthesis was tested by comparing the theoretical and the actual amount of radiolabeled oleic acid that was associated with triacylglycerol as a function of time. The radioactive oleic acid found in triacylglycerol at various intervals was derived from phospholipids via a diacylglycerol intermediate. Assuming no phospholipid desaturation, the potential or theoretical amounts of [(14)C]oleic acid that could be transferred to triacylglycerol from phospholipids was defined by a system of differential equations. The results demonstrated that the decline in [(14)C]oleic acid from phospholipid after long chase intervals was equal to the total amount of radioactive unsaturated fatty acids found in neutral lipids. The difference between the theoretical and actual amounts of [(14)C]oleic acid present in triacylglycerol after long time intervals was equal to the amount of radioactivity present in polyunsaturated fatty acids. Based upon those findings in soybeans, the desaturation of oleic acid associated with phospholipids was highly probable.     

Fatty acid specificity of hormone-sensitive lipase. Implication in the selective hydrolysis of triacylglycerols.

The selective mobilization of fatty acids from white fat cells depends on their molecular structure, in particular the degree of unsaturation. The present study was designed to examine if the release of fatty acids by hormone-sensitive lipase (HSL) in vitro i) is influenced by the amount of unsaturation, ii) depends on the temperature, and iii) could explain the selective pattern of fatty acid mobilization and notably the preferential mobilization of certain highly unsaturated fatty acids. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 35 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation from 0 to 6 double bonds was measured. Fatty acid composition of in vitro released NEFA was compared with that of fat cell triacylglycerols (TAG), the ratio % NEFA/% TAG being defined as the relative hydrolysis. The relative hydrolysis of individual fatty acids differed widely, ranging from 0.44 (24:1n-9) to 1.49 (18:1n-7) with rat HSL, and from 0.38 (24:1n-9) to 1.67 (18:1n-7) with human HSL. No major difference was observed between rat and human HSL. The relative release was dependent on the number of double bonds according to chain length. The amount of fatty acid released by recombinant rat HSL was decreased but remained robust at 4 degrees C compared with 37 degrees C, and the relative hydrolysis of some individual fatty acids was affected. The relative hydrolysis of fatty acids moderately, weakly, and highly mobilized by adipose tissue in vivo was similar and close to unity in vitro. We conclude that i) the release of fatty acids by HSL is only slightly affected by their degree of unsaturation, ii) the ability of HSL to efficiently and selectively release fatty acids at low temperature could reflect a cold adaptability for poikilotherms or hibernators when endogenous lipids are needed, and iii) the selectivity of fatty acid hydrolysis by HSL does not fully account for the selective pattern of fatty acid mobilization, but could contribute to explain the preferential mobilization of some highly unsaturated fatty acids compared with others.     

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.

     

Effect of freezing and cold storage on phospholipids in developing soybean cotyledons

Freezing of plant tissue adversely affects lipid composition. Immature soybean cotyledons (Glycine max L. Merr.) var. “Harosoy 63” were frozen with liquid N₂, dry ice, or stored in a freezer (−20 C) before lipid extraction. The effects of freezing temperature, thawing rate, and cold storage on the lipid composition of frozen tissue revealed significantly higher levels of phosphatidic acid, and diminished levels of phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine from the control. Regardless of freezing temperature, phosphatidic acid levels increased from 4.7 mole% to nearly 50 mole% of the total phospholipid when frozen tissues were stored 10 days at −20 C. During the same period, N-acylphosphatidylethanolamine decreased from 54.1 mole% to 6.6 mole% phospholipid. At least 8 mole% of the phosphatidic acid increase occurred during slow thawing of the frozen tissues. In autoclaved samples, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine levels were not different from the control. Labeling of the lipid-glycerol with ³H, and fatty acids with ¹⁴C, demonstrated the degradation product was primarily phosphatidic acid. Apparently enzymic destruction of the phospholipids occurred during freezing, cold storage, and thawing.     

Glyoxylate cycle enzymes of the glyoxysomal membrane from cucumber cotyledons.

Glyoxysomes were isolated from etiolated cotyledons of cucumber seedlings. After separation of matrix proteins from the glyoxysomal membranes, enzymes were solubilized from the membranes by 100 mm MgCl₂ and purified by sedimentation velocity centrifugation, ion exchange chromatography, and separation on hydroxylapatite. Malate synthase, citrate synthase, and malate dehydrogenase the three enzymes of the glyoxylate cycle which were primarily membrane bound in this type of microbody-were thus obtained in a homogeneous form, as judged by sodium dodecyl sulfate-gel electrophoresis. Enzymatically active malate synthase, as obtained by solubilization of membrane proteins, behaved on Sepharose 6B columns as a protein with a molecular weight of about 70,000 and is characterized by an acidic isoelectric point. Malate synthase aggregates in the presence of Mg²⁺ and glyoxylate, yielding an active octamer with an alkaline isoelectric point and a molecular weight of about 540,000. Upon sodium dodecyl sulfate-gel electrophoresis, a subunit molecular weight of 63,000 was estimated. Citrate synthase exists as a dimer (molecular weight of 100,000) and tetramer (molecular weight of 200,000) and exhibits the same subunit molecular weight as the liver enzyme (46,000). Malate dehydrogenase was found to have a molecular weight similar to the microbody catalase (about 225,000), while for the single peptide chain a value of approximately 34,000 was determined.     

Biosynthesis and intracellular transport of glyoxysomal malate dehydrogenase in germinating pumpkin cotyledons

Glyoxysomal malate dehydrogenase was synthesized as a larger molecular mass precursor in germinating pumpkin cotyledons. In pulse-chase experiments, the radioactive larger molecular mass precursor (38 kDa) disappeared and was converted to the mature form (33 kDa) of the enzyme. When the radiolabeled cotyledon was fractionated into cytosolic and organellar fractions, the larger molecular mass precursor was first recovered in the cytosolic fraction and then only after a 20 min chase the mature form was found in the organellar fraction. This indicates that the higher molecular mass precursor is synthesized in the cytosol and the processing of the transient precursor is coupled to the transport into glyoxysomes.     

Purification and properties of glyoxysomal lipase from castor bean

The alkaline lipase in the glyoxysomes from the endosperm of young castor bean seedlings, an integral membrane component, was solubilized in deoxycholate:KCl and purified to apparent homogeneity. The molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 62,000 daltons. The enzyme reaction was markedly stimulated by salts and inhibited by detergents. Triricinolein, the endogenous storage lipid, was hydrolyzed by the purified enzyme which is therefore a true lipase. Treatment of intact glyoxysomes with trypsin strongly diminished the lipase activity but did not affect matrix enzymes. An antibody preparation raised in a rabbit against the purified enzyme inhibited the purified enzyme and that in glyoxysomal membranes.     

Effects of exogenous methionine on storage protein composition of soybean cotyledons cultured in vitro

Supplemental methionine in a complete culture medium increased the methionine content of the protein fraction of cultured soybean (Glycine max L. Merrill) cotyledons (Thompson, Madison, Muenster 1981 Phytochemistry 20: 941-945). To explain the observed increase in protein methionine, we have measured the amounts and subunit compositions of 7S and 11S storage proteins and determined the amino acid compositions of the three major protein fractions (2-5S, 7S, 11S) of seeds developed on plants and of cultured cotyledons grown in the presence or absence of supplemental l-methionine. Development of cultured cotyledons was representative of development of seeds on plants. The ratios of 11S to 7S proteins, the subunit contents, and amino acid compositions of their storage protein fractions were similar, but not identical. Supplemental methionine increased the mole percent methionine in each of the three protein fractions of cultured cotyledons and changed the amounts of several other amino acids. Supplemental methionine inhibited expression of the 7S beta-subunit gene. Concomitant with the absence of the beta-subunit, which contains no methionine, was an increase in the ratio of 11S to 7S proteins, and an increase in the methionine content of the subunits composing these fractions. Inhibition of beta-subunit gene expression by methionine in cultured cotyledons provides a reproducible, easily controlled system for the study of eucaryotic gene expression.     

Citrate metabolism in. soybean cotyledons

Citrate metabolism and the citrate cleavage enzyme were investigatedin soybean (Glycine max (L.), Merr.) cotyledons throughout developmentand during the first 5 days of germination. It was noted thatboth the lipid synthesizing and the acetyl CoA generating systemsare present when the soybean seed matures, and that the activityof these systems declines throughout development as citrateincreases. Citrate represents 1% of the cotyledon dry weightat seed maturity. During the first 24 hr of germination, therewas an activation of the citrate cleavage enzyme and a concomitantdrop of some 60% in citrate content. Accompanying the drop incitrate content is the de novo synthesis of fatty acids foruse in the production of phospholipids. All of the data areconsistent with the hypothesis that acetyl CoA for lipid synthesisis supplied by the citrate molecule via the citrate cleavageenzyme and that activity of this system is necessary both duringseed development and during germination. ¹ Research was supported by cooperative investigations of theAgricultural Research Service, United States Department of Agriculture,and Illinois Agricultural Experiment Station. ² This research represents partial fulfillment of Ph.D. degree. (Received September 20, 1976; )     

Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings

The aim of this study was to design a convenient, specific, sensitive, and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs were used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent because more than half of the fatty acids from Parinari oil are known to contain 9,11,13, 15-octadecatetraenoic acid (parinaric acid) in its esterified form. The presence of detergents (sodium taurodeoxycholate, CHAPS, Sulfobetaine SB12, Tween 20, Brij 35, Dobanol, n-dodecylglucoside) above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase in the fluorescence intensity is linear with time and proportional to the amount of lipase added. This new method, performed under non-oxidative conditions, was applied successfully to detecting low lipase levels in crude protein extracts from plant seeds and could be scaled down to microtiterplate measurements. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8). Lipase activity can also be assayed in acidic media (pH 5) using human gastric lipase. This simple and continuous assay is compatible with a high sample throughput and might be applied to detecting true lipase activities in various biological samples.     

Development of enzymes in the cotyledons of watermelon seedlings

Changes in hypocotyl length, cotyledon weight, lipid content, chlorophyll content, and capacity for photosynthesis have been described in seedlings of Citrullus vulgaris, Schrad. (watermelon) growing at 30 C under various light treatments. Corresponding changes in the levels of 19 enzymes in the cotyledons are described, with particular emphasis on enzymes of microbodies, since during normal greening, enzymes of the glyoxysomes are lost and those of leaf peroxisomes appear. In complete darkness enzymes of the glyoxysomes reach a peak at 4 days and decline as the fat is depleted. Enzymes of mitochondria and of glycolytic pathways also peak at 4 to 5 days and either remain unchanged or decline to a lesser extent. Exposure to light at 4 days, when the cotyledons emerge, results in a selectively greater destruction of enzymes of the glyoxylate cycle; chlorophyll synthesis and capacity for photosynthesis increase in parallel, and there is a striking increase in the activities of chloroplast enzymes and in those of the leaf peroxisomes, hydroxypyruvate reductase and glycolate oxidase. The reciprocal changes in enzymes of the glyoxysomes and of leaf peroxisomes can be temporally dissociated, since even after 10 days in darkness, when malate synthetase and isocitrate lyase have reached very low levels, hydroxypyruvate reductase and glycolate oxidase increase strikingly on exposure to light and the cotyledons become photosynthetic. Furthermore, the parallel development of enzymes of leaf peroxisomes and functional chloroplasts is not immutable, since hydroxypyruvate reductase and glycolate oxidase activity can be elicited in darkness following a 5-minute exposure to light at day 4 while chlorophyll does not develop under these conditions.     

Translocation of iron from soybean cotyledons

Soybean seeds, Glycine max L. Merrill, were produced by plants treated from anthesis to seed maturity with ⁵⁹Fe supplied as ferric ethylenediaminedi (o-hydroxyphenylacetate). Seed coats accounted for 7.4% of dry seed weight and had Fe concentrations 5 times greater than the embryos. After germinating 2 days, cotyledons contained 69.6% and radicles 5.0% of original seed Fe. Fractions of seed Fe unavailable to seedlings were: 19.8% in seed coats, 1.7% in germination paper, 0.1% in the water under germinating seeds, and 3.8% unaccounted for. Every 3 days seedlings received nutrient solution without Fe or with 10 μm ferric ethylenediaminedi (o-hydroxyphenylacetate) and developed as deficient Fe or normal Fe plants. The deficient Fe cotyledons on day 18 retained 13% of the labeled Fe originally present. Cotyledons of normal Fe plants retained 50 to 70% of their original Fe. Moreover, cotyledons of the normal Fe plants accumulated externally supplied Fe and finally contained twice the quantity of Fe originally present. Stem exudate collected above cotyledons of deficient Fe plants contained 5.3 μm⁵⁹Fe. Electrophoresis of exudate showed that most of the ⁵⁹Fe migrated anodically as a single band and was in the position of ferric citrate.     

Involvement of gibberellins in expression of a cysteine proteinase (SH-EP) in cotyledons of Vigna mungo seedlings.

The expression of a papain-type proteinase, designated SH-EP, in cotyledons of Vigna mungo seedlings has been shown to require some factors in the embryonic axes. Gibberellin A1 (GA(1)) and GA(20) were identified by GC-MS in embryonic axes of V. mungo seedlings. The level of accumulation of SH-EP in cotyledons of V. mungo seedlings was greatly reduced by treatment of the seeds with uniconazole-P, an inhibitor for GA biosynthesis. The reduced level of accumulation of SH-EP in cotyledons by uniconazole-P was recovered by exogenous application of GA(1) and GA(20) to the seedlings.     

A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings

Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds. During germination, stored reserves are used as a source of nutrients by the plant seedling. Phytic acid is degraded by the activity of phytases to yield inositol and free phosphate. Due to the lack of phytases in the non-ruminant digestive tract, monogastric animals cannot utilize dietary phytic acid and it is excreted into manure. High phytic acid content in manure results in elevated phosphorus levels in soil and water and accompanying environmental concerns. The use of phytases to degrade seed phytic acid has potential for reducing the negative environmental impact of livestock production. A phytase was purified to electrophoretic homogeneity from cotyledons of germinated soybeans (Glycine max L. Merr.). Peptide sequence data generated from the purified enzyme facilitated the cloning of the phytase sequence (GmPhy) employing a polymerase chain reaction strategy. The introduction of GmPhy into soybean tissue culture resulted in increased phytase activity in transformed cells, which confirmed the identity of the phytase gene. It is surprising that the soybean phytase was unrelated to previously characterized microbial or maize (Zea mays) phytases, which were classified as histidine acid phosphatases. The soybean phytase sequence exhibited a high degree of similarity to purple acid phosphatases, a class of metallophosphoesterases.