Ureide assay for measuring nitrogen fixation by nodulated soybean calibrated by N methods.

We report experiments to quantify the relationships between the relative abundance of ureide-N in root-bleeding sap, vacuum-extracted sap, and hot water extracts of stems and petioles of nodulated soybean (Glycine max [L.] Merrill cv Bragg) and the proportion of plant N derived from nitrogen fixation. Additional experiments examined the effects of plant genotype and strain of rhizobia on these relationships. In each of the five experiments reported, plants of cv Bragg (experiment 1), cv Lincoln (experiments 3, 4, 5), or six cultivars/genotypes (experiment 2) were grown in a sand:vermiculite mixture in large pots in a naturally lit, temperature-controlled glasshouse during summer. Pots were inoculated at sowing with effective Bradyrhizobium japonicum CB1809 (USDA 136) or with one of 21 different strains of rhizobia. The proportions of plant N derived from nitrogen fixation were determined using (15)N dilution. In one experiment with CB1809, plants were supplied throughout growth with either N-free nutrients or with nutrients supplemented with 1, 2, 4, or 8 millimolar (15)N-nitrate and harvested on eight occasions between V6 and R7 for root-bleeding sap, vacuum-extracted sap, stems (including petioles), and whole plant dry matter. Analyses of the saps and stem extracts for ureides (allantoin plus allantoic acid), alpha-amino-N, and nitrate, and of dry matter for N and (15)N, indicated a positive effect of nitrate supply on concentrations of nitrate in saps and extracts and a negative effect on ureides and on the proportion of plant N derived from nitrogen fixation. The relative abundance of ureide-N in root-bleeding sap, vacuum-extracted sap (100 [ureide-N]/[ureide-N+ alpha-amino-N + nitrate-N]) and stem extracts (100 [ureide-N]/[ureide-N + nitrate-N]) and the proportion of plant N, derived from nitrogen fixation between successive samplings were highly correlated (r = 0.97-1.00). For each variable, two standard curves were prepared to account for the shifts in the compositions of N solutes of xylem saps and extracts after flowering which were not related to a change in nitrogen fixation. Relationships between relative ureide-N and the proportion of plant N derived from nitrogen fixation were not affected by plant genotype or by strain of rhizobia. Therefore, assessment of nitrogen fixation by soybean using the ureide technique should now be possible with the standard curves presented, irrespective of genotype or strain of rhizobia occupying the nodules.

Measurement of nitrogen fixation by soybean in the field using the ureide and natural N abundance methods.

Nitrogen fixation by field-grown soybean (Glycine max [L.] Merrill) was assessed by the natural (15)N abundance and ureide methods. The field sites (five) and genotypes (six, plus two levels of inoculation on Bragg) were chosen to provide a range of proportions of plant N derived from nitrogen fixation (P). Genotypes K466, K468, nts1007, and nts1116 and Davis were included on the basis of their reported tolerance of the suppressive effects of nitrate on nodulation and nitrogen fixation. Bragg was included as a ;nitrate-sensitive' genotype. Seeds of all genotypes were inoculated at sowing with Bradyrhizobium japonicum CB1809 (USDA136). Amounts of nitrate in the soil profile (0-1.2 meter depth) at sowing ranged from 70 (site 3) to 278 kilograms per hectare (site 5), resulting in large effects on plant nodulation, on the delta(15)N values of nodulated plants, on the relative abundance of ureide-N in vacuum-extracted sap (VES) and stem extracts, and finally on the estimates of P. There was no relationship between amount of soil nitrate at sowing and the delta(15)N of the plant-available soil N. Correlation matrices of the measured and calculated parameters indicated generally weak correlations between crop growth (dry matter and N) and the parameters of symbiotic activity (nodule weight, delta(15)N, relative ureide-N); correlations were strong and highly significant between nodulation and the measures of nitrogen fixation (delta(15)N, relative ureide-N; r = 0.79-0.92). Estimates of P ranged between 0 and 68% (delta(15)N) and between 6 and 56% (ureide) and were highly correlated (r = 0.97). Results indicated that the ureide method can be used with confidence to assess P by field-grown crops of soybean.

Relative abundance of ureides and nitrate in plant tissues of soybean as a quantitative assay of nitrogen fixation.

The relationship between the relative abundance of ureides ([ureide-N/ureide-N plus nitrate-N] x 100) in the shoot axis (stems plus petioles), nodulated roots and leaflets of "Bragg" soybean (Glycine max [L.] Merrill) and the symbiotic dependence of these plants was examined under glass-house conditions. Plants, inoculated with effective Rhizobium japonicum CB1809, were grown with their roots exposed continuously to a nutrient solution containing either 0, 1.5, 3.0, 6.0 or 12.0 millimolar NO(3)-N per liter. Nodulation and N(2)-acetylene fixation were correlated inversely with the level of nitrate. Seasonal acetylene reduction profiles for each of the nitrate treatments were integrated and the symbiotic dependence ([N(2) fixed per total plant N] x 100) determined using a conversion ratio of 1.5:1 (acetylene reduced:N(2) fixed), calculated from the zero NO(3) treatment. Examination of the nitrogenous solutes of the shoot axis and nodulated roots showed linear relationships between the relative abundance of ureides and the symbiotic dependence of the plants. Two standard curves, depicting these relationships during vegetative and reproductive growth, were drawn for each plant part. The overriding effect of plant age invalidated any attempt to develop a standard relationship for leaflets. Data from two diurnal studies suggested that relative ureides were insensitive to diurnal fluctuations, thus simplifying sampling procedures. Plant material could be stored at ambient temperatures (20-30 degrees C) for up to 24 h without affecting the relative concentration of ureides and nitrate. It is suggested that the shoot axis provides the most suitable target organ when using this technique as a quantitative assay for N(2) fixation because of ease of sampling of these tissues, especially with field-grown plants.

Use of the ureide technique to describe the nitrogen economy of field-grown soybeans.

Nitrogen fixation was estimated in ;Bragg,' ;Forrest,' and ;Bethel' soybean (Glycine max [L.] Merrill) from seven locations northwest of New South Wales, Australia, by relating ureide and nitrate contents of plant parts sampled at regular intervals during growth to standard curves derived under controlled nitrate regimes. Estimates were combined with data on crop growth and mineral N contents of soils to (a) determine the total requirements for N by the crops, (b) determine the contributions of N(2) fixation to crop growth, and (c) relate symbiotic dependence ([N(2) fixed/total plant N] x 100) of the crops to levels of mineral N in the soil at sowing. At two locations, Myall Vale and Glenara, levels of ureides in the shoot axes and roots of unnodulated seedlings were surprisingly high at the first time of sampling, perhaps reflecting effects of uptake of ammonium-N by the soybeans or breakdown and remobilization of cotyledonary protein. Ureides in plant parts declined significantly by the second (V5 to V7 growth stage) sampling. Subsequently, ureide contents increased whereas levels of nitrate in plant parts decreased. The relative abundance of ureides ([ureide-N/ureide-N + nitrate-N] x 100) in the shoot axes and nodulated roots of both crops increased linearly from almost zero during mid-vegetative growth (V5 to V7) to virtually 100% during late reproductive growth (R4 to R5, Myall Vale and R6, Glenara). The data suggest a steady transition in soybeans at both locations from dependence upon mineral N for early growth to complete reliance upon fixed N during late reproductive growth. Estimates of seasonal N(2) fixation for soybeans at the seven locations ranged from 73 to 288 kilograms per hectare N (shoot axes ureides) and from 147 to 337 kilograms per hectare N ha (nodulated roots ureides).

Synthesis, Storage, and Utilization of Amino Compounds in White Lupin (Lupinus albus L.).

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.

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 (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.

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

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