Search

Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation ((15)N(2) fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. Light and gas phase oxygen concentration had marked interactive effects on activity, with higher (up to 100-fold) rates of acetylene reduction and (15)N(2) fixation in light. The relationship between ethylene formation and N(2)-fixation varied in the freshly isolated cyanobacteria from 4 to 7 nanomoles of C(2)H(4) per nanomole (15)N(2). Intact coralloid roots, incubated in darkness and ambient air, showed a value of 4.3. Maximum rates of nitrogenase activity occurred at about 0.6% O(2) in light, while in darkness there was a broad optimum around 5 to 8% O(2). Inhibition of nitrogenase, in light, by pO(2) above 0.6% was irreversible. Measurements of light-dependent O(2) evolution and (14)CO(2) fixation indicated negligible photosynthetic electron transport involving photosystem II and, on the basis of inhibitor studies, the stimulatory effect of light was attributed to cyclic photophos-phorylation. Nitrogenase activity of free-living culture of an isolate from Macrozamia (Nostoc PCC 73102) was only slightly inhibited by O(2) levels above 6% O(2) and the inhibition was reversible. These cells showed rates of light-dependent O(2) evolution and (14)CO(2) fixation which were 100- to 200-fold higher than those by the freshly isolated symbiont. Furthermore, nitrogenase activity was dependent on both photosynthetic electron transport and photophosphorylation. These data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O(2), and a direct dependence on the cycad for substrates to support nitrogenase activity.

Cytokinin-like activity in samples of xylem and phloem sap collected from field-grown plants of white lupin (Lupinus albus L.) over a period of 9 to 24 weeks after sowing was measured using the soybean hypocotyl callus bioassay following paper chromatographic separation. The phloem sap was collected from shallow incisions made at the base of the stem, the base of the inflorescence (e.g. stem top), the petioles, and the base and tip of the fruit. Xylem sap was collected as root exudate from the stump of plants severed a few centimeters above ground level. Concentration of cytokinin-like substances was highest in phloem sap collected from the base of the inflorescence and showed an increase over the entire sampling period (from week 10 [61 nanogram zeatin equivalents] to week 24 [407 nanogram zeatin equivalents]). Concentrations in the xylem sap and in the other phloem saps were generally lower. Relatively high concentrations of cytokinin-like substances in petiole phloem sap (70 to 130 nanogram zeatin equivalents per milliliter) coincided in time with high concentrations in sap from the base of the inflorescence (see above). Concentrations in sap (phloem or xylem) from the base of the stem were very much lower. This finding is consistent with movement of cytokinins from leaves into the developing inflorescence and fruit, rather than direct input to the fruit from xylem sap. However, an earlier movement of cytokinins from roots into leaves via the xylem cannot be ruled out. Sap collected at an 18-week harvest was additionally separated by sequential C(18) reversed-phase high performance liquid chromatography --> NH(2) normal phase high performance liquid chromatography, bioassayed, and then analyzed by electron impact gas chromatography-mass spectrometry. Identification of zeatin riboside and dihydrozeatin as two of the major cytokinins in combined sap samples was accomplished by gas chromatography-mass spectrometry-selected ion monitoring.

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.

The ureides, allantoin and allantoic acid, represented major fractions of the soluble nitrogen pool of nodulated plants of cowpea (Vigna unguiculata [L.] Walp. cv. Caloona) throughout vegetative and reproductive growth. Stem and petioles were the principal sites of ureide accumulation, especially in early fruiting.Labeling studies using (14)CO(2) and (15)N(2) and incubation periods of 25 to 245 minutes indicated that synthesis of allantoin and allantoic acid in root nodules involved currently delivered photosynthate and recently fixed N, and that the ureides were exported from nodule to shoot via the xylem. From 60 to 80% of xylem-borne N consisted of ureides; the remainder was glutamine, asparagine, and amino acids. Allantoin predominated in the soluble N fraction of nodules and fruits, allantoin and allantoic acid were present in approximately equal proportions in xylem exudate, stems, and petioles.Extracts of the plant tissue fraction of nitrogen-fixing cowpea nodules contained glutamate synthase (EC 2.6.1.53) and glutamine synthetase (EC 6.3.1.2), but little activity of glutamate dehydrogenase (EC 1.4.1.3). High levels of uricase (EC 1.7.3.3) and allantoinase (EC 3.5.2.5) were also detected. Allantoinase but little uricase was found in extracts of leaflets, pods, and seeds.Balance sheets were constructed for production, storage, and utilization of ureide N during growth. Virtually all (average 92%) of the ureides exported from roots was metabolized on entering the shoot, the compounds being presumably used as N sources for protein synthesis.

Allopurinol (1H-pyrazolo-[3,4-d]pyrimidine-4-ol), an inhibitor of xanthine oxidation in ureide-producing nodulated legumes, was taken up from the rooting medium, translocated in xylem, and transferred to nodules of both the ureide-forming cowpea (Vigna unguiculata L. Walp.) and the amide-forming white lupin (Lupinus albus L.). Cowpea suffered severe nitrogen deficiency, extreme chlorosis, and reduced growth, whereas lupin was unaffected by the inhibitor. Similar results were obtained with oxypurinol (1H-pyrazolo-[3,4-d]pyrimidine-4,6-diol). Xylem composition of symbiotic cowpea was markedly changed by allopurinol. Ureides fell to a very low level, but xanthine and, to a lesser extent, hypoxanthine increased markedly. Xylem glutamine was also reduced, but there was little change in other amino acids. Nitrogenase (EC 1.7.99.2) activity of intact nodulated plants or nodulated root segments of plants treated with allopurinol or oxypurinol for 24 hours or more was severely inhibited in cowpea but unaffected in lupin for periods of exposure up to 9 days. Nitrogenase activity of slices of nodules prepared from allopurinol-treated cowpea showed inhibition comparable to that of intact plants. Breis prepared from nodules of treated plants showed no reduction in nitrogenase, nor was there reduction in activity of breis following addition of allopurinol, xanthine, or a range of purine pathway intermediates. Increasing the O(2) concentration in assays above 20% (volume/volume) reversed inhibition of nitrogenase by allopurinol in intact nodulated roots. It was concluded for cowpea that allopurinol not only inhibited ureide synthesis but also caused inhibition of nitrogenase activity, thereby leading to progressive dysfunction and eventual senescence of nodules. The mechanistic relationships between inhibition of ureide biosynthesis, changes in gaseous diffusion resistance, and reduced nitrogenase activity remain obscure.

The nutritional economy of the developing fruit of Pisum sativum L. (cv. Greenfeast) was studied in terms of intake of translocate, incorporation of C and N into dry matter, transpiration, and CO(2) exchanges of the fruit with its external and internal atmospheres. The environmental conditions were 12-hr days (22 C, 850 mueinsteins m(-2) sec(-1) at fruit level); 12-hr nights of 15 C.Between 6 and 30 days after anthesis, pod photosynthesis resulted in small gains of CO(2) from the external atmosphere, and assimilated most of the CO(2) respired by the fruit during the day. From then until maturity (40 days) the fruit lost CO(2) during the day. Night losses of CO(2) increased with fruit age.The gas cavity of the fruit contained 0.15 to 1.5% (v/v) CO(2). Lower levels were maintained in the day than at night. CO(2) levels were influenced by fruit age, radiant flux, and temperature. Labeled CO(2) injected into the gas cavity was fixed by the pod but not by seeds in the light, and by neither pod nor seeds in darkness. Dark-to-light or light-to-dark transfer of a fruit promoted rapid changes in CO(2) and O(2) levels of the gas space, consistent with a shift in the assimilation-respiration balance of the pod.The fruit transpired 27.6 cm(3) H(2)O per gram dry matter accumulated. Daytime ventilation was greatest 12 to 15 days after anthesis and declined as pod photosynthesis became increasingly involved in the retrieval of CO(2) respired by pod and seeds. Most, 69% by weight, of the translocate from the parent plant was converted to dry matter of seeds; nearly half, 45%, to useful seed reserves (sugar plus starch-protein-oil, 45:20:1). Illumination resulted in a fruit requiring 16% less translocate than if laying down an equal amount of dry matter in darkness.

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.

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.

The pod wall of pea (Pisum sativum L.) was shown to contain two distinct photosynthetic layers. The outer, comprising chlorenchyma of the mesocarp, captured CO(2) from the outside atmosphere; the inner, a chloroplast-containing epidermis lining the pod gas cavity, was involved in photoassimilation of the CO(2) released from respiring seeds.Structural features of the pod included the thick cuticle and stomata of the outer epidermis, the inward projecting veinlets of the vascular network in the mesocarp, the sparsity of air spaces, the fiber and parenchyma layers of the endocarp, and the abundant chloroplasts, thin cuticle, and rounded outer contours of cells of the inner epidermis.The inner epidermis showed high specific activities of ribulose 1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) and phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31), contained up to 20% of the pod's chlorophyll, and was capable of fixing 66% of the CO(2) released during the photoperiod to the pod gas space by the seeds of a fully grown fruit.The in vitro carboxylation capacity of the pod exceeded the estimated gross photosynthesis of the fruit for all but the last few days of development. Chlorophyll content and carboxylation activity declined more markedly in the outer photosynthetic layers than in the inner epidermis.The ratio of activities of RuDP carboxylase to PEP carboxylase in pod extracts varied from 2.4:1 to 12:1 as against 48:1 to 156:1 in extracts of leaves.Structural and physiological properties of the pod were related to its capacity to conserve respired CO(2) and provide photosynthate to developing seeds.

The principal forms of amino nitrogen transported in xylem were studied in nodulated and non-nodulated peanut (Arachis hypogaea L.). In symbiotic plants, asparagine and the nonprotein amino acid, 4-methyleneglutamine, were identified as the major components of xylem exudate collected from root systems decapitated below the lowest nodule or above the nodulated zone. Sap bleeding from detached nodules carried 80% of its nitrogen as asparagine and less than 1% as 4-methyleneglutamine. Pulse-feeding nodulated roots with (15)N(2) gas showed asparagine to be the principal nitrogen product exported from N(2)-fixing nodules. Maintaining root systems in an N(2)-deficient (argon:oxygen, 80:20, v/v) atmosphere for 3 days greatly depleted asparagine levels in nodules. 4-Methyleneglutamine represented 73% of the total amino nitrogen in the xylem sap of non-nodulated plants grown on nitrogen-free nutrients, but relative levels of this compound decreased and asparagine increased when nitrate was supplied. The presence of 4-methyleneglutamine in xylem exudate did not appear to be associated with either N(2) fixation or nitrate assimilation, and an origin from cotyledon nitrogen was suggested from study of changes in amount of the compound in tissue amino acid pools and in root bleeding xylem sap following germination. Changes in xylem sap composition were studied in nodulated plants receiving a range of levels of (15)N-nitrate, and a (15)N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N(2) or fed nitrate. The abundance of asparagine in xylem sap and the ratio of asparagine:nitrate fell, while the ratio of nitrate:total amino acid rose as plants derived less of their organic nitrogen from N(2). Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N(2) and nitrate are being used in peanuts.

Changes in total N and in free amino compounds were followed during growth of nodulated white lupin. Leaflets contained the greatest fraction of plant N but had lower proportions (1 to 4%) of their N in soluble amino form than stem + petioles (10 to 27%) and reproductive parts (15 to 33%). Mobilization of free amino compounds from plant parts to fruits contributed at most only 7% of the total N intake of fruits, compared with 50% in mobilization of other forms of N and 43% from fixation during fruiting. Asparagine was usually the most abundant free amino compound in plant parts, followed by glutamine and alanine. Valine, glycine, isoleucine, aspartic acid and gamma-aminobutyric acid comprised the bulk of the remaining soluble amino N. Composition of tissue pools of amino-N closely resembled that of xylem and phloem exudates. Data on N flow and utilization were combined with information on composition of transport fluids to quantify syntheses, exchanges, and consumptions of asparagine, glutamine, aspartic acid, and valine by organs of the 51- to 58-day plant. These amino compounds carried 56, 29, 5, and 2%, respectively, of the N exported from nodules and contributed in roughly commensurate proportions to transport exchanges and N increments of plant parts. There were, however, more than expected involvements of glutamine and valine in mobilization of N from lower leaves, of asparagine in xylem to phloem transfer, and of aspartic acid in cycling of N through the root, and there was a less than expected participation of aspartic acid in xylem to phloem transfer and in phloem translocation to the shoot apex. The significance of these differences is discussed.

Exchanges of CO(2) and changes in content of C and N were studied over the life of a leaf of Lupinus albus L. These data were combined with measurements of C:N weight ratios of xylem (upper stem tracheal) and phloem (petiole) sap to determine net fluxes of C and N between leaf and plant. Phase 1 of leaf development (first 11 days, leaf to one-third area) showed increasing net import of C and N, with phloem contributing 61% of the imported C and 18% of the N. (14)C feeding studies suggested the potential for simultaneous import and export through phloem over the period 9 to 12 days. Phase 2 (11-20 days, leaf attaining maximum area and net photosynthesis rate) exhibited net import through xylem and increasing export through phloem. Eighty-two% of xylem-delivered N was consumed in leaf growth, the remainder exported in phloem. Phase 3 (20-38 days) showed high but declining rates of photosynthesis, translocation, and net export of N. Phase 4 (38-66 days) exhibited substantial losses of N and declining photosynthesis and translocation of C. C:N ratio of xylem sap remained constant (2.3-2.6) during leaf life; petiole phloem sap C:N ratio varied from 25 to 135 over leaf development. The relationships between net photosynthesis and N import in xylem were: phase 1, 4.8 milligrams C per milligram N; phase 2, 24.7 milligrams C per milligram N; phase 3, 91.9 milligrams C per milligram N; and phase 4, 47.7 milligrams C per milligram N.

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.

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.

Asparagine accounted for 50 to 70% of the nitrogen carried in translocatory channels serving fruit and seed of white lupin (Lupinus albus L.). Rates of supply of the amide always greatly exceeded its incorporation as such into protein. An asparaginase (l-asparagine amido hydrolase EC 3.5.1.1) was demonstrated in crude extracts of seeds. In vitro activity was up to 5 mumoles of aspartate formed per hour per gram fresh weight at the apparent Km(Asn) value of 10 mM, and this more than accounted for the estimated rates of asparagine utilization in vivo. Asparaginase activity per seed increased 10-fold in the period 5 to 7 weeks after anthesis, coinciding with early stages of storage protein synthesis in the cotyledons.Double labeled ((14)C (U), (15)N (amide)) asparagine was fed to fruiting shoots through the transpiration steram. Fruit phloem sap analysis indicated that virtually all of the label was translocated to seeds in the form of asparagine. In young seeds (15)N from asparagine breakdown was traced to the ammonia, glutamine, and alanine of endospermic fluid, the (14)C appearing mainly in nonamino compounds. In the cotyledon-filling stage the C and N of asparagine was contributed to a variety of amino acid residues of protein.

The economy of functioning of the developing fruit of white lupin (Lupinus albus L.) is assessed quantitatively in relation to intake and usage of carbon, nitrogen, and water. Of every 100 units of carbon imported from the parent plant, 52 are incorporated into seeds, 37 into nonmobilizable material of the pod, and the remaining 11 lost as CO(2) to the atmosphere. An illuminated fruit can make net gains of CO(2) from the atmosphere during the photoperiods of all but the last 2 weeks of its life, suggesting that it is active in assimilation of CO(2) respired from pods and seeds. This conservation activity is important to carbon economy.Phloem supplies 98% of the fruit's carbon and 89% of its nitrogen. Most of the xylem's contribution enters early in development. Xylem and phloem supply similar sets of amino compounds, amides predominating. Ninety-six per cent of the fruit's nitrogen becomes incorporated into seeds. Sixteen per cent of the seed's nitrogen is mobilized from the senescing pod.The transpiration ratio of the fruit is 22.5 ml per gram dry matter accumulated. Xylem supplies 60% of a fruit's total water requirement and the equivalent of two-thirds of its transpiration loss. Phloem becomes prominent as a water donor once the seeds start to fill.The fruit exhibits a 31% conversion by weight of organic imports into food reserves of seeds. This entails an intake through vascular channels of 1756 mg sucrose and 384 mg amino compounds and an accumulation in seeds of 412 mg protein, 132 mg oil, and 110 mg perchloric acid-soluble carbohydrate.

Net balances of amino acids were constructed for stages of development of a leaf of white lupin (Lupinus albus L.) using data on the N economy of the leaf, its exchanges of amino acids through xylem and phloem, and net changes in its soluble and protein-bound amino acids. Asparagine, aspartate, and gamma-aminobutyrate were delivered to the leaf in excess of amounts consumed in growth and/or phloem export. Glutamine was supplied in excess until full leaf expansion (20 days) but was later synthesized in large amounts in association with mobilization of N from the leaf. Net requirements for glutamate, threonine, serine, proline, glycine, alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were met mainly or entirely by synthesis within the leaf. Amides furnished the bulk of the N for amino acid synthesis, asparagine providing from 24 to 68%. In vitro activity of asparaginase (EC 3.5.1.1) exceeded that of asparagine:pyruvate aminotransferase (EC 2.6.1.14) during early leaf expansion, when in vivo estimates of asparagine metabolism were highest. Thereafter, aminotransferase activity greatly exceeded that of asparaginase. Rates of activity of one or both asparagine-utilizing enzymes exceeded estimated rates of asparagine catabolism throughout leaf development. In vitro activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1) were consistently much higher than that of glutamate dehydrogenase (EC 1.4.1.3), and activities of the former two enzymes more than accounted for estimated rates of ammonia release in photorespiration and deamidation of asparagine.

Empirically based models depicting exchanges of C, N, and H(2)O in phloem and xylem among organs of nodulated white lupin (Lupinus albus cv Ultra) were constructed for the interval 51 to 58 days after sowing. Information was incorporated on the economy of C, N, and H(2)O in plant parts, the solute composition of transport fluids collected at selected sites on the plant, and the photosynthetic inputs, transpirational losses, and translocatory activities of different age groups of leaflets and stem + petiole segments of the shoot. Partitioning of C and N showed preferential transfer of N to the shoot apex, which imported 13 milligrams C per milligram N, compared with 54 milligrams C per milligram N for the nodulated root. Leaves translocated assimilates at a C:N weight ratio of 43 to 59, and older leaves serving the roots produced the translocate most rich in N relative to C. The shoot apex was enriched with N, additional to its intake from leaves, by direct uptake of xylem fluid (C:N ratio, 2.4) and receipt of nitrogenous solutes transferred from xylem to upward-moving phloem streams in upper regions of the stem. The models for flow of N and H(2)O indicated that xylem streams passing to leaves were substantially less rich in N than the adjacent stream moving through the body of the stem and that a progressive increase in concentration of N occurred within stem xylem elements from base to top of the shoot. This apparently resulted from an abstraction of N from xylem of departing leaf traces, possibly by xylem transfer cells, and a subsequent feedback of this N to xylem streams passing on up the shoot. Upper leaves and shoot apex, therefore, acquired more N from xylem per unit of H(2)O transpired than lower parts of the shoot.

Early growth, nodule development, and nitrogen fixation by two cultivars of cowpea (Vigna unguiculata L. Walp), one large-seeded (Vita 3; 146.0 +/- 0.9 milligrams seed dry weight, 4.1 +/- 0.2 milligrams seed N), the other small-seeded (Caloona; 57.5 +/- 2.5 milligrams seed dry weight, 1.8 +/- 0.1 milligrams seed N), were compared under conditions of sand culture with nutrient solution free of combined N. The seed stocks used had been obtained from plants uniformly labeled with (15)N, thus enabling changes with time in distribution of cotyledon and fixed N among plant parts to be measured by isotope dilution. Caloona, but not Vita 3, showed physiological symptoms of ;N hunger,' i.e. transient loss of chlorophyll (visible yellowing) and N from the first-formed unifoliolate leaves at or around the onset of symbiotic functioning and N(2) fixation. The smaller-seeded Caloona showed higher early nitrogenase activity than the larger-seeded Vita 3 and by 28 days had fixed 6.6 milligrams of N per milligram of seed N [mg N . (mg seed N)(-1)] versus only 3.5 mg N . (mg seed N)(-1) in Vita 3. Both cultivars lost around 30% of their initial seed N at germination, mostly as fallen cotyledons. Abscised cotyledons of Caloona contained 1.21 +/- 0.17% N; those of Vita 3 contained 2.61 +/- 0.37% N. When compared on the basis of cotyledon N available for seedling growth, Caloona was shown to have fixed 10.6 mg N . (mg seed N)(-1) and Vita 3 only 5.3 mg N . (mg seed N)(-1). Most of the cotyledon N withdrawn from the unifoliolate leaf pair of Caloona during ;N-hunger' was committed to early nodule growth and, in total, 20 to 25% of the cotyledon N resource of this cultivar was ultimately invested in establishment of symbiosis compared with only 7% in Vita 3.

The response of nonnodulated white lupin (Lupinus albus L. cv. Ultra) plants to a range of NO(3) levels in the rooting medium was studied by in vitro assays of extracts of plant parts for NO(3) reductase (EC 1.6.6.1) activity, measurements of NO(3)-N in plant organs, and solute analyses of root bleeding (xylem) sap and phloem sap from stems and petioles. Plants were grown for 65 days with 5 millimolar NO(3) followed by 10 days with 1, 5, 15, or 30 millimolar NO(3). NO(3) reductase was substrate-induced in all tissues. Roots contained 76, 68, 62 and 31% of the total NO(3) reductase activity of plants fed with 1, 5, 15, and 30 millimolar NO(3), respectively. Stem, petioles, and leaflets contained virtually all of the NO(3) reductase activity of a shoot, the activity in extracts of fruits amounting to less than 0.3% of the total enzyme recovered from the plant. Xylem sap from NO(3)-grown nonnodulated plants contained the same organic solutes as from nodulated plants grown in the absence of combined N. Asparagine accounted for 50 to 70% and glutamine 10 to 20% of the xylem-borne N. The level of NO(3) in xylem sap amounted to 4, 13, 12, and 17% of the total xylem N at 1, 5, 15, and 30 millimolar NO(3), respectively. Xylem to phloem transfer of N appeared to be quantitatively important in supplying fruits and vegetative apices with reduced N, especially at low levels of applied NO(3). NO(3) failed to transfer in any quantity from xylem to phloem, representing less than 0.3% of the phloem-borne N at all levels of applied NO(3). Shoot organs were ineffective in storing NO(3). Even when NO(3) was supplied in great excess (30 millimolar level) it accounted for only 8% of the total N of stem and petioles, and only 2 and 1% of the N of leaflets and fruits, respectively.

Xylem sap composition was examined in nodulated and nonnodulated cowpea (Vigna unguiculata [L.] Walp.) plants receiving a range of levels of NO(3) and in eight other ureide-forming legumes utilizing NO(3) or N(2) as sole source of nitrogen. A (15)N dilution technique determined the proportions of plant nitrogen derived from N(2) in the nodulated cowpeas fed NO(3). Xylem sap composition of NO(3)-fed, nodulated cowpea varied predictably with the relative extents to which N(2) and NO(3) were being utilized. The ratios of asparagine to glutamine (N/N) and of NO(3) to ureide (N/N) in xylem sap increased with increasing dependence on NO(3) whereas per cent of xylem nitrogen as ureide and the ratio of ureide plus glutamine to asparagine plus NO(3) (N/N) in xylem sap increased with increasing dependence on N(2) fixation. The amounts of NO(3) and ureides stored in leaflets, stems plus petioles, and roots of cowpea varied in a complex manner with level of NO(3) and the presence or absence of N(2) fixation. All species showed higher proportions of organic nitrogen as ureide and several-fold lower ratios of asparagine to glutamine in their xylem sap when relying on N(2) than when utilizing NO(3). In nodulated (minus nitrate) cowpea and mung bean (Vigna radiata [L.] Wilczek) the percentage of xylem nitrogen as ureide remained constant during growth but the ratio of asparagine to glutamine varied considerably. The biochemical significance of the above differences in xylem sap composition was discussed.

Collections of xylem exudate of root stumps or detached nodules, and of phloem bleeding sap from stems, petioles, and fruits were made from variously aged plants of Lupinus albus L. relying on nodules for their N supply. Sucrose was the major organic solute of phloem, asparagine, glutamine, serine, aspartic acid, valine, lysine, isoleucine, and leucine, the principal N solutes of both xylem and phloem. Xylem sap exhibited higher relative proportions of asparagine, glutamine and aspartic acid than phloem sap, but lower proportions of other amino acids. Phloem sap of petioles was less concentrated in asparagine and glutamine but richer in sucrose than was phloem sap of stem and fruit, suggesting that sucrose was unloaded from phloem and amides added to phloem as translocate passed through stems to sinks of the plant. Evidence was obtained of loading of histidine, lysine, threonine, serine, leucine and valine onto phloem of stems but the amounts involved were small compared with amides. Analyses of petiole phloem sap from different age groups of leaves indicated ontogenetic changes and effects of position on a shoot on relative rates of export of sucrose and N solutes. Diurnal fluctuations were demonstrated in relative rates of loading of sucrose and N solutes onto phloem of leaves. Daily variations in the ability of stem tissue to load N onto phloem streams were of lesser amplitude than, or out of phase with fluctuations in translocation of N from leaves. Data were related to recent information on C and N transport in the species.

Partitioning and utilization of assimilated C and N were compared in nonnodulated, NO(3)-fed and nodulated, N(2)-fed plants of white lupin (Lupinus albus L.). The NO(3) regime used (5 millimolar NO(3)) promoted closely similar rates of growth and N assimilation as in the symbiotic plants. Over 90% of the N absorbed by the NO(3)-fed plants was judged to be reduced in roots. Empirically based models of C and N flow demonstrated that patterns of incorporation of C and N into dry matter and exchange of C and N among plant parts were essentially similar in the two forms of nutrition. NO(3)-fed and N(2)-fed plants transported similar types and proportions of organic solutes in xylem and phloem. Withdrawal of NO(3) supply from NO(3)-fed plants led to substantial changes in assimilate partitioning, particularly in increased translocation of N from shoot to root. Nodulated plants showed a lower (57%) conversion of C or net photosynthate to dry matter than did NO(3)-fed plants (69%), and their stems were only half as effective as those of NO(3)-fed plants in xylem to phloem transfer of N supplied from the root. Below-ground parts of symbiotic plants consumed a larger share (58%) of the plants' net photosynthate than did NO(3)-fed roots (50%), thus reflecting a higher CO(2) loss per unit of N assimilated (10.2 milligrams C/milligram N) by the nodulated root than by the root of the NO(3)-fed plant (8.1 milligrams C/milligram N). Theoretical considerations indicated that the greater CO(2) output of the nodulated root involved a slightly greater expenditure for N(2) than for NO(3) assimilation, a small extra cost due to growth and maintenance of nodule tissue, and a considerably greater nonassimilatory component of respiration in root tissue of the symbiotic plant than in the root of the NO(3)-fed plant.

Amino compounds representative of the major N solutes of xylem sap were pulse-fed (10 to 20 minutes) singly in (14)C-labeled form to cut transpiring shoots of white lupin (Lupinus albus L.). (14)C distribution was studied by autoradiography and radioassays of phloem sap, leaflet tissues, and shoot parts harvested at intervals after labeling. Primary distribution of N by xylem was simulated using a 20-minute labeling pulse followed by a 30-minute chase in unlabeled xylem sap. Shoots fed (14)C-labeled asparagine, glutamine, valine, serine, or arginine showed intense labeling of leaflet veins and marked retention (35 to 78%) of (14)C by stem + petioles. Shoots fed (14)C-labeled aspartic acid or glutamic acid showed heaviest (14)C accumulation in interveinal regions of leaflets and low uptake (11 to 20%) of (14)C by stem + petioles. Departing leaf traces were major sites of uptake of all amino compounds, and the implications of this were evaluated. Fruits acquired only 1 to 5% of the fed label directly from xylem, but more than doubled their intake during the period 30 to 160 minutes after feeding through receipt of (14)C transferred from xylem to phloem in stem and leaves. (14)C-Labeled asparagine and valine transferred directly from xylem to phloem, but the (14)C of (14)C-labeled aspartic acid and arginine appeared in phloem mainly as metabolic products of the fed compound. The labeling of the soluble pool of leaflets reflected these differences. The significance of heterogeneity in distribution and metabolism of xylem amino compounds in the shoot was discussed.

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.

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.

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.

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.

Nodulated root systems of white lupin (Lupinus albus L. cv Ultra: Rhizobium strain WU425) were exposed to Ar:O(2) (80:20, v/v) or Ar:N(2):O(2) (70:10:20, v/v/v) and C and N partitioning were examined over a 9- or 10-day period in comparison with control plants with nodulated roots retained in air. Accumulation of N ceased in plants exposed to Ar:O(2) or was much reduced in plants exposed to Ar:N(2):O(2), but net C assimilation rates and profiles of C utilization remained similar to those of control N(2)-fixing plants. There was, however, a proportional reduction in CO(2) evolution from nodulated roots of the Ar:O(2) treatment. Xylem N levels fell rapidly after application of Ar:O(2). C:N ratios of phloem sap of petioles and of stem base rose during the first day of Ar:O(2) treatment and then fell progressively back to levels close to that of control plants as leaf reserves of N became available for loading of phloem. Stem top phloem sap increased progressively in C:N ratio throughout Ar:O(2) treatment, presumably due to increasing shortage of xylem derived N for xylem to phloem exchange. Reexposure of Ar:O(2)-treated nodulated root systems to air prompted a rapid recovery of N(2) fixation and restoration of plant N status. Rates of N(2) fixation in plants whose roots were exposed to a range of N(2) concentrations indicated an apparent K(m) of 10% N(2) for the attached intact white lupin nodule.

The response of non-nodulated cowpea (Vigna unguiculata (L.) Walp. cv Caloona) to a wide range of NO(3) levels in the rooting medium was studied 40 days after sowing by in vitro assays of plant organs for NO(3) reductase (EC 1.6.6.1) and analyses of root bleeding (xylem) sap for nitrogenous solutes. Plants fed 1, 5, 10, 20, and 40 millimolar NO(3) showed, respectively, 64, 92, 94, and 91% of their total reductase activity in shoots and 34, 30, 66, 62, and 58% of the total N of their xylem sap as NO(3). These data, and the absence in the plants of significant pools of stored NO(3), indicated that shoots were major organs of NO(3) assimilation, especially at levels of NO(3) (10 to 40 millimolar) that maintained plant growth at near maximum rates. Partitioning and utilization of C and N were studied in nodulated, minus NO(3) plants and non-nodulated plants fed 10 or 20 millimolar NO(3), the levels of NO(3) which gave rates of growth and N assimilation closest to those of the symbiotic plants. The conversion of the C of net photosynthate to dry matter was similar in nodulated plants (67%) and NO(3)-grown plants (64%), but greater proportions of photosynthate were translocated to below ground parts of nodulated plants (37%) than of NO(3)-fed plants (23 to 26%). Greater photosynthate consumption by nodulated roots was associated with proportionately greater root growth and respiration and 2-fold greater export of C in xylem than in the NO(3)-fed plants. Theoretical considerations suggest that the elevated CO(2) output of nodulated roots was due not only to CO(2) loss associated with nodule function, but also to a much greater nonassimilatory component of respiration in the supporting root of the nodulated plant compared to roots of the NO(3)-fed plants. Data are compared with previously published information from other legumes.

An empirical modeling technique was developed for depicting quantitatively the transport and partitioning of photosynthetically fixed C and symbiotically fixed N during 10-day intervals of a 40-day period in the growth of nodulated plants of white lupin (Lupinus albus L. cv. Ultra). Model construction utilized data for C and N consumption of plant parts and C:N weight ratios of the xylem and phloem fluids serving specific plant organs. Formulas were derived from calculating the net transport of C and N between plant parts in xylem and phloem. The models provided quantitative information on the dependence of growing organs on xylem and phloem for their supply of C and N, the cycling of N through leaflets and of C through nodules, the extent of direct incorporation of fixed N into growing nodules, and the involvement of N from shoot translocate in the nutrition of the nodulated root. Stem plus petioles abstracted considerably more N from xylem than expected from their transpirational activity. Xylem to phloem transfer of recently fixed N in mature stem and petioles was substantiated by the models, being depicted as a device for dispensing N to growing parts of the shoot extra to that attracted transpirationally in xylem or received as translocate from leaflets.

In the presence of 0.5 millimolar allopurinol (4-hydroxypyrazolo [3,4-d]pyrimidine), an inhibitor of NAD:xanthine oxidoreductase (EC 1.2.3.2), intact attached nodules of cowpea (Vigna unguiculata L. Walp. cv Vita 3) formed [(15)N]xanthine from (15)N(2) at rates equivalent to those of ureide synthesis, confirming the direct assimilation of fixed nitrogen into purines. Xanthine accumulated in nodules and was exported in increasing amounts in xylem of allopurinol-treated plants. Other intermediates of purine oxidation, de novo purine synthesis, and ammonia assimilation did not increase and, over the time course of experiments (4 hours), allopurinol had no effect on nitrogenase (EC 1.7.99.2) activity. Negligible (15)N-labeling of asparagine from (15)N(2) was observed, suggesting that the significant pool (up to 14 micromoles per gram of nodule fresh weight) of this amide in cowpea nodules was not formed directly from fixation but may have accumulated as a consequence of phloem delivery.

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.

The synthesis of (14)C-labeled xanthine/hypoxanthine, uric acid, allantoin, allantoic acid, and urea from [8-(14)C]guanine or [8-(14)C]hypoxanthine, but not from [8-(14)C]adenine, was demonstrated in a cell-free extract from N(2)-fixing nodules of cowpea (Walp.). The (14)C recovered in the acid/neutral fraction was present predominantly in uric acid and allantoin (88-97%), with less than 10% of the (14)C in allantoic acid and urea. Time courses of labeling in the cell-free system suggested the sequence of synthesis from guanine to be uric acid, allantoin, and allantoic acid. Ureide synthesis was confined to soluble extracts from the bacteroid-containing tissue, was stimulated by pyridine nucleotides and intermediates of the pathways of aerobic oxidation of ureides, but was completely inhibited by allopurinol, a potent inhibitor of xanthine dehydrogenase (EC 1.2.1.37). The data indicated a purine-based pathway for ureide synthesis by cowpea nodules, and this suggestion is discussed.

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.