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PubMed1983

Diurnal variation in the functioning of cowpea nodules.

Nitrogenase (EC 1.7.99.2) activity of nodules of cowpea (Vigna unguiculata [L.] Walp), maintained under conditions of a 12-hour day at 30 degrees C and 800 to 1,000 microeinsteins per square meter per second (photosynthetically active radiation) and a 12-hour night at 20 degrees C, showed a marked diurnal variation with the total electron flux through the enzyme at night being 60% of that in the photoperiod. This diurnal pattern was, however, due to changes in hydrogen evolution. The rate of nitrogen fixation, measured by short-term (15)N(2) assimilation or estimated from the difference in hydrogen evolution in air or Ar:O(2) (80:20; v/v), showed no diurnal variation. Carbon dioxide released from nodules showed a diurnal variation synchronized with that of nitrogenase functioning and, as a consequence, the apparent ;respiratory cost' of nitrogen fixation in the photoperiod was almost double that at night (9.74 +/- 0.38 versus 5.70 +/- 0.90 moles CO(2) evolved per mole N(2) fixed). Separate carbon and nitrogen balances constructed for nodules during the photoperiod and dark period showed that, at night, nodule functioning required up to 40% less carbohydrate to achieve the same level of nitrogen fixation as during the photoperiod (2.4 versus 1.4 moles hexose per mole N(2) fixed).Stored reserves of nonstructural carbohydrate of the nodule only partly satisfied the requirement for carbon at night, and fixation was dependent on continued import of translocated assimilates at all times. Measurements of the soluble nitrogen pools of the nodule together with (15)N studies indicated that, both during the day and night, nitrogenous products of fixation were effectively translocated to all organs of the host plant despite low rates of transpiration at night. Reduced fluxes of water through the plant at night were apparently counteracted by increased concentration of nitrogen, especially as ureides, in the xylem stream.

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PubMed1983

Binding of butyl gallate to isolated mung bean mitochondria : relationship to inhibition of the alternative pathway.

The binding of radioactively labeled butyl gallate to sucrose gradient-purified mung bean (Vigna radiata L.) mitochondria was studied. Titrations showed the binding of [(14)C]butyl gallate to the mitochondria consisted of both reversible and irreversible components. The reversible component bound with a dissociation constant of approximately 1 micromolar which was comparable to the observed inhibition constant for the inhibition of the alternative pathway by butyl gallate. The reversible binding of labeled butyl gallate was also prevented by addition of excess, unlabeled salicylhydroxamic acid. The concentration of binding sites associated with reversible butyl gallate binding was around 0.5 nanomole per milligram of mitochondrial protein. These results were consistent with the reversible binding site being associated with the butyl gallate site of inhibition of the cyanide-resistant, alternative electron transfer pathway in mung bean mitochondria. In addition to the reversible butyl gallate binding site, a nonspecific, irreversible association of butyl gallate with the mitochondrial membrane was observed. The latter binding did not readily saturate at high butyl gallate concentrations and was not correlated with butyl gallate inhibition of the alternative pathway.

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PubMed1983

Effect of temperature on nitrogenase functioning in cowpea nodules.

Nitrogenase (EC 1.7.99.2) activity of a cowpea (Vigna unguiculata (L.) Walp cv Caloona) symbiosis formed with a Rhizobium strain (176A27) lacking uptake hydrogenase and maintained under conditions of a 12-hour day at an air temperature of 30 degrees C (800-1000 microeinsteins per square meter per second) and a 12-hour night at an air temperature of 20 degrees C showed a marked diurnal variation in ratio of nitrogen fixed to hydrogen evolved. As little as 0.3 micromole nitrogen was fixed per micromole hydrogen evolved in the photoperiod versus up to 0.6 in the dark period. In plants maintained under the same diurnal illumination regime but at constant (day and night) air temperature (30 degrees C), this difference was abolished and a relatively constant ratio of nitrogen fixed to hydrogen evolved (around 0.3 micromole per micromole) was observed day and night. Exposure of nodulated roots to a range of temperatures maintained for 2 hours in a single photoperiod indicated that, whereas hydrogen evolution increased with increasing temperature from 15 degrees C to a maximum around 35 degrees C, nitrogen fixation was largely unaffected over this temperature range. Both functions of the enzyme declined sharply at temperatures above 38 degrees C. A similar general response of nitrogen fixation to root temperature was observed in glasshouse-grown, sand-cultured plants maintained under a range of temperatures (from 15 to 35 degrees C) for a 14-day period in mid vegetative growth. The effect of temperature on the proportion of electrons allocated to proton reduction compared with nitrogen reduction showed a linearly increasing relationship (correlation coefficient = 0.96) between 15 degrees C and 47 degrees C.

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PubMed1983

Abscission: quantification of light control.

Low level red light treatments prevented dark-induced leaf abscission in mung bean (Vigna radiata L. Wilczek cv Jumbo) cuttings. The amount of inhibition depended upon the level and length of the light treatment. The red light inhibition could be reversed by a brief exposure of tissue to far-red light.

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PubMed1983

A pathway for photosynthetic carbon flow to mannitol in celery leaves : activity and localization of key enzymes.

In the polyol producing plant, celery (Apium graveolens L.), mannitol is a major photosynthetic product and a form in which carbohydrate is translocated. Measurements of whole leaf extracts of celery indicated substantial activity of the following enzymes: mannose-6-P reductase, mannose-6-P isomerase, mannitol-1-P phosphatase, and nonreversible glyceraldehyde-3-P dehydrogenase. The activities of these enzymes were either undetectable or very low in the nonpolyol producing plants, Secale cereale L. (rye) and Vigna mungo (L.) Hepper (black gram).Mesophyll protoplasts were enzymically isolated from celery leaves, broken with a Yeda press and the intracellular localization of the above enzymes for mannitol synthesis studied following differential and/or sucrose density gradient centrifugation of the protoplast extract. These data suggested the enzymes involved in mannitol synthesis are exclusively localized in the cytoplasm. Ninety-five to 100% of the activity of these enzymes, along with the cytoplasmic marker enzyme phosphoenolpyruvate carboxylase, was found in the cytosolic fraction.We propose the pathway of photosynthetic carbon flow from triose-P to mannitol in celery occurs via fructose-6-P, mannose-6-P, and mannitol-1-P; these final reactions being catalyzed by the cytoplasmic enzymes, mannose-6-P isomerase, NADPH-dependent mannose-6-P reductase, and mannitol-1-P phosphatase, respectively. The requirement for NADPH may be met via the cytoplasmically located NADP-linked nonreversible glyceraldehyde-3-P dehydrogenase.

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PubMed1984

Comparison of water potentials measured by in situ psychrometry and pressure chamber in morphologically different species.

Leaf water potentials measured by in situ psychrometry were compared with leaf water potentials measured by the pressure chamber technique at various values of water potential in Helianthus annuus, Helianthus nuttallii, Vigna unguiculata, Nerium oleander, Pistacia vera, and Corylus avellana. In V. unguiculata, the leaf water potentials measured by the in situ psychrometer oscillated at the same periodicity as, and proportional to, the leaf conductance. In all species, potentials measured by in situ psychrometers operating in the psychrometric mode were linearly correlated with potentials measured with the pressure chamber. However, the in situ psychrometers underestimated the leaf water potential in the two Helianthus species at low water potentials and overestimated the water potential in P. vera, N. oleander, and C. avellana. The underestimation in the two Helianthus species at low water potentials resulted from differences in water potential across the leaf. The overestimation in P. vera, N. oleander, and C. avellana was considered to arise from low epidermal conductances in these species even after abrasion of the cuticle. Pressure-volume studies with Lycopersicon esculentum showed that less water was expressed from distal than proximal leaflets when the whole leaf was slowly pressurized. The implication of this for water relations characteristics obtained by pressure-volume techniques is discussed. We conclude that in situ psychrometers are suitable for following dynamic changes in leaf water potential, but should be used with caution on leaves with low epidermal conductances.

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PubMed1984

Spontaneous Phloem bleeding from cryopunctured fruits of a ureide-producing legume.

The vasculature of the dorsal suture of cowpea (Vigna unguiculata [L.] Walp) fruits bled a sugar-rich exudate when punctured with a fine needle previously cooled in liquid N(2). Bleeding continued for many days at rates equivalent to 10% of the estimated current sugar intake of the fruit. A phloem origin for the exudate was suggested from its high levels (0.4-0.8 millimoles per milliliter) of sugar (98% of this as sucrose) and its high K(+) content and high ratio of Mg(2+) to Ca(2+). Fruit cryopuncture sap became labeled with (14)C following feeding of [(14)C]urea to leaves or adjacent walls of the fruit, of (14)CO(2) to the pod gas space, and of [(14)C] asparagine or [(14)C]allantoin to leaflets or cut shoots through the xylem. Rates of translocation of (14)C-assimilates from a fed leaf to the puncture site on a subtended fruit were 21 to 38 centimeters per hour. Analysis of (14)C distribution in phloem sap suggested that [(14)C]allantoin was metabolized to a greater extent in its passage to the fruit than was [(14)C] asparagine. Amino acid:ureide:nitrate ratios (nitrogen weight basis) of NO(3)-fed, non-nodulated plants were 20:2:78 in root bleeding xylem sap versus 90:10:0.1 for fruit phloem sap, suggesting that the shoot utilized NO(3)-nitrogen to synthesize amino acids prior to phloem transfer of nitrogen to the fruit. Feeding of (15)NO(3) to roots substantiated this conclusion. The amino acid:ureide ratio (nitrogen weight basis) of root xylem sap of symbiotic plants was 23:77 versus 89:11 for corresponding fruit phloem sap indicating intense metabolic transfer of ureide-nitrogen to amino acids by vegetative parts of the plant.

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PubMed1984

Abscission: characterization of light control.

Exposure of mung bean (Vigna radiata [L.] Wilczek cv Jumbo) cuttings to low level red light inhibits dark-induced leaf abscission. A 12-hour daily light requirement for maximum inhibition of abscission was equally effective as a continuous red light treatment or shorter light-dark cycles. Transfer of cuttings from dark to light stopped the abscission process at the time of transfer. The available evidence suggests a light receptor located in the leaves with an abscission inhibitor translocated from lighted leaf to the abscission zone.

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PubMed1984

Adsorption of slow- and fast-growing rhizobia to soybean and cowpea roots.

Roots of soybean (Glycine max [L.] Merr. cv Hardee) and cowpea (Vigna unguiculata [L.] Walp. cv Pink Eye Purple Hull) were immersed in suspensions containing 10(4)Rhizobium cells per milliliter of a nitrogen-free solution. After 30 to 120 minutes the roots were rinsed, and the distal 2-centimeter segments excised and homogenized. Portions of the homogenates then were plated on a yeast-extract mannitol medium for bacterial cell counts. The adsorption capacities of four slow-growing rhizobia and a fast-growing R. meliloti strain varied considerably. Adsorption was independent of plant species and of the abilities of the Rhizobium strains to infect and nodulate. R. lupini 96B9 had the greatest adsorption capacity, and Rhizobium sp. 3G4b16 the least. Rhizobium sp. 229, R. japonicum 138, and R. meliloti 102F51 were intermediate, except on cowpea, where the adsorption of strain 102F51 was similar to that of strain 3G4b16. The initial adsorption rates of bacteria cultured in synthetic media and in the presence of soybean roots were about the same. Addition of soybean lectin to the bacterial inoculum failed to influence initial adsorption rates. Both treatments, however, reduced the numbers of bacteria that bound after incubation with roots for 120 minutes. The relationship between the logarithm of the number of strain 138 cells bound per soybean root segment and the logarithm of the density of bacteria in the inoculum was linear over five orders of magnitude. Binding of strain 138 to soybean roots was greatest at room temperature (27 degrees C) and substantially attenuated at both 4 and 37 degrees C. Although R. lupini 96B9 strongly rejected a model hydrophobic plastic surface, there were no simple correlations between bacterial binding to model hydrophobic and hydrophilic plastic surfaces and bacterial adsorption to roots.

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PubMed1984

Adenine nucleotide regulation of malate oxidation in isolated mung bean hypocotyl mitochondria.

We have studied the effects of ATP and ADP on the oxidation of malate by coupled and uncoupled mitochondria prepared from etiolated hypocotyls of mung bean (Vigna radiata L.).In coupled mitochondria, ATP (1 millimolar) increased pyruvate production and decreased oxaloacetate formation without altering the rate of oxygen consumption. ATP also significantly decreased oxaloacetate production and increased pyruvate production in mitochondria that were uncoupled by carbonyl cyanide p-trifluoromethoxyphenyl hydrazone plus oligomycin.In coupled mitochondria, ADP (1 millimolar) increased the production of both pyruvate and oxaloacetate concomitantly with the acceleration of oxygen uptake to the state 3 rate. The effects of ADP were largely eliminated in uncoupled mitochondria. These results indicate that, whereas the ADP stimulation of oxaloacetate and pyruvate production in the coupled mitochondria is brought about primarily as the result of the accelerated rates of electron transport and NADH oxidation by the respiratory chain in state 3, ATP has significant regulatory effects independent of those that might be exerted by control of electron transport.

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PubMed1984

A Simple Technique of Studying Water Deficit Effects on Nitrogen Fixation in Nodules without Influencing the Whole Plant.

Cowpea (Vigna unguiculata L. Walp cv C-152) plants were grown in a system in which watering was withheld from the soil zone containing nodules, while the plants were able to maintain normal water status. The system was developed in a pot by making two soil zones, an upper and a lower separated by a gravel column between these two zones. Plants extended their roots into the lower layer of soil and were able to absorb water. The dry matter accumulation, photosynthesis rate, and leaf area development of the plant were not affected when the upper soil zone was dried, but the water potential of the nodules was lower than in the nodules in fully irrigated pots. Nitrogenase activity in the nodules obtained from plants stressed in the upper zone only was lower than in nodules obtained from fully irrigated plants. The present technique is helpful in distinguishing the direct water stress effects on nitrogen fixation compared to those mediated via photosynthate availability.

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PubMed1984

Nickel in higher plants: further evidence for an essential role.

Soybeans (Glycine max [L.] Merr.) grown in Ni-deficient nutrient solutions accumulated toxic urea concentrations which resulted in necrosis of their leaflet tips, a characteristic of Ni deficiency. Estimates of the Ni requirement of a plant were made by using seeds produced with different initial Ni contents. When compared to soybeans grown from seeds containing 2.5 nanograms Ni, plants grown from seeds containing 13 nanograms Ni had a significantly reduced incidence of leaflet tip necrosis. Plants grown from seeds containing 160 nanograms Ni produced leaves with almost no leaflet tip necrosis symptoms. Neither Al, Cd, Sn, nor V were able to substitute for Ni.In other experiments, a small excess of EDTA was included in the nutrient solution in addition to that needed to chelate micronutrient metals. Under these conditions, nodulated nitrogen-fixing soybeans had a high incidence of leaflet tip necrosis, even when 1 micromolar NiEDTA was supplied. However, in nutrient solutions containing inorganic sources of N, 1 micromolar NiEDTA almost completely prevented leaflet tip necrosis, although no significant increase in leaf urease activity was observed. Cowpeas (Vigna unguiculata [L.] Walp) grown in Ni-deficient nutrient solutions containing NO(3) and NH(4) also developed leaflet tip necrosis, which was analogous to that produced in soybeans, and 1 micromolar NiEDTA additions prevented these symptoms.These findings further support our contention that Ni is an essential element for higher plants.

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PubMed1984

Effects of short-term n(2) deficiency on N metabolism in legume nodules.

The study aimed to test the hypothesis that ammonia production by Rhizobium bacteroids provides not only a source of nitrogen for growth but has a central regulatory role in maintaining the metabolic activity and functional integrity of the legume nodule. Production of ammonia in intact, attached nodules was interrupted by short-term (up to 3 days) exposure of the nodulated root systems of cowpea (Vigna unguiculata L. Walp cv Vita 3: Rhizobium CB 756) and lupin (Lupinus albus L. cv Ultra: Rhizobium WU 425) to atmospheres of argon:oxygen (80:20; v/v). Treatment did not affect nodule growth, levels of plant cell and bacteroid protein, leghaemoglobin content, or nitrogenase (EC 1.7.99.2) activity (acetylene reduction) but severely reduced (by 90%) synthesis and export of the major nitrogenous solutes produced by the two symbioses (ureides in cowpea, amides in lupin). Glutamine synthetase (EC 6.3.1.2) and NAD:glutamate oxidoreductase (EC I.4.1.2) were more or less stable to Ar:O(2) treatment, but activities of the glutamine-utilizing enzymes, glutamate synthase (EC 2.6.1.53), asparagine synthetase (EC 6.3.5.4) (lupin only), and de novo purine synthesis (cowpea only), were all markedly reduced. Production and export of nitrogenous solutes by both symbioses resumed within 2 hours after transferring Ar:O(2)-treated plants back to air. In each case the major exported product of fixation after transfer was initially glutamine, reflecting the relative stability of glutamine synthetase activity. Subsequently, glutamine declined and products of its assimilation became predominant consistent with resurgence of enzymes for the synthesis of asparagine in lupin and ureides in cowpea. Enzymes not directly involved with either ammonia or glutamine assimilation (purine synthesis, purine oxidation, and carbon metabolism of both bacteroids and plant cells) also showed transient changes in activity following interruption of N(2) supply. These data have been interpreted to indicate a far-reaching effect of the production of ammonia by bacteroids on a wide range of enzymes, possibly through control of protein turnover, rather than a highly specific effect of ammonia, or some product of its assimilation, on a few enzyme species.

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PubMed1985

Economy of water, carbon, and nitrogen in the developing cowpea fruit.

The nutritional economy of the fruit of cowpea (Vigna unguiculata (L.) Walp cv Vita 3) was assessed quantitatively from intake and utilization of carbon, nitrogen, and water. Fruits failed to make net gains of CO(2) from the atmosphere during daytime, although pod photosynthesis did play a role in the fruit's carbon economy by refixing a proportion of the fruit's respired CO(2). Of every 100 units by weight of carbon entering the fruit, 70.4 were finally incorporated into seeds, 10.3 remained as nonmobilizable material in pod walls, and the remaining 19.3 were lost in fruit respiration. Phloem supplied 97% of the fruit's carbon and 72% of its nitrogen. The xylem contribution of nitrogen occurred mainly in early growth. Ninety-six% of the fruit's nitrogen was incorporated into seeds, approximately 10% of this mobilized from the senescing pod. The mean transpiration ratio of the fruit was very low-8 milliliters water transpired per gram dry matter accumulated. Models of carbon, nitrogen, and water flow were constructed for the two consecutive 11 day periods of fruit development, and indicated a considerably greater entry of water through xylem and phloem than could be accounted for in changes in fruit tissue water and transpiration loss. This discrepancy was greater in the second half of fruit growth and was interpreted as evidence that a significant fraction of the water entering the fruit through phloem cycled back to the parent plant via the xylem.

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PubMed1985

Diurnal water balance of the cowpea fruit.

The vascular network of the cowpea (Vigna unguiculata [L.] Walp.) fruit exhibits the anatomical potential for reversible xylem flow between seeds, pod, and parent plant. Feeding of cut shoots with the apoplast marker acid fuchsin showed that fruits imported regularly via xylem at night, less frequently in early morning, and only rarely in the afternoon. The dye never entered seeds or inner dorsal pod strands connecting directly to seeds. Root feeding (early morning) of intact plants with (32)PO(4) or (3)H(2)O rapidly (20 min) labeled pod walls but not seeds, consistent with uptake through xylem. Weak subsequent (4 hours) labeling of seeds suggested slow secondary exchange of label with the phloem stream to the fruit. Vein flap feeding of subtending leaves with [(14)C]sucrose, (3)H(2)O, and (32)PO(4) labeled pod and seed intensely, indicating mass flow in phloem to the fruit. Over 90% of the (14)C and (3)H of fruit cryopuncture phloem sap was as sucrose and water, respectively. Specific (3)H activities of transpired water collected from fruits and peduncles were assayed over 4 days after feeding (3)H(2)O to roots, via leaf flaps, or directly to fruits. The data indicated that fruits transpired relatively less xylem-derived (apoplastic) water than did peduncles, that fruit and peduncle relied more heavily on phloem-derived (symplastic) water for transpiration in the day than at night, and that water diffusing back from the fruit was utilized in peduncle transpiration, especially during the day. The data collectively support the hypothesis of a diurnally reversing xylem flow between developing fruit and plant.

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PubMed1985

Nitrogen nutrition and metabolic interconversions of nitrogenous solutes in developing cowpea fruits.

Budgets for import and utilization of ureide, amides, and a range of amino acids were constructed for the developing first-formed fruit of symbiotically dependent cowpea (Vigna unguiculata [L.] Walp. cv Vita 3). Data on fruit total N economy, and analyses of the xylem and phloem streams serving the fruit, were used to predict the input of various solutes while the compositions of the soluble and protein pools of pod, seed coat, and embryo were used to estimate the net consumption of compounds. Ureides and amides provided virtually all of the fruit's N requirements for net synthesis of amino compounds supplied inadequately from the parent plant. Xylem was the principal source of ureide to the pod, while phloem was the major source of amides to pod and seed. All fruit parts showed in vitro activity of urease (EC 3.5.1.5), allantoinase (EC 3.5.2.5), asparaginase (EC 3.5.11), ammonia-assimilating enzymes and aspartate and alanine aminotransferases (EC 2.61.1 and EC 2.6.1.1.2). Asparagine:pyruvate aminotransferase (EC 2.6.1.14) was recovered only from the pod. The pod was initially the major site for processing and incorporating N; later seed coats and finally embryos became predominant. Ureides were broken down mainly in the pod and seed coat. Amide metabolism occurred in all fruit organs, but principally in the embryo during much of seed growth. Seed coats released N to embryos mainly as histidine, arginine, glutamine, and asparagine, hardly at all as ureide. Amino compounds delivered in noticeably deficient amounts to the fruit were arginine, histidine, glycine, glutamate, and aspartate, while seeds received insufficient arginine, histidine, serine, glycine, and alanine. Quantitatively based schemes are proposed depicting the principal metabolic transformation accompanying N-flow between seed compartments during development.

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PubMed1985

Transport and Partitioning of CO(2) Fixed by Root Nodules of Ureide and Amide Producing Legumes.

Nodulated and denodulated roots of adzuki bean (Vigna angularis), soybean (Glycine max), and alfalfa (Medicago sativa) were exposed to (14)CO(2) to investigate the contribution of nodule CO(2) fixation to assimilation and transport of fixed nitrogen. The distribution of radioactivity in xylem sap and partitioning of carbon fixed by nodules to the whole plant were measured. Radioactivity in the xylem sap of nodulated soybean and adzuki bean was located primarily (70 to 87%) in the acid fraction while the basic (amino acid) fraction contained 10 to 22%. In contrast, radioactivity in the xylem sap of nodulated alfalfa was primarily in amino acids with about 20% in organic acids. Total ureide concentration was 8.1, 4.7, and 0.0 micromoles per milliliter xylem sap for soybean, adzuki bean, and alfalfa, respectively. While the major nitrogen transport products in soybeans and adzuki beans are ureides, this class of metabolites contained less than 20% of the total radioactivity. When nodules of plants were removed, radioactivity in xylem sap decreased by 90% or more. Pulse-chase experiments indicated that CO(2) fixed by nodules was rapidly transported to shoots and incorporated into acid stable constituents. The data are consistent with a role for nodule CO(2) fixation providing carbon for the assimilation and transport of fixed nitrogen in amide-based legumes. In contrast, CO(2) fixation by nodules of ureide transporting legumes appears to contribute little to assimilation and transport of fixed nitrogen.

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PubMed1985

Polyamines and Root Formation in Mung Bean Hypocotyl Cuttings : II. Incorporation of Precursors into Polyamines.

The incorporation of [(14)C]arginine and [(14)C]ornithine into various polyamines was studied in mung bean (Vigna radiata [L.] Wilczek) hypocotyl cuttings with respect to the effect of indole-3-butyric acid on adventitious root formation.Both [(14)C]arginine and [(14)C]ornithine are rapidly incorporated into putrescine, spermidine, and spermine, with similar kinetics, during 5- to 24-hour incubation periods. The incorporation of arginine into putrescine is generally higher than that of ornithine. The biosynthesis of putrescine and spermidine from the precursors, in the hypocotyls, is closely related to the pattern of root formation: a first peak at 0 to 24 hours corresponding to the period of root primordia development, and a second peak of putrescine biosynthesis at 48 to 72 hours corresponding to root growth and elongation. Indole-3-butyric acid considerably enhances putrescine biosynthesis in both phases, resulting in an increase of the putrescine/spermidine ratio.It is concluded that the promotive effect of indole-3-butyric acid on putrescine biosynthesis, from both arginine and ornithine, supports the hypothesis that auxin-induced root formation may require the promotion of polyamine biosynthesis.

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