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PubMed1980

Effects of Several Tunicamycin-like Antibiotics on Glycoprotein Biosynthesis in Mung Beans and Suspension-cultured Soybean Cells.

The antibiotics Streptovirudin and 24010 were tested to determine their effects on the formation of lipid-linked saccharide intermediates associated with glycoprotein biosynthesis in mung bean (Vigna radiata) and suspension-cultured soybean cells (Glycine max cv. Mandarin). In vitro both compounds strongly inhibited the transfer of N-acetyl[(3)H]glucosamine from UDP-N-[(3)H]acetylglucosamine to N-acetylglucosaminyl-pyrophosphoryl-polyisoprenol and lipid-linked oligosaccharides, although they had no apparent effect on the incorporation of [(14)C]mannose from GDP-[(14)C]mannose into mannosyl-phosphoryl-dolichol with a small inhibition into lipid-linked oligosaccharides. In vivo, Streptovirudin and tunicamycin dramatically inhibited the incorporation of N-[(14)C]acetylglucosamine and [(3)H]mannose into Pronase-released material (glycoproteins), whereas there was no effect on [(3)H]leucine incorporation into Pronase-released material (protein). Because the action of Streptovirudin and antibiotic 24010 in plants and other systems is similar to that for tunicamycin, these antibiotics are believed to be closely related. The use of tunicamycin is discussed with respect to its importance in studying glycoprotein biosynthesis and function in animal and plant systems.

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PubMed1980

Nitrate Effect on Nitrogen Fixation (Acetylene Reduction): ACTIVITIES OF LEGUME ROOT NODULES INDUCED BY RHIZOBIA WITH VARIED NITRATE REDUCTASE ACTIVITIES.

The effect of nitrate on symbiotic nitrogen fixation by root nodules of cowpea (Vigna unguiculata L., Walp., cv. California Blackeye) and lupine (Lupinus augustifolius L., cv. Frost) plants inoculated with nitrate reductase-expressing and nitrate reductase-nonexpressing Rhizobium strains were examined. Nitrate reductase of Rhizobium bacteroids in the nodules of cowpea and lupine reduced nitrate to nitrite. Both cowpea and lupine nodules accumulated nitrite when grown in the presence of 15 millimolar nitrate and induced by Rhizobium strains which express nitrate reductase activity (Rhizobium sp. 32H1 and 127E15). The nitrogen fixation (acetylene reduction) activities of cowpea and lupine nodules were inhibited by nitrate whether the nodules were induced by Rhizobium strains that express (Rhizobium sp. 32H1 and 127E15) or do not express (Rhizobium sp. 127E14 and R. lupini ATCC 10318) nitrate reductase activity. These findings indicate that nitrite, the product of bacteroid nitrate reductase, may not play a role in the inhibitory effect of nitrate on nitrogen fixation activities of legume root nodules. However, the degree of inhibition on the fixation activity by nitrate varied in different legume-Rhizobium combinations.

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PubMed1980

Characteristics and subcellular localization of phospholipase d and phosphatidic Acid phosphatase in mung bean cotyledons.

Mixed micelles of (32)P-labeled phosphatidylcholine or phosphatidic acid (PA) and the nonionic detergent octylphenol polyethylene oxide (NP-40 Nonidet) were used to assay the activities of phospholipase D and PA phosphatase in crude extracts of mung bean (Vigna radiata) cotyledons. Together these enzymes degrade phosphatidylcholine to free choline, inorganic phosphate, and sn-1,2-diacylglycerol. Both enzymes have pH optima around 5.0. The enzymes are present in fully imbibed cotyledons and increase in activity during seedling growth. Fractionation of cotyledon extracts on sucrose gradients showed that the cells contain two PA phosphatases. One enzyme with a pH optimum of 7.5 has the same distribution on sucrose gradient as the endoplasmic reticulum marker enzyme NADH-cytochrome c reductase. The other, PA phosphatase, with a pH optimum of 5.0, was present in a protein body-rich fraction and in the load portion of the gradient. Fractionation of broken protoplasts on Ficoll gradients (a method which allows for the isolation of a high proportion of intact protein bodies) indicates that most of the cellular phospholipase D and PA phosphatase (pH 5.0) are associated with the protein bodies. Using column chromatography (DEAE-cellulose and Sephadex G-200), PA phosphatase (pH 5.0) was found to be a different enzyme from the major acid phosphatase in the cotyledons. Apparent molecular weights of phospholipase D and PA phosphatase were 150,000 and 37,000, respectively. The activity of phospholipase D was not affected by free choline, but was markedly inhibited by the choline analog and plant growth retardant isopropyl 4'-(trimethylammonium chloride-5'-methylphenyl piperidine-1-carboxylate (AMO 1618). The finding that these acid hydrolases are located in the protein bodies supports the conclusion that protein bodies form the general lytic compartment in the storage parenchyma cells.

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PubMed1981

Exclusion of selenium from proteins of selenium-tolerant astragalus species.

Protein fractions from three selenium-tolerant and three selenium-sensitive Astragalus species, grown in the presence of [(75)Se]selenate, were analyzed for their selenium content. Though tolerant species are known to accumulate considerably more selenium than do sensitive plants, protein fractions from the three selenium accumulators were found to contain significantly less selenium (0.46 to 0.57 picomoles selenium per milligram protein) than did protein fractions from the three nonaccumulators (4.17 to 5.02 picomoles selenium per milligram protein). Under similar conditions, seedlings of Vigna radiata (L.) Wilczek had taken up selenium (6.31 picomoles selenium per milligram protein) at levels comparable to those observed in the proteins of the nonaccumulator Astragali. These results establish that the ability to tolerate and to circumvent the toxic effects of selenium, characteristic of the accumulator species of Astragalus, is associated with a reduced incorporation of this element into protein.

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PubMed1981

Differential compartmentation of gibberellin a(1) and its metabolites in vacuoles of cowpea and barley leaves.

The metabolism and efflux of gibberellin A(1) (GA(1)) taken up by leaves of cowpea (Vigna sinensis cv. Blackeye pea No. 5), as well as the distribution of GA(1) metabolites in the protoplasts and vacuoles of cowpea and barley (Hordeum vulgare L. cv. Numar), were studied.GA(1) is metabolized rapidly in cowpea leaf discs to products tentatively identified as gibberellin A(8) (GA(8)) and gibberellin A(8) glucoside (GA(8)-glu). After labeling leaf discs with [(3)H]GA(1) for 1 hour, the release of radioactivity from the leaf was followed. Over a 12-hour period, the level of radioisotope in the tissue declined to about 35% of the original, after which no further release was observed. At this time, almost all of the radioactivity remaining in the leaf was GA(8)-glu, while most of the radioactivity which had been released was unmetabolized GA(1).Mesophyll protoplasts and vacuoles were isolated from cowpea and barley leaves previously fed [(3)H]GA(1). These protoplasts retain the ability to metabolize GA(1), indicating that neither the leaf structure nor the cell wall is necessary for this metabolism. A higher proportion of GA(8)-glu was found in the vacuoles relative to the entire protoplasts. The results obtained suggest that GA(1) metabolites are preferentially compartmentalized in the vacuoles relative to GA(1).

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PubMed1981

Transient susceptibility of root cells in four common legumes to nodulation by rhizobia.

Root cells of four common legumes were found to remain susceptible to nodulation by rhizobia for only a short period of time. Delayed inoculation experiments conducted with these legume hosts indicated that the initially susceptible region of the root became progressively less susceptible if inoculations were delayed by a few hours. Profiles of the frequency of nodule formation relative to marks indicating the regions of root and root hair development at the time of inoculation indicated that nodulation of Vigna sinensis (L.) Endl. cv California Black Eye and Medicago sativa L. cvs Moapa and Vernal roots was inhibited just below the region that was most susceptible at the time of inoculation. This result suggests the existence of a fast-acting regulatory mechanism in these hosts that prevents overnodulation. Nodulation in white clover may occur in two distinct phases. In addition to the transient susceptibility of preemergent and developing root hair cells, there appeared to be an induced susceptibility of mature clover root hair cells. A cell-free bacterial exudate preparation from Rhizobium trifolii cells was found to render mature root hair cells of white clover more rapidly susceptible to nodulation.

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PubMed1981

Solute leakage resulting from leaf desiccation.

The leakage of solutes from foliar tissue is utilized as a dynamic measure of apparent changes in membrane integrity in response to desiccation. It is found that rehydrating leaf discs of cowpea (Vigna sinensis [L.] Endl.) show increasing leakiness in proportion to the extent of prior desiccation, whereas Selaginella lepidophylla Spring., a resurrection plant, does not. The elevated leakage rate of cowpea after desiccation recovers with time, and the passage of time in the stressed condition results in reduced subsequent leakiness. These characteristics are interpreted as suggesting that the leakage of solute reflects the condition of cellular membranes, and that desiccation stress leads to lesions in the membranes. The kinetics of solute leakage is suggested as a simple means of following changes in membrane lesions and associated features of membrane repair and hardening.

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PubMed1982

Callose induction in cowpea by uridine diphosphate glucose and calcium phosphate-boric Acid treatments.

Calcium phosphate-boric acid treatments and UDP-glucose both elicited aniline blue fluorescent, periodic acid-Schiff's reagent-resistant, deposits in association with the cell walls of cowpea (Vigna sinensis [Torner] Savi cv. Early Ramshorn) tissue. Those deposits induced by calcium phosphateboric acid treatment ultrastructurally resembled the "wound callose" commonly triggered by cell damage; they were formed in seemingly intact cells of stems and leaves and their formation was associated with an increase in the surface density of rough endoplasmic reticulum in the cell cytoplasm. In contrast, UDP-glucose induced a more rapid accumulation of aniline blue fluorescent material, but only at the cut edges of stem slices. Comparative light and electron microscopy indicated that the material was incorporated into the walls of the damaged cells, even when such cells were devoid of organized cytoplasm. These results indicate a difference in the mode and site of synthesis between wound callose and that elicited by exogenous UDP-glucose. They support the hypothesis that externally supplied UDP-glucose cannot be utilized by intact cells.

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PubMed1982

Arginine and ornithine decarboxylases, the polyamine biosynthetic enzymes of mung bean seedlings.

General properties and relative activities of l-arginine decarboxylase (ADC) (EC 4.1.1.19) and l-ornithine decarboxylase (ODC) (EC 4.1.1.17), two important enzymes in putrescine and polyamine biosynthesis, were investigated in mung bean (Vigna radiata L.) tissues. Both activities increase linearly with increasing concentrations of crude enzyme, but the increase in ADC activity is considerably greater. The decarboxylation reaction is linear for up to 30 to 60 minutes, and both enzymes have a pH optimum of 7.2. alpha-Difluoromethyl-ornithine inhibits ODC activity of excised roots, while increasing ADC activity.High specific activity of both enzymes is detected in terminal buds and leaves, while root and hypocotyl activity is low. Different ADC-to-ODC activity ratios are found in various tissues of mung bean plants. Substantial increase in the activity of both enzymes is detected in incubated sections as compared with intact plants. A comparison of several plant species indicates a wide range of ADC-to-ODC activity ratio.It is suggested that both ADC and ODC are active in plant tissues and that their relative contribution to putrescine biosynthesis is dependent upon the type of tissue and growth process.

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PubMed1982

Cytokinin modification of mitochondrial function.

6-Benzylaminopurine, 6-(Delta(2)-isopentenylamino)purine, 6-furfurylaminopurine, rotenone, and antimycin A inhibited oxidation of NADH by mitochondrial sonicates or submitochondrial particles (but not by intact mitochondria) from pea (Pisum sativum L., cult. Alaska) stems and mung bean (Vigna radiata L. Wilczak) hypocotyls. The above purine cytokinins can interfere with electron transport from NADH to the cytochrome system in the inner mitochondrial membrane. Adenine did not inhibit oxidation by sonicated mitochondria, and zeatin was almost ineffective. Zeatin scarcely inhibited state 3 malate respiration by intact mitochondria, but the O-formyl and O-n-propionyl esters of zeatin and the O-acetyl ester of 2-chlorozeatin were more active. Perhaps zeatin is ineffective because it does not get into the inner membranes of the isolated mitochondria, whereas the esters and other cytokinins mentioned above do. N-4-(2-chloropyridyl)-N'-Phenylurea, which has cytokinin-like effects on plant growth and development, inhibited NADH oxidation by sonicated mitochondria. It also inhibited malate, succinate, and NADH oxidation by intact mitochondria; in contrast, the latter two oxidations were not decreased by purine cytokinins.The benzyl, isopentenyl, and furfuryl aminopurines inhibited malate (but not succinate) oxidation by rat liver mitochondria, if ADP was present. In the absence of ADP, these cytokinins promoted succinate (but not malate) oxidation. Zeatin and adenine had slight, if any, effect in either situation.

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PubMed1982

Stereospecific conversion of 1-aminocyclopropanecarboxylic Acid to ethylene by plant tissues : conversion of stereoisomers of 1-amino-2-ethylcyclopropanecarboxylic Acid to 1-butene.

Inasmuch as the molecule of 1-aminocyclopropanecarboxylic acid (ACC) possesses reflective symmetry but lacks rotational symmetry, the two chemically alike methylene groups can be distinguished by a stereospecific enzyme. To determine whether ACC conversion to ethylene by plant tissues proceeds in a stereospecific fashion, the four stereoisomers of 1-amino-2-ethylcyclopropanecarboxylic acid (AEC) were administered to postclimacteric apple (Malus sylvestris Mill., var. Golden Delicious), excised preclimacteric cantaloupe (Cucumis melo L., var. reticulatis Naud cv. PMR-45), and etiolated mung bean (Vigna radiata L., Wilczek, var. Berken) hypocotyls. In each case (1R,2S)-AEC was the preferred substrate yielding 1-butene. In contrast, all AEC isomers were converted equally well to butene by chemical oxidation using NaOCl. Both ACC and AEC appear to be substrates for the same enzyme since both reactions are inhibited in parallel by N(2) or Co(2+), both reactions are induced in parallel by excision, and when both substrates are present simultaneously each will act as an inhibitor with respect to the other. The aforementioned observations indicate that ACC is stereospecifically converted to ethylene. For AEC to be the most active precursor of 1-butene, the ethyl substituent should be trans to the carboxyl group and the pro-(S) methylene group should be unsubstituted. This observation leads to the suggestion that the enzyme interacts with amino, carboxyl, and pro-(S) methylene groups, a configuration corresponding to a l-amino acid. This view is consistent with the observation that the l-forms of alanine and methionine inhibit the conversion of ACC to ethylene more than the corresponding d-amino acids in the mung bean hypocotyl system.

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PubMed1982

N abundance of nodules as an indicator of N metabolism in n(2)-fixing plants.

This paper expands upon previous reports of (15)N elevation in nodules (compared to other tissues) of N(2)-fixing plants. N(2)-Fixing nodules of Glycine max (soybeans), Vigna unguiculata (cowpea), Phaseolus vulgaris (common bean), Phaseolus coccineus (scarlet runner bean), Prosopis glandulosa (mesquite), and Olneya tesota (desert ironwood) were enriched in (15)N. Nodules of Vicia faba (fava beans), Arachis hypogaea (peanut), Trifolium pratense (red clover), Pisum sativum (pea), Lathyrus sativus (grass pea), Medicago sativa (alfalfa), and Lupinus mutabilis (South American lupine) were not; nor were the nodules of nine species of N(2)-fixing nonlegumes. The nitrogen of ineffective nodules of soybeans and cowpeas was not enriched in (15)N. Thus, (15)N elevation in nodules of these plants depends on active N(2)-fixation. Results obtained so far on the generality of (15)N enrichment in N(2)-fixing nodules suggest that only the nodules of plants which actively fix N(2) and which transport allantoin or allantoic acid exhibit (15)N enrichment.

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PubMed1982

Metabolism and translocation of allantoin in ureide-producing grain legumes.

Transfer of the nitrogen and carbon of allantoin to amino acids and protein of leaflets, stems and petioles, apices, peduncles, pods, and seeds of detached shoots of nodulated cowpea (Vigna unguiculata L. Walp. cv. Caloona) plants was demonstrated following supply of [2-(14)C], [1,3-(15)N]allantoin in the transpiration stream. Throughout vegetative and reproductive growth all plant organs showed significant ureolytic activity and readily metabolized [2-(14)C]allantoin to (14)CO(2). A metabolic pathway for ureide nitrogen utilization via allantoic acid, urea, and ammonia was indicated. Levels of ureolytic activity in extracts from leaves and roots of nodulated cowpea were consistently maintained at higher levels than in non-nodulated, NO(3) (-) grown plants.[(14)C]Ureides were recovered in extracts of aphids (Aphis craccivora and Macrosiphum euphorbieae) feeding at different sites on cowpea plants supplied with [2-(14)C]allantoin through the transpiration stream or to the upper surface of single leaflets. The data indicated that the ureides were effectively transferred from xylem or leaf mesophyll to phloem, and then translocated in phloem to fruits, apices, and roots.

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PubMed1982

Enzymological basis for herbicidal action of glyphosate.

The effects of 1 millimolar glyphosate (N-[phosphonomethyl]glycine) upon the activities of enzymes of aromatic amino acid biosynthesis, partially purified by ion-exchange chromatography from mung bean seedings (Vigna radiata [L.] Wilczek), were examined. Multiple isozyme species of shikimate dehydrogenase, chorismate mutase, and aromatic aminotransferase were separated, and these were all insensitive to inhibition by glyphosate. The activities of prephenate dehydrogenase and arogenate dehydrogenase were also not sensitive to inhibition. Two molecular species of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase were resolved, one stimulated several-fold by Mn(2+) (DAHP synthase-Mn), and the other absolutely dependent upon the presence of Co(2+) for activity (DAHP synthase-Co). Whereas DAHP synthase-Mn was invulnerable to glyphosate, greater than 95% inhibition of DAHP synthase-Co was found in the presence of glyphosate. Since Co(2+) is a V(max) activator with respect to both substrates, glyphosate cannot act simply by Co(2+) chelation because inhibition is competitive with respect to erythrose-4-phosphate. The accumulation of shikimate found in glyphosate-treated seedlings is consistent with in vivo inhibition of both 5-enolpyruvylshikimic acid 3-phosphate synthase and one of the two DAHP synthase isozymes. Aromatic amino acids, singly or in combination, only showed a trend towards reversal of growth inhibition in 7-day seedlings of mung bean. The possibilities are raised that glyphosate may act at multiple enzyme targets in a given organism or that different plants may vary in the identity of the prime enzyme target.

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PubMed1982

Polyamines and root formation in mung bean hypocotyl cuttings : I. Effects of exogenous compounds and changes in endogenous polyamine content.

The effect of several polyamines (putrescine, spermidine, and spermine), their precursors (l-arginine and l-ornithine), and some analogs and metabolic inhibitors (l-canavanine, l-canaline, and methylglyoxal-bis [guanylhydrazone]) on root formation have been studied in mung bean (Vigna radiata [L.] Wilczek) hypocotyl cuttings.Exogenously applied polyamines did not promote adventitious root formation. Rooting was inhibited by l-canavanine and l-canaline, and this inhibition was reversed by the corresponding amino acids l-arginine and l-ornithine. Methylglyoxal-bis (guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase and polyamine biosynthesis, was also found to inhibit root formation. All compounds at concentrations of >10(-4) molarity completely inhibited natural root formation, whereas at <10(-5) molarity only the indole-butyric acid-induced root formation was inhibited.Indole-butyric acid-induced root formation was accompanied by a considerable increase in polyamine levels, more than 2-fold of the control. Whereas senescing (unrooted) cuttings evinced a rapid decline in polyamine content during 48 hours, indole-butyric acid treatment resulted in elevated levels of putrescine and increased putrescine to spermidine ratio. The changes in polyamines were dependent on indole-butyric acid concentration and were organ specific.

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PubMed1982

Succinate dehydrogenase : a partial purification from mung bean hypocotyls and soybean cotyledons.

A procedure was developed for the partial purification of succinate dehydrogenase from mung bean (Vigna radiata L.) hypocotyls and soybean (Glycine max [L] Merr. v. Ransom) cotyledons. The procedure utilized a Triton X-100 extraction followed by ammonium sulfate precipitation. The final fraction was enriched in two polypeptides with approximate molecular weights of 67,000 and 30,000 daltons, exhibited a pH optima of 7.0 to 7.5, contained a b-type cytochrome, and exhibited the characteristic ferredoxin-type and high potential iron-sulfur protein-type electron paramagnetic resonance signals reported for the iron-sulfur centers of mammalian succinate dehydrogenase. Inhibition constants of 1.15 and 24.6 micromolar for oxaloacetate and malonate, respectively, were obtained.

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PubMed1983

Significance of hydrogen evolution in the carbon and nitrogen economy of nodulated cowpea.

The carbon and nitrogen economies of a single cultivar of cowpea (Vigna unguiculata (L.) Walp.cv Caloona) nodulated with either a high H(2)-evolving strain (176A27) or a low H(2)-evolving strain (CB756) of Rhizobium were compared. The two symbioses did not differ in total dry matter production, seed yield, nitrogen fixed, the spectrum of nitrogenous solutes produced by nodules for export, or the partitioning of net photosynthate within the plant throughout the growth cycle. Detailed examination of the carbon and nitrogen economy of the nodules, however, showed a significant difference between the symbioses. Nodules formed with CB756 lost less CO(2) in respiration compared to the higher H(2)-evolving symbioses and this could have been largely responsible for a 36% better economy of carbon use in CB756 nodules during the period of maximum H(2) evolution (48-76 days) and over the whole growth period (20-90 days), a 16% economy. In terms of overall net photosynthate generated by the plant, these economies were equivalent to 5% and 2% of the carbon utilized in the two periods, respectively. From the differences in H(2) evolution and CO(2) production by nodules of the two symbioses, the cost of H(2) evolution was found to be 3.83+/-0.6 millimoles CO(2)/millimoles H(2) for plants grown in sand culture and 1.69 +/- 0.48 millimoles CO(2)/millimoles H(2) for those in water culture. In both symbioses, the ratio of H(2) evolution to N(2) fixed varied markedly during ontogeny, indicating a significant variation in the relative efficiency and thus metabolic cost of N(2) fixation at different stages during development.

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PubMed1983

Amino Acid Sequence of Mung Bean Trypsin Inhibitor and Its Modified Forms Appearing during Germination.

The amino acid sequence of the major trypsin inhibitor, F, of ungerminated mung beans (Vigna radiata [L.] Wilczek) was determined by a combination of automatic solid phase and manual sequencing techniques. F is a typical Bowman-Birk-type proteinase inhibitor with 80 amino acid residues and exhibits a high degree of identity with the other sequenced members of the Bowman-Birk family of inhibitors. Thin layer peptide maps of mung bean inhibitors E and C (which appear during germination) indicate that both are derived from inhibitor F by limited specific proteolysis. Loss of the carboxyl-terminal residues 77 to 80 from F produces inhibitor E, while the loss of an additional two carboxyl-terminal residues, the loss of the amino-terminal residues 1 to 8, and an internal cleavage at Ala(35)-Asp(36) produces inhibitor C from E. Another inhibitor species, E', was isolated from ungerminated seeds. It differs from F in the loss of residues 1 to 6. The majority of the proteolytic cleavages noted in the F-E-C-E' system are at peptide bonds involving aspartyl residues.

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