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.

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 (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.

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.

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

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

Diurnal water balance of the cowpea fruit.

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

The extrafloral nectaries of cowpea (Vigna unguiculata (L.) Walp.) II. Nectar composition, origin of nectar solutes, and nectary functioning.

Nectar was collected from the extrafloral nectaries of leaf stipels and inflorescence stalks, and phloem sap from cryopunctured fruits of cowpea plants. Daily sugar losses as nectar were equivalent to only 0.1-2% of the plant's current net photosynthate, and were maximal in the fourth week after anthesis. Sucrose:glucose:fructose weight ratios of nectar varied from 1.5:1:1 to 0.5:1:1, whereas over 95% of phloem-sap sugar was sucrose. [(14)C]Sucrose fed to leaves was translocated as such to nectaries, where it was partly inverted to [(14)C]glucose and [(14)C]fructose prior to or during nectar secretion. Invertase (EC 3.2.1.26) activity was demonstrated for inflorescence-stalk nectar but not stipel nectar. The nectar invertase was largely associated with secretory cells that are extruded into the nectar during nectary functioning, and was active only after osmotic disruption of these cells upon dilution of the nectar. The nectar invertase functioned optimally (phloem-sap sucrose as substrate) at pH 5.5, with a starting sucrose concentration of 15% (w/v). Stipel nectar was much lower in amino compounds relative to sugars (0.08-0.17 mg g(-1) total sugar) than inflorescence nectar (22-30 mg g(-1)) or phloem sap (81-162 mg g(-1)). The two classes of nectar and phloem sap also differed noticeably in their complements of organic acids. Xylem feeding to leaves of a range of (14)C-labelled nitrogenous solutes resulted in these substrates and their metabolic products appearing in fruit-phloem sap and adjacent inflorescence-stalk nectar. (14)C-labelled asparagine, valine and histidine transferred freely into phloem and appeared still largely as such in nectar. (14)C-labelled glycine, serine, arginine and aspartic acid showed limited direct access to phloem and nectar, although labelled metabolic products were transferred and secreted. The ureide allantoin was present in phloem, but absent from both types of nectar. Models of nectary functioning are proposed.

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

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

Nitrogen nutrition and the development and senescence of nodules on cowpea seedlings.

Cowpea (Vigna unguiculata (L.) Walp cv. Vita 3) seedlings inoculated with Rhizobium strain CB756 were cultured with their root systems maintained in air or in Ar: O2 (80:20, v/v) during early nodule development (up to 24 d after sowing). Compared with those in air, seedlings in Ar:O2 showed progressive N deficiency with inhibited shoot growth, reduced ribulose-1,5-bisphosphate carboxylase and total protein levels and loss of chlorophyll in the leaves. Nodule initiation, differentiation of infected and uninfected nodule tissues and the ultrastructure of bacteriod-containing cells were similar in the air and Ar: O2 treatments up to 16 d after sowing. Thereafter the Ar: O2 treatment caused cessation of growth and development of nodules, reduced protein levels in bacteroids and nodule plant cells, and progressive degeneration of nodule ultrastructure leading to premature senescence of these organs. Provision of NO 3 (-) (0.1-0.2 mM) to Ar: O2-grown seedlings overcame the abovementioned consequences of N2 deficiency on nodule and plant growth, but merely delayed the degenerative effects of Ar: O2 treatment on nodule structure and senescence. Treatment of Ar: O2-grown seedlings with NO 3 (-) greatly increased the protein level of nodules but the increase was largely restricted to the plant cell fraction as opposed to the bacteroids. By contrast, NO 3 (-) treatment of air-grown seedlings increased protein of bacteroid and host nodule fractions to the same relative extents when compared with air-grown plants not supplemented with NO 3 (-) . These findings, taken together with studies of the distribution of N in nodules of symbiotically effective plants grown from (15)N-labeled seed, indicate that direct incorporation of fixation products by bacteroids may be a critical feature in the establishment and continued growth of an effective symbiosis in the cowpea seedling.

Amino Acid transport and metabolism in relation to the nitrogen economy of a legume leaf.

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.

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-(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.

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.) : II. Chloroplast senescence and the degradation of ribulose-1,5-bisphosphate carboxylase.

The flag leaf of wheat was examined for changes in quantity and activity of ribulose-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39), in the proteolytic degradation of RuBPCase and other native proteins, and in the ultrastructure of the leaf cells during grain development. Proteolytic degradation of RuBPCase at pH 4.8 increased until 8-10 d after anthesis, then declined, and increased again 16-18 d after anthesis. The second peak coincided with the onset of a preferential loss of immunologically recognizable RuBPCase. The specific activity and number of active sites per molecule of RuBPCase did not change during senescence. Examination of ultrastructure with the electron microscope showed little change in the appearance of the mitochondria as the flag leaf aged. Prominent cristae were still evident 35 d after anthesis. In contrast, the chloroplasts showed a progressive disruption of the thylakoid structure and an increasing number of osmiophilic glubules. The double membrane envelope surrounding the chloroplast appeared intact until at least 20 d after anthesis. The tonoplast also appeared intact up to 20 d. At later stages of senescence of the leaf the outer membrane of the chloroplast adjacent to the tonoplast appeared to break but the inner membrane of the envelope appeared intact until at least 35 d after anthesis.

Nitrogen redistribution during grain growth in wheat (Triticum aestivum L.) : I. Peptide hydrolase activity and protein breakdown in the flag leaf, glumes and stem.

The activity of a range of endo- and exopeptidase enzymes have been measured in the glumes, flag leaf and stem during the period of grain development in wheat. The enzymes show a sequential pattern of appearance with activity peaks occurring at a number of intervals from anthesis until just prior to the cessation of grain growth. Of the enzymes studied only the haemoglobin- and casein-degrading activity and alanylglycine-dipeptidase activity increased during the period of rapid protein loss, while aminopeptidase, carboxypeptidase and leucyltyrosine dipeptidase reached maximum activity prior to this period.

Proteolysis in the axis of the germinating pea seed : I. Changes in protein degrading enzyme activities of the radicle and primary root.

Autoproteolytic, caseolytic and haemoglobin degrading activities, carboxypeptidase and aminopeptidase activities have all been measured in extracts prepared from the radicle of germinating pea seeds (Pisum sativum L.). With increasing time from the beginning of imbibition, the spectrum of protein degrading enzyme activities changed in a complex manner. As a proportion of total autoproteolytic activity, acid proteinases declined, while sulphydryl-and serine-active site endopeptidases accounted for increased proportions of the total activity. The distribution of protein degrading enzyme activities in the root tip compared with the balance of the root was determined after 4 days, at the onset of cell division in the root apex. On a fresh weight basis the tip was enriched ca. 2-fold in protein concentration and all of the exopeptidases. Autoproteolytic activity was concentrated in the tip to a lesser degree, and haemoglobin degrading activity not at all. In contrast, the root tip was depleted in caseolytic activity.

Degradation of ribulose-1,5-bisphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves.

In crude extracts from the primary leaf of wheat seedlings, Triticum aestivum L., cv. Olympic, maximum proteinase activity, as determined by measuring the rate of release of amino nitrogen from ribulose-bisphosphate carboxylase (RuBPCase), was found to be obtained only when EDTA and L-cysteine were included in the extraction buffer. Highest proteinase activity was obtained by grinding at pH 6.8, although the level of activity was similar in the pH range 5.6 to 8.0; this range also coincided with maximum extractability of protein. The lower amount of RuBPCase degrading proteinase extracted at low pH was not due to an effect of pH on enzyme stability. The optimum temperature of reaction was 50° C and reaction rates were linear for at least 120 min at this temperature. In the absence of substrate the proteinase was found to be very sensitive to temperatures above 30° C, with even short exposures causing rapid loss of activity. The relation between assay pH and RuBPCase degradation indicated that degradation was restricted to the acid proteinase group of enzymes, with a pH optimum of 4.8, and no detectable activity at a pH greater than 6.4. The levels of extractable RuBPCase proteinase exhibited a distinct diurnal variation, with activity increasing during the latter part of the light period and then declining once the lights were turned off. The effect of leaf age on the level of RuBPCase, RuBPCase proteinase and total soluble protein was investigated. Maximum RuBPCase activity occurred 9 days after sowing as did soluble protein. After the maximum level was obtained, the pattern of total soluble protein was shown to be characterised by three distinct periods of protein loss: I (day 9-13) 125 ng leaf(-1) day(-1); II (day 15-27) 11 ng leaf(-1) day(-1); III (day 29-49) 22 ng leaf(-1) day(-1). Comparison of the pattern of RuBPCase activity and total protein suggest that the loss of RuBPCase may be largely responsible for the high rate of protein loss during period I. Proteinase activity increased sharply during the period of most rapid loss of RuBPCase activity, and because the specific activity of RuBPCase also declined, we concluded that RuBPCase was being degraded more rapidly than the other proteins. Once the majority of the RuBPCase was lost, there did not appear to be a direct relation between RuBPCase proteinase activity and rate of total soluble protein loss, since the proteinase exhibited maximum activity during the slowest period of protein loss (II), and was declining in activity while the rate of protein loss remained stable during the third and final period of total protein loss.