Transport of nitrogen in the xylem of soybean plants

Experiments were conducted to characterize the distribution of N compounds in the xylem sap of nodulated and nonnodulated soybean plants through development and to determine the effects of exogenous N on the distribution of N compounds in the xylem. Xylem sap was collected from nodulated and nonnodulated greenhouse-grown soybean plants (Glycine max [L.] Merr. “Ransom”) from the vegetative phase to the pod-filling phase. The sum of the nitrogen in the amino acid, nitrate, ureide (allantoic acid and allantoin), and ammonium fractions of the sap from both types of plants agreed closely with total N as assayed by a Kjeldahl technique. Sap from nodulated plants supplied with N-free nutrient solution contained seasonal averages of 78 and 20% of the total N as ureide-N and amino acid-N, respectively. Sap from nonnodulated plants supplied with a 20 millimolar KNO₃ nutrient solution contained seasonal averages of 6, 36, and 58% of total N as ureide-N, amino acid-N, and nitrate-N, respectively. Allantoic acid was the predominant ureide in the xylem sap and asparagine was the predominant amino acid. When well nodulated plants were supplied with 20 millimolar KNO₃, beginning at 65 days, C₂H₂ reduction (N₂ fixation) decreased relative to nontreated plants and there was a concomitant decrease in the ureide content of the sap. A positive correlation (r = 0.89) was found between the ureide levels in xylem sap and nodule dry weights when either exogenous nitrate-N or urea-N was supplied at 10 and 20 millimolar concentrations to inoculated plants. The results demonstrate that ureides play a dominant role in N transport in nodulated soybeans and that the synthesis of ureides is largely dependent upon nodulation and N₂ fixation.     

Allantoin and Allantoic Acid in the Nitrogen Economy of the Cowpea (Vigna unguiculata [L.] Walp.)

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 ¹⁴CO₂ and ¹⁵N₂ 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.

     

Nitrogen Nutrition and Xylem Sap Composition of Peanut (Arachis hypogaea L. cv Virginia Bunch)

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 ¹⁵N₂ gas showed asparagine to be the principal nitrogen product exported from N₂-fixing nodules. Maintaining root systems in an N₂-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 N₂ 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 ¹⁵N-nitrate, and a ¹⁵N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N₂ 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 N₂. Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N₂ and nitrate are being used in peanuts.     

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 N₂. 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 Mg²⁺ to Ca²⁺. Fruit cryopuncture sap became labeled with ¹⁴C following feeding of [¹⁴C]urea to leaves or adjacent walls of the fruit, of ¹⁴CO₂ to the pod gas space, and of [¹⁴C] asparagine or [¹⁴C]allantoin to leaflets or cut shoots through the xylem. Rates of translocation of ¹⁴C-assimilates from a fed leaf to the puncture site on a subtended fruit were 21 to 38 centimeters per hour. Analysis of ¹⁴C distribution in phloem sap suggested that [¹⁴C]allantoin was metabolized to a greater extent in its passage to the fruit than was [¹⁴C] asparagine. Amino acid:ureide:nitrate ratios (nitrogen weight basis) of NO₃-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 NO₃-nitrogen to synthesize amino acids prior to phloem transfer of nitrogen to the fruit. Feeding of ¹⁵NO₃ 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.     

Inhibition of nodule functioning in cowpea by a xanthine oxidoreductase inhibitor, allopurinol

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 O₂ 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.     

Waterlogging effect on xylem sap glutamine of nodulated soybean

Waterlogging of soybean plants (Glycine max L.) led to impaired symbiotic N₂ fixation and a marked decline in glutamine (Gln) concentration in xylem bleeding sap. Xylem Gln concentration increased during the growth cycle of the plant and was correlated with nodule formation. Treatments known to impair N₂ fixation, such as exposing the root system to pure N₂ gas or a mixture of Ar and O₂ (80:20; v/v), led to specific declines in xylem sap Gln. The decrease in Gln observed during waterlogging was also seen on transfer of nodulated plants to aerated hydroponics, where the decline was highly correlated with ureide content in the xylem sap. Upon flooding the nodulated root system, the specific decline in xylem sap Gln could be detected within 10 min and reached a minimum within 60 min, indicating that waterlogging has an immediate effect on N₂ fixation. It is concluded that xylem Gln arises directly from N₂-fixation and is a useful indicator of N₂ fixation activity of symbiotic soybean plants.     

Effect of NO3 on N2 fixation and nitrogenous solutes of xylem in two nodulated West African geocarpic legumes,Kersting's bean (Macrotyloma geocarpum L.) and Bambara groundnut (Vigna subterranea L.)

Nodulation, nitrogen (N₂) fixation and xylem sap composition were examined in sand cultured plants of Bambara groundnut (Vigna subterranea L.) and Kersting's bean (Macrotyloma geocarpum L.) inoculated with Bradyrhizobium strain CB756 and supplied via the roots for a 4 week period from the third week onwards with different levels of (¹⁵N)-nitrate (0–15 mM). The separate contributions of nitrate and N₂ to plant nitrogen were measured by isotope dilution. Increasing levels of nitrate inhibited nodule growth (measured as dry matter or nodule N) of both species parallel with decreased dependence on symbiotically-fixed N. Specific nodule activity (N₂ fixed g nodule dry⁻¹ d⁻¹ of nodules) was reduced progressively with time in V. subterranea at higher (5 or 15 mM) levels of NO₃, but this was not so for M. geocarpum. Root xylem bleeding sap of both species showed ureides (allantoin and allantoic acid) as predominant (>90%) solutes of nitrogen when plants were relying solely on atmospheric N. Levels of ureide and glutamine decreased and those of asparagine and nitrate in xylem increased with increasing level of applied nitrate. Relative levels of xylem ureide-N were positively correlated (R²=0.842 for M. geocarpum and 0.556 for V. subterranea), and the ratio of asparagine to glutamine in xylem exudate negatively correlated (R²=0.955 for M. geocarpum and 0.736 for V. subterranea) with plant reliance on nitrogen fixation. The data indicate that xylem sap analyses might be useful for indirect field assays of nitrogen fixation by the species and that Kersting's bean might offer some potential as a symbiosis in which N₂ fixation is relatively tolerant of soil N.     

Evaluation of the Relative Ureide Content of Xylem Sap as an Indicator of N(2) Fixation in Soybeans: GREENHOUSE STUDIES

The use of the relative ureide content of xylem sap [(ureide-N/total N) × 100] as an indicator of N₂ fixation in soybeans (Merr.) was examined under greenhouse conditions. Acetylene treatments to inhibit N₂ fixation were imposed upon the root systems of plants totally dependent upon N₂ fixation as their source of N and of plants dependent upon both N₂ fixation and uptake of exogenous nitrate. Significant decreases in the total N concentration of xylem sap from plants of the former type were observed, but no significant decrease was observed in the total N concentration of sap from the latter type of plants. In both types of plants, acetylene treatment caused significant decreases in the relative ureide content of xylem sap. The results provided further support for a link between the presence of ureides in the xylem and the occurrence of N₂ fixation in soybeans. The relative ureide content of xylem sap from plants totally dependent upon N₂ fixation was shown to be insensitive to changes in the exudation rate and total N concentration of xylem sap brought about by diurnal changes in environmental factors. There was little evidence of soybean cultivars or nodulating strains affecting the relative ureide content of xylem sap. `Ransom' soybeans nodulated with Rhizobium japonicum strain USDA 110 were grown under conditions to obtain plants exhibiting a wide range of dependency upon N₂ fixation. The relative ureide content of xylem sap was shown to indicate reliably the N₂ fixation of these plants during vegetative growth using a ¹⁵N method to measure N₂ fixation activity. The use of the relative ureide content of xylem sap for quantification of N₂ fixation in soybeans should be evaluated further.     

Economy of Carbon and Nitrogen in Nodulated and Nonnodulated (NO(3)-grown) Cowpea [Vigna unguiculata (L.) Walp.

The response of non-nodulated cowpea (Vigna unguiculata (L.) Walp. cv Caloona) to a wide range of NO₃ levels in the rooting medium was studied 40 days after sowing by in vitro assays of plant organs for NO₃ 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 NO₃ 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 NO₃. These data, and the absence in the plants of significant pools of stored NO₃, indicated that shoots were major organs of NO₃ assimilation, especially at levels of NO₃ (10 to 40 millimolar) that maintained plant growth at near maximum rates. Partitioning and utilization of C and N were studied in nodulated, minus NO₃ plants and non-nodulated plants fed 10 or 20 millimolar NO₃, the levels of NO₃ 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 NO₃-grown plants (64%), but greater proportions of photosynthate were translocated to below ground parts of nodulated plants (37%) than of NO₃-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 NO₃-fed plants. Theoretical considerations suggest that the elevated CO₂ output of nodulated roots was due not only to CO₂ 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 NO₃-fed plants. Data are compared with previously published information from other legumes.     

Assimilation and Transport of Nitrogen in Nonnodulated (NO(3)-grown) Lupinus albus L

The response of nonnodulated white lupin (Lupinus albus L. cv. Ultra) plants to a range of NO₃ levels in the rooting medium was studied by in vitro assays of extracts of plant parts for NO₃ reductase (EC 1.6.6.1) activity, measurements of NO₃-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 NO₃ followed by 10 days with 1, 5, 15, or 30 millimolar NO₃. NO₃ reductase was substrate-induced in all tissues. Roots contained 76, 68, 62 and 31% of the total NO₃ reductase activity of plants fed with 1, 5, 15, and 30 millimolar NO₃, respectively. Stem, petioles, and leaflets contained virtually all of the NO₃ 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 NO₃-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 NO₃ in xylem sap amounted to 4, 13, 12, and 17% of the total xylem N at 1, 5, 15, and 30 millimolar NO₃, 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 NO₃. NO₃ 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 NO₃. Shoot organs were ineffective in storing NO₃. Even when NO₃ 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.     

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.     

Pathways of Nitrogen Assimilation in Cowpea Nodules Studied using N(2) and Allopurinol

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 [¹⁵N]xanthine from ¹⁵N₂ 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 ¹⁵N-labeling of asparagine from ¹⁵N₂ 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.     

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 ¹⁴CO₂ to investigate the contribution of nodule CO₂ 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 CO₂ fixed by nodules was rapidly transported to shoots and incorporated into acid stable constituents. The data are consistent with a role for nodule CO₂ fixation providing carbon for the assimilation and transport of fixed nitrogen in amide-based legumes. In contrast, CO₂ fixation by nodules of ureide transporting legumes appears to contribute little to assimilation and transport of fixed nitrogen.     

Amino acid content in xylem sap of regrowing alfalfa (Medicago sativa L.): Relations with N uptake,N2 fixation and N remobilization

During vegetative regrowth of Medicago sativa L., soil N, symbiotically fixed N₂ and N reserves meet the nitrogen requirements for shoot regrowth. Experiments with nodulated or non-nodulated plants were carried out to investigate the changes in N flows originating from the different N sources and in xylem transport of amino acids during regrowth. Exogenous N uptake, N₂ fixation and endogenous N remobilization were estimated by ¹⁵N labelling and amino acids in xylem sap were analysed. Removal of shoots resulted in great declines of exogenous N flows derived either from N₂ or from NH₄NO₃ during the first week of regrowth, thereafter recovery increased linearly. Mineral N uptake as well as N₂ fixation occurred mainly between the 10th and 18th day after removal of shoots while exogenous N assimilation in intact plants remained at a steady level. Nitrogen remobilization rates in defoliated plants increased by at least three to five-fold, especially during the first 10 days following shoot removal. Compared to control plants, contents of amino acids in xylem sap, during the first 10 days of regrowth, were reduced by about 72% and 82% in NH₄NO₃ grown and in N₂ fixing plants, respectively. Asparagine was the main amino acid transported in xylem sap of both treated plants. Its relative contents during this period significantly decreased from 75% to 59% and from 67% to 36% respectively in non-nodulated plants and in nodulated ones. This decline was accompanied by compensatory increase in the relative contents of aspartate and glutamine.     

The Effect of Nitrogen Source on Transport and Metabolism of Nitrogen in Fruiting Plants of Cowpea (Vigna unguiculata (L.) Walp.)

Nitrogen metabolism and transport were studied during reproductivedevelopment of cowpea (Vigna unguiculata (L.) Walp. cv. Vita3) under three contrasting nitrogen regimes: (1) nitrate suppliedcontinuously (plants non-nodulated), (2) symbiotic N₂ fixation(no combined nitrogen), (3) nitrogenstarvation post-anthesisof previously N₂-fixing plants. The last treatment involveddaily flushing of the root systems with 100% oxygen which suppressedpost-anthesis N₂-fixation by 76–79%, thereby making fruitgrowth almost entirely reliant upon mobilization of previouslyaccumulated nitrogen. The bulk of the xylem nitrogen (root bleedingsap or peduncle tracheal sap) of nitrate-fed plants was nitrateand amide, that of symbiotic and O₂-treated plants largely ureide.The composition of fruit cryopuncture phloem sap, however, wasclosely similar in all treatments, with most nitrogen as amidesand amino acids. The evidence suggested intense metabolic transferof root derived nitrate-N or ureide-N to amino acids by vegetativeplant parts prior to translocation to fruits. All tissues offruits showed patterns of development of enzymic activitiesconsistent with release of nitrogen from both ureides and amidesand re-assimilation of ammonia to form amino acids. Althoughthe levels of enzyme activities varied between treatments thedifferences could not be readily associated with individualpatterns of nitrogen transport in the treatments. Nitrogen sufficiencyin the NO₃-fed plants was marked by elevated vegetative biomassand low harvest indices for dry matter and nitrogen, while nitrogendeficiency of the O₂-treated plants was associated with seedabortion, small seed size and low seed nitrogen concentration,and efficient mobilization of nitrogen from vegetative partsto fruits. Key words: Nitrogen, Translocation, Cowpea     

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 CO₂ and N₂, respiratory losses of C, and the production of dry matter and protein.     

Metabolism and translocation of allantoin in ureide-producing grain legumes

Transfer of the nitrogen and carbon of allantoin to amino acids and protein of leaflets, stems and petioles, apices, peduncles, pods, and seeds of detached shoots of nodulated cowpea (Vigna unguiculata L. Walp. cv. Caloona) plants was demonstrated following supply of [2-¹⁴C], [1,3-¹⁵N]allantoin in the transpiration stream. Throughout vegetative and reproductive growth all plant organs showed significant ureolytic activity and readily metabolized [2-¹⁴C]allantoin to ¹⁴CO₂. 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, NO₃⁻ grown plants.

[¹⁴C]Ureides were recovered in extracts of aphids (Aphis craccivora and Macrosiphum euphorbieae) feeding at different sites on cowpea plants supplied with [2-¹⁴C]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.

     

Effect of nitrogen source on ureides in soybean

In field-grown soybeans (Glycine max L. Merr. cv Harosoy), the percentage of N in the xylem as ureides increased with increasing N₂ fixation. During a 9-week collection period, the ureide content varied from 9.0 to 69.2% of the xylary N. Between 9 and 11 weeks (early pod fill), there was a good correlation (r = 0.93) between C₂H₂ reduction and the per cent N in xylem as ureides. The per cent N as ureides, however, does not always indicate the reliance of the plant on symbiotic N₂ fixation. This ureide content also depended on the level of NO₃⁻ available to the roots. Non-nodulated soybeans given from 0 to 200 kilogram N per hectare produced xylem sap which averaged from 31.8% to 9.0% N, respectively, in the xylem as ureides over the 9-week period.

Feeding of ¹⁵N₂, ¹⁵NH₄, or ¹⁵NO₃ to greenhouse-grown soybeans indicated substantial differences in the initial distribution of N by the xylem stream, but the ultimate distribution of N between plant parts and grain did not vary with available N or percentage of xylary N as ureides. Amino acids, not ureides, were the major source of N in the phloem. The soybeans maintained a similar composition in phloem irrespective of the xylem sap constituents, with N derived from N₂, NH₄, or NO₃ being equally accessible to the phloem stream.

     

Recovery of nitrogen fixation after short-term flooding of the nodulated root system of soybean

Nitrogen fixation of terrestrial legumes is strongly and rapidly diminished under flooding. Although recovery is possible with the formation of aerenchyma, information is scarce regarding recovery after draining following short-term flooding, before the appearance of morphological adaptations. This study used soybean (Glycine max) plants nodulated with Bradyrhizobium elkanii to determine xylem sap glutamine as an indication of nitrogen fixation activity during recovery from different periods of flooding. Xylem glutamine levels showed rapid recovery (within 90 min) following periods of flooding up to 4 h. Recovery was progressively slower after longer periods of flooding. After 48 h flooding very little recovery could be observed within the first 120 min after draining but recovery was possible within 48 h. Consistent with the changes in xylem glutamine, direct measurements of apparent nitrogenase activity carried out immediately on draining revealed rapid recovery after flooding for 1 h and slow recovery following 48 h of flooding. In the latter case, nitrogenase activity largely recovered 24 h after draining. Experiments with ¹⁵N₂ incorporation into amino acids exported in the xylem sap revealed that glutamine was by far the most highly labelled amino acid in sap collected over the first 30 min of exposure to the isotope. This is conclusive evidence that xylem sap glutamine is an immediate product of N₂ fixation and export. The changes in xylem sap glutamine seen on flooding (decline) and after draining (recovery) can therefore be attributed to changes in nitrogenase activity. The data show that xylem sap glutamine is a useful means for assessing changes in nitrogenase activity, especially when the root system is submersed in water and activity cannot be measured directly.     

Allantoin and Allantoic Acid in Tissues and Stem Exudate from Field-grown Soybean Plants

Samples of stem exudate and plant tissue collected from field-grown soybean (Glycine max [L.] Merr.) plants were analyzed for allantoin and allantoic acid. Nitrogen in nitrate plus amino acids exceeded ureide N concentration in stem exudate prior to flowering. During all of reproductive development (from about 40 days after planting until maturity), ureide N concentration was two to six times greater than amino acid plus nitrate N concentration. Allantoin and allantoic acid, not asparagine, are the principal forms of nitrogen transported from nodulated roots to shoots of the soybean plant. During pod and seed development ureide N comprised as high as 2.3, 37.7, and 15.8% of total N in leaf blades, stems + petioles, and fruits, respectively. The concentration of ureide in stems and fruits declined to nearly zero at maturity.