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PubMed2001

Homo-phytochelatins are synthesized in response to cadmium in azuki beans.

In a recent report, it was claimed that azuki beans (Vigna angularis) do not synthesize phytochelatins (PCs) upon exposure to cadmium, although glutathione (GSH), the substrate for PC synthesis, is present in this plant. This legume species thus would be the first exception in the plant kingdom that would fail to complex heavy metals by PCs. Here, we report that not GSH, but only homoglutathione can be detected in this plant and that homo-phytochelatins are formed when azuki beans are challenged with heavy metals such as cadmium. We also show that the 5,5'-dithiobis(2-nitrobenzoic acid)-oxidized GSH reductase recycling assay, used for GSH quantification in the recent study of heavy metal tolerance in azuki beans, reacts both with GSH and homoglutathione and therefore cannot be used when biological samples should be analyzed exclusively for GSH.

Cadmium↗
PubMed1966

Effects of ethylene on auxin transport.

The effect of ethylene on the uptake, distribution and polar transport of C(14) from indole-3-acetic acid-2-C(14) and naphthalene acetic acid-1-C(14) in tissue sections was studied. Test species were cotton (Gossypium hirsutum, L.) and cowpea (Vigna sinensis, Endl.). Generally, incubation of tissue or intact plants with ethylene reduced the degree of polar auxin transport. Ethylene inhibited the movement of both auxins in stem tissue and IAA in petiole tissue of cotton. The effect of ethylene on auxin movement in cow-peas was more complex. Ethylene apparently inhibited transport in younger petiole and stem tissue, but stimulated the process to a small but significant degree in basal petiole segments.Ethylene, in some experiments, reduced C(14) (auxin) uptake. This reduction was consistently smaller than the inhibition of transport. Effects upon transport were observed when uptake was not different. Differences in uptake declined as the period of incubation with auxin was lengthened, but transport was inhibited for up to 23 hours.It is proposed that ethylene may, through its effect on transport, cause localized shortages and surpluses of auxin which in turn contribute to symptoms now associated with the response of sensitive species to ethylene.

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PubMed1966

Variation of nicotinamide adenine dinucleotide phosphate level in bean hypocotyls in relation to o(2) concentration.

Slices of hypocotyls from 3-day-old seedlings of Vigna sesquipedalis (L.) Fruwirth in the germination stage were incubated under various gaseous conditions. The NADP+NADPH level in the hypocotyl slices changed with the oxygen tension. A high NADP+NADPH level was observed under aerobic conditions and a low NADP+NADPH level under anaerobic conditions.The 100 x NADH/NAD+NADH ratio increased greatly under anaerobic conditions. In general a low NADP + NADPH level corresponded with a high 100 x NADH/NAD+NADH ratio. On the basis of the results given in the following paper, it was discussed that the slowness of NADH oxidation in hypocotyl tissue due to anaerobic conditions results in the inhibition of NADP formation.The variation of the NADP+NADPH level was considered to produce a modification of the carbohydrate metabolism.The NADP+NADPH level in E. coli cells suspended in glucose solution also changed with the oxygen tension.

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PubMed1966

Enzymes of the glyoxylate cycle in rhizobia and nodules of legumes.

The relatively high level of fatty acids in soybean nodules and rhizobia from soybean nodules suggested that the glyoxylate cycle might have a role in nodule metabolism. Several species of rhizobia in pure culture were found to have malate synthetase activity when grown on a number of different carbon sources. Significant isocitrate lyase activity was induced when oleate, which presumably may act as an acetyl CoA precursor, was utilized as the principle carbon source. Malate synthetase was active in extracts of rhizobia from nodules of bush bean (Phaseolus vulgaris L.), cowpea (Vigna sinensis L.), lupine (Lupinus angustifolius L.) and soybean (Glycine max L. Merr.). Activity of malate synthetase was, however, barely detectable in rhizobia from alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.) and pea (Pisum sativum L.) nodules. Appreciable isocitrate lyase activity was not detected in rhizobia from nodules nor was it induced by depletion of endogenous substrates by incubation of excised bush bean nodules. Although rhizobia has the potential for the formation of the key enzymes of the glyoxylate cycle, the absence of isocitrate lyase activity in bacteria isolated from nodules indicated that the glyoxylate cycle does not operate in the symbiotic growth of rhizobia and that the observed high content of fatty acids in nodules and nodule bacteria probably is related to a structural role.

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PubMed1971

The Coding Properties of Lysine-accepting Transfer Ribonucleic Acids from Black-eyed Peas.

Lysine-accepting transfer RNA from ungerminated and germinated embryo axes of black-eyed peas (Vigna sinensis L. Savi) was fractionated on benzoylated diethylaminoethyl cellulose and reverse phase Freon columns. Cochromatography indicated the presence of two similar lysyl transfer RNA fractions in each tissue. Ribosome binding studies revealed that the larger of the two fractions in each case is specific for the AAG codon, while the smaller one recognizes AAA and AAG. Possible implications of this difference in quantities of isoacceptors in translation of genetic information are discussed.

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PubMed1972

Auxin transport: a new synthetic inhibitor.

The new synthetic plant growth regulator DPX1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo [5,1-a] isoindol-8-one) was examined for its effects on auxin transport. At a concentration of 0.5 mm in the receiver agar cylinders DPX1840 significantly inhibited the basipetal transport of naphthaleneacetic acid-1-(14)C in stem sections of Vigna sinensis Endl., Pisum sativum L., Phaseolus vulgaris L., Glycine max L., Helianthus annuus L., Gossypium hirsutum L., and Zea mays L. without significantly reducing total auxin uptake or recovery. The time sequence of the effect varied with the plant species. A similar inhibition of the basipetal movement of indoleacetic acid-1-(14)C was observed in intact seedlings of Phaseolus vulgaris L. In contrast to basipetal auxin transport DPX1840 had no significant effect on the acropetal movement of indoleacetic acid-1-(14)C in stem sections of Gossypium hirsutum L. Qualitatively the effect of DPX1840 on basipetal auxin transport was similar to that of other known auxin transport inhibitors. Quantitative differences, however, suggested the following order of activity: Naptalam>morphactin[unk]DPX1840>2,3,5-triiodobenzoic acid.DPX1840 also inhibited the lateral displacement of auxin. In horizontally placed stem sections of Helianthus annuus L. pretreated with DPX1840, the ratio of radioactivity from indoleacetic acid-1-(14)C in the upper versus the lower halves of the sections following basipetal indoleacetic acid-1-(14)C transport was approximately 50:50, whereas in the corresponding controls it was approximately 40:60.The data indicate that many of the characteristic effects of DPX1840 on plants, especially those which are known to involve auxin (e.g., epinasty, abscission, apical dominance, tropism), are due, at least in part, to its effects on auxin transport.

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PubMed1973

Asparate transcarbamylase activity in etiolated cowpea hypocotyls treated with 2,4-dichlorophenoxyacetic Acid.

Treating etiolated cowpea (Vigna unguiculata) seedlings with 2,4-dichlorophenoxyacetic acid resulted in 2.5-, 3.9-, and 6.5-fold increases in DNA, soluble protein, and RNA, respectively, over untreated controls 84 hours after treatment. Aspartate transcarbamylase activity increased within 12 hours after treatment, and by 84 hours it was almost 12-fold greater than that in the untreated controls. During that time, activity in untreated controls dropped 60%. The assay used (14)C-aspartate, which was then separated from the (14)C-ureidosuccinate product by Dowex 50 (H(+) form) column chromatography. Thin layer chromatography of the reaction product indicated that most of the carbamyl-phosphate-dependent radioactivity co-chromatographed with ureidosuccinate. The reaction has a pH optimum near 10.0 and is inhibited by uridine 5'-phosphate and succinate. The data suggest that aspartate transcarbamylase is important in pyrimidine biosynthesis in 2,4-dichlorophenoxyacetic acid-treated seedlings.

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PubMed1974

Mechanism of Indole-3-acetic Acid Conjugation: No Induction by Ethylene.

Formation of indole-3-acetic acid-aspartate in detached primary leaves of cowpea (Vigna sinensis Endl.) floating on (14)C-indole-3-acetic acid (3 muc; 3.15 mum, phosphate-citrate buffer, pH 4.75), almost doubled when leaves were pretreated with 31.5 mum(12)C-indole-3-acetic acid for 17 hr and then transferred to (14)C-indole-3-acetic acid for 4 hours as compared with leaves preincubated in buffer only. When leaves were preincubated with ethylene (11.0 and 104 mul/l) instead of (12)C-indole-3-acetic acid, no induction of indole-3-acetylaspartic acid formation was observed, and the rate of indole-3-acetylaspartic acid formation decreased as compared with control leaves. Rhizobitoxine (1.87 mum) inhibited indole-3-acetic acid-induced ethylene production but did not prevent the formation of indole-3-acetylaspartic acid. In view of the similarity of these results and those previously obtained with alpha-naphthaleneacetic acid, it is concluded that ethylene has no role in the auxin-induced indole-3-acetylaspartic acid formation in cowpea leaves.

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PubMed1976

Effects of inhibitors of RNA and protein synthesis on aspartate transcarbamylase activity in etiolated plant tissue.

Aspartate transcarbamylase (ATCase) activity declines in etiolated cowpea (Vigna unguiculata L. Walp.) and soybean (Glycine max L. Merr.) hypocotyls between 3 and 11 days after planting. Treating cow-pea hypocotyls with cycloheximide (CH), actinomycin D (AMD), 6-methyl purine (6-MP), or cordycepin increases ATCase activity up to 740, 350, 465, and 305%, respectively, over water-treated controls 48 to 72 hours after treatment. In contrast erythromycin had no effect, and d-threo-chloramphenicol (CHL) reduced ATCase activity nearly 40%. CH, AMD, and CHL, whose effects were further characterized, each markedly reduced total RNA synthesis and protein synthesis. Respiration was stimulated by CH and AMD and reduced by CHL. In soybean, CHL-treated tissues and water-treated controls had comparable ATCase activities 48 hours after treatment, while AMD, 6-MP, and CH treatments reduced activities 29, 37, and 78%, respectively. The results suggest that the level of ATCase activity in etiolated cowpea hypocotyls is regulated by a mechanism or mechanisms that are interfered with by inhibition of RNA and protein synthesis. Possibly the mechanism is absent from etiolated soybean hypocotyls.

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PubMed1977

Temperature-induced Changes in Hill Activity of Chloroplasts Isolated from Chilling-sensitive and Chilling-resistant Plants.

The effect of temperature on Hill activity has been compared in chilling-sensitive and chilling-resistant plants. The Arrhenius activation energy (Ea) for the photoreduction of 2,6-dichlorophenolindophenol by chloroplasts isolated from two chilling-sensitive plants, mung bean (Vigna radiata L. var. Mungo) and maize (Zea mays L. cv. PX 616), increased at low temperatures, below 17 C for mung bean and below 11 C for maize. However, the Ea for this reaction in pea (Pisum sativum L. cv. Massay Gem), a chilling-resistant plant, likewise increased at temperatures below 14 C. A second change in Ea occurred at higher temperatures. The Ea decreased above about 28 C for mung bean, 30 C for maize, and 25 C for pea. At temperatures approaching 40 C, thermal inactivation of Hill activity occurred. These results, when taken together with previous results obtained with the chilling-resistant plant barley, indicate that chloroplasts from both chilling-sensitive and chilling-resistant plants can undergo a change in chloroplast membrane activity at low temperatures above freezing and that the presence of such a change in chloroplast membranes is not necessarily correlated with chilling sensitivity.

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PubMed1977

Hydrogen reactions of nodulated leguminous plants: I. Effect of rhizobial strain and plant age.

The ATP-dependent evolution of H(2) catalyzed by nitrogenase and the hydrogenase-catalyzed oxidation of H(2) have been implicated as factors influencing the efficiency of energy utilization in the N(2) fixation process. The effects of rhizobial strain and plant age on the H(2)-evolving and H(2)-utilizing activity of leguminous root nodules are described in this manuscript. Two classes of legume-Rhizobium combinations were observed in studies with soybeans (Glycine max L. Merr.) and cowpeas (Vigna unguiculata L. Walp.). One group evolved H(2) in air; the other group did not exhibit net evolution of H(2). The latter group metabolized H(2) formed within the nodule through the action of a hydrogenase. The capacity to oxidize H(2) was strongly linked to the strain of Rhizobium used to inoculate cowpeas and soybeans. Although the magnitude of H(2) evolution in air changed during vegetative growth of a given symbiont, the ratio of H(2) evolved in air to total nitrogenase activity was not appreciably altered during this period. No consistent difference in nitrogenase activity as measured by the C(2)H(2) reduction assay was observed between symbionts with an active hydrogenase and those that apparently lack the enzyme and evolve H(2). The effects of the two reactions of H(2) on total N(2) fixation and yield must now be established.

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PubMed1977

Utilization of net photosynthate for nitrogen fixation and protein production in an annual legume.

The economy of C and N in nodulated cowpea (Vigna unguiculata [L.] Walp.) was described in terms of fixation of CO(2) and N(2), respiratory losses of C, and the production of dry matter and protein.Net daytime gain of C by the shoot (net photosynthesis) rose to a maximum at flowering and then declined sharply due to abscission of leaves. Maximum N fixation occurred 10 days prior to maximum net photosynthesis. Shedding of nodules reduced fixation to zero by midfruiting. Fifty per cent of the plant's N and 37% of its net photosynthate were assimilated before flowering; 39% of plant N was incorporated into seed dry matter.Respiration of nodules and roots utilized 24% of the C from net photosynthate assimilated over the growth cycle; night respiration of shoots, 20%; dry matter production in seeds, 17%; and dry matter production in other plant parts, 39%. The proportion of net photosynthate translocated to the nodulated root decreased from 41 to 14% during growth. Developing fruits were major competitors for translocate. Nodules consumed 9% of the C from the plant's total net photosynthate, 43% of which was respired, 6% made into dry matter, and 51% returned to the shoot with N fixation products.For every 1 g N fixed, net photosynthate equivalent to 6.8 g carbohydrate was consumed by nodules, 25.7 g carbohydrate by the nodulated root. Translocate was used most efficiently for N fixation in late vegetative growth when nodules were most active and their carbohydrate supply still adequate.During vegetative growth and early flowering (0 to 78 days after sowing) cowpea consumed 17.2 g net photosynthate (as carbohydrate) for every gram of protein synthesized in its shoot. The comparable conversion in seed production was 32.5 g net photosynthate/g seed protein or 6.6 g/g seed dry matter.

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PubMed1978

Hydrogen Reactions of Nodulated Leguminous Plants: II. Effects on Dry Matter Accumulation and Nitrogen Fixation.

The interaction between the ATP-dependent evolution of H(2) catalyzed by nitrogenase and the oxidation of H(2) via a hydrogenase has been postulated to influence the efficiency of the N(2)-fixing process in nodulated legumes. A comparative study using soybean (Glycine max L. Merr.) cv. Anoka inoculated with either Rhizobium japonicum strain USDA 31 or USDA 110 and cowpea (Vigna unguiculata L. Walp.) cv. Whippoorwill inoculated with Rhizobium strain 176A27 or 176A28 cultured on a N-free medium was conducted to address this question. Nodules from the Anoka cultivar inoculated with USDA 31 evolved H(2) in air and the H(2) produced accounted for about 30% of the energy transferred to the nitrogenase system during the period of active N(2) fixation. In contrast the same soybean cultivar inoculated with USDA 110 produced nodules with an active hydrogenase and consequently did not evolve H(2) in air. A comparison of Anoka soybeans inoculated with the two different strains of R. japonicum showed that mean rates of C(2)H(2) reduction and O(2) consumption and mean mass of nodules taken at four times during vegetative growth were not significantly different.When compared to Anoka inoculated with USDA 31, the same cultivar inoculated with USDA 110 showed increases in total dry matter, per cent nitrogen, and total N(2) fixed of 24, 7, and 31%, respectively. Cowpeas in symbiosis with the hydrogenase-producing strain 176A28 in comparison with the same cultivar inoculated with the H(2)-evolving strain 176A27 produced increases in plant dry weight and total N(2) fixed of 11 and 15%, respectively. This apparent increase in the efficiency of N(2) fixation for nodulated legumes capable of reutilizing the H(2) evolved from nitrogenase is considered and it is concluded that provision of conclusive evidence of the role of the H(2)-recycling process in N(2)-fixing efficiency of legumes will require comparison of Rhizobium strains that are genetically identical with the exception of the presence of hydrogenase.

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PubMed1978

Trypsin inhibitor in mung bean cotyledons: purification, characteristics, subcellular localization, and metabolism.

Trypsin inhibitor was purified to homogeneity from seeds of the mung bean (Vigna radiata [L.] Wilczek). The protease inhibitor has the following properties: inhibitory activity toward trypsin, but not toward chymotrypsin; isoelectric point at pH 5.05; molecular weight of 11,000 to 12,000 (sodium dodecyl sulfate gel electrophoresis) or 14,000 (gel filtration); immunological cross-reactivity against extracts of black gram and black-eyed pea, but not against soybean; no inhibitory activity against vicilin peptidohydrolase, the principal endopeptidase in the cotyledons of mung bean seedlings.The trypsin inhibitor content of the cotyledons declines in the course of seedling growth and the presence of an inactivating factor can be demonstrated by incubating crude extracts in the presence of beta-mercaptoethanol. This inactivating factor may be a protease as vicilin peptidohydrolase rapidly inactivates the trypsin inhibitor. Removal of trypsin inhibitory activity from crude extracts by means of a trypsin affinity column does not result in an enhancement of protease activity in the extracts.The intracellular localization of trypsin inhibitor was determined by fractionation of crude extracts on isopycnic sucrose gradients and by cytochemistry with fluorescent antibodies. Both methods indicate that trypsin inhibitor is associated with the cytoplasm and not with the protein bodies where reserve protein hydrolysis occurs. No convincing evidence was obtained which indicates that the catabolism of trypsin inhibitor during germination and seedling growth is causally related to the onset of reserve protein breakdown.

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PubMed1978

Phytochrome-induced Increase of Fluorescein Translocation in Mung Bean Hypocotyls.

Moderate doses of red (660 nanometer) irradiation cause a rapid increase in the translocation of fluorescein in dark-grown mung bean hypocotyl (Vigna radiata L.) segments. The increase fails to appear following large doses of red (660 nanometers) irradiation. The red induced increase is prevented by a subsequent far red (730 nanometer) irradiation. Reversibility suggests the participation of phytochrome in the process. The increase in translocation is attributed to the generation of a positive electrostatic charge in the plasma membrane by some action of phytochrome on membrane molecules.

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PubMed1978

Influence of the axis on the enzymes of protein and amide metabolism in the cotyledons of mung bean seedlings.

The growth of the mung bean (Vigna radiata) seedling is accompanied by the biosynthesis and accumulation of the endopeptidase vicilin peptidohydrolase and the catabolism of the reserve proteins in the cotyledons. If the axis is removed from the dry seeds and the cotyledons incubated on moist sand the accumulation of vicilin peptidohydrolase is reduced by 77% and the catabolism of reserve proteins slowed to 25% of the rate in intact seedlings. The cotyledons and the cotyledon exudate are rich in asparagine and this amino acid accounts for more than half of the reduced nitrogen exported from the cotyledons. Glutamine synthetase and asparagine synthetase, two key enzymes in the pathway of asparagine synthesis, are under temporal control in the cotyledons. Their activities increase 3.5- and 10-fold, respectively, then decline again. These increases in enzyme activity occur to the same extent in excised cotyledons and are prevented when the cotyledons are incubated in 5 micromolar cycloheximide. The results indicate that the axis may control certain key metabolic events in the cotyledons, such as the synthesis of vicilin peptidohydrolase, while many other anabolic activities may not depend on a growing axis.

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PubMed1978

Spectral characterization of five chlorophyll-protein complexes.

Sodium dodecyl sulfate-solubilized chloroplast internal membranes of higher plants (cowpea [Vigna unguiculata L. Walp], chinese cabbage [Brassica chinensi L.], and tobacco [Nicotiana tabacum L.]) are resolved by polyacrylamide gel electrophoresis into two chlorophyll a- and three chlorophyll a,b-proteins. A small portion (about 15%) of the membrane chlorophyll migrates as a component of high electrophoretic mobility and presumably consists of detergent-complexed, protein-free pigment.One of the chlorophyll a-proteins is qualitatively similar to the P(700) chlorophyll a-protein but contains a much larger proportion of total chlorophyll (about 30%) than previously reported. The second chlorophyll a-protein is a recently discovered component of the membrane and accounts for about 7% of the total chlorophyll. The absorption and fluorescence emission spectra of these two chlorophyll a-proteins differ.The three chlorophyll a,b-proteins are components of the chloroplast membrane chlorophyll a,b-light-harvesting complex which was previously resolved as a single chlorophyll-protein band. The two additional chlorophyll a,b-proteins observed in our work probably represent larger aggregates contained within that membrane complex which are preserved under the solubilization and electrophoretic conditions used here.

Journal Article↗
PubMed1979

Economy of Photosynthate Use in Nitrogen-fixing Legume Nodules: Observations on Two Contrasting Symbioses.

The economy of C use by root nodules was examined in two symbioses, Vigna unguiculata (L.) Walp. (cv. Caloona):Rhizobium CB756 and Lupinus albus L. (cv. Ultra):Rhizobium WU425 over a 2-week period in early vegetative growth. Plants were grown in minus N water culture with cuvettes attached to the nodulated zone of their primary roots for collection of evolved CO(2) and H(2). Increments in total plant N and in C and N of nodules, and C:N weight ratios of xylem and phloem exudates were studied by periodic sampling from the plant populations. Itemized budgets were constructed for the partitioning and utilization of C in the two species. For each milligram N fixed and assimilated by the cowpea association, 1.54 +/- 0.26 (standard error) milligrams C as CO(2) and negligible H(2) were evolved and 3.11 milligrams of translocated C utilized by the nodules. Comparable values for nodules of the lupin association were 3.64 +/- 0.28 milligrams C as CO(2), 0.22 +/- 0.05 milligrams H(2), and 6.58 milligrams C. More efficient use of C by cowpea nodules was due to a lesser requirement of C for synthesis of exported N compounds, a smaller allocation of C to nodule dry matter, and a lower evolution of CO(2). The activity of phosphoenolpyruvate carboxylase in nodule extracts and the rate of (14)CO(2) fixation by detached nodules were greater for the cowpea symbiosis (0.56 +/- 0.06 and 0.22 milligrams C as CO(2) fixed per gram fresh weight per hour, respectively) than for the lupin 0.06 +/- 0.02 and 0.01 milligrams C as CO(2) fixed per gram fresh weight per hour. The significance of the data was discussed in relation to current information on theoretical costs of nitrogenase functioning and associated nodule processes.

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