Non-nodulated Plants Research Articles

Early Nodulation Signals of the Wild Type and Symbiotic Mutants of Soybean (Glycine max)

The results indicate that the autoregulatory and nonnodulating plant mutants do not have significantly different levels of bacterial nod gene signaling molecules compared to wild-type plants. It appears unlikely that alterations in levels of plant signals are responsible for the mutant symbiotic plant phenotypes examined

Characteristics and carbon metabolism of mesophyll and paraveinal mesophyll protoplasts from leaves of non-nodulated Glycine max

The paraveinal mesophyll (PVM) is a spatially, morphologically and ultrastructurally specialized tissue in soybean leaves. It accounts for about 18% of the mesophyll volume as determined by stereological measurements and represents a significant cellular compartment, equal in size to that of the spongy mesophyll. Mesophyll cells (MC), their protoplasts (MP) and PVM protoplasts (PVMP) were isolated from leaves of non-nodulated plants and their physical characteristics and relative activity in some aspects of carbon metabolism were determined. PVMP were about twice the diameter and seven times the volume of MP: they had smaller and 2 3 fewer chloroplasts, and about one third the chlorophyll content of MP. Ribulose 1,5-biphosphate carboxylase (RuBPCase) activity and 14CO 2 fixation rates of PVMP were 40% and 30%, respectively, of the values measured in MP on a protoplast basis. PVMP showed net oxygen consumption (240 nmol O 2 (10 6 protoplasts) −1 h −1) in the light with 10 mM bicarbonate, while MP showed the expected photosynthetic oxygen evolution (89 nmol O 2 (10 6 protoplasts) −1 h −1) under similar conditions. The dark respiration rate of PVMP was about six times greater on a chlorophyll basis, three times greater on a protoplast basis and one point seven times greater on a cytoplasmic volume basis than the rate measured in MP. The data show that the PVM plays a minor role in carbon fixation in soybean leaves and supports the hypothesis that one of its primary roles is as a cellular pathway for metabolite transport within the leaf.

Differences in the distribution of mannose- and galactose-binding lectins in peanut tissues

A mannose/glucose-binding lectin (ML), previously purified from peanut ( Arachis hypogaea L.) nodules, was detected in other tissues using an enzyme-linked immunoassay system and its distribution in nodulated and non-nodulated plants compared with that of the galactose-binding lectin (GL) of peanut. Unlike GL, ML was not detection seed but was present in cotyledons and roots of 5-day-old seedlings at planting and in all tissues throughout growth of plants receiving fixed or inorganic N. Concentrations of ML declined to base levels only in tissues of plants deprived of N whereas GL declined from high levels in seed to less than 0.2 μg/g in all plant tissues irrespective of conditions of N-supply. Levels of GL rose slightly after 6 weeks but remained below 1 μg/g except in pods where levels reached 3–6 μg/g at 10 weeks. Both lectins were detected in Bradyrhizobium-incited nodules at 2 weeks and their pattern of synthesis was similar in this tissue although levels of ML were higher than those of GL. Appropriate sugar haemagglutination inhibition tests confirmed the presence of each lectin in tissue extracts. Neither purified nodule lectin reacted with cells or bacteroids of symbiotic peanut rhizobia.

Temporal water stress effects on nodulation, nitrogen accumulation and growth of soybean

The N accumulation and growth of regularly watered soybean (Glycine max. L.) plants were compared with those grown under various durations of drought stress varying from 14 to 47 days (D). The stresses were imposed between the V3 (three nodes) and R7 (physiological maturity) growth stages. The stress was defined as eithermild, moderate orsevere, corresponding to (i), a single drought cycle from V3 to R2 (18 D) or R5 to R7 (14 D), (ii) double cycle from V3 to R5 (33 D) or R2 to R7(29 D), and (iii) three continuous cycles from V3 to R7 (47 D), respectively. Plants were harvested at the R7 stage (76 days after planting). The A-values measured by the non-nodulated plants under all treatments were similar (P<0.05), indicating that the available amount of soil N was not changed by drought. However, the A-values assessed by the nodulated plants (which included the N2 fixed), differed significantly among treatments. Except under the longest stress condition, all plants (in both genotypes) absorbed similar amounts of soil N, but N2 fixed in the nodulated plants differed, significantly among treatments. The regularly-watered plants derived the highest amount (68 mg plant−1) and proportion (46.1%) of N from fixation, and the water stresses resulted in significant reductions in N2 fixed. Although the growth of both nodulated and non-nodulated plants, was adversely affected by water stress, this was not as great as was the effect on N2 fixation. Nitrogen fixation was the most sensitive parameter to drought, followed by plant growth, and the least sensitive was soil N uptake.

Evaluating alder-endophyte (Alnus acuminata-Frankia-Mycorrhizae) interactions

The objective of this study was to compare the interaction betweenFrankia and vesicular-arbuscular mycorrhizae (VAM) on the growth and N-fixing response ofAlnus acuminata seedlings under three different phosphorus levels.A. acuminata is an actinorhizal tree, commonly associated with pastures on upland areas. Seedlings were grown in sterile vermiculite, and inoculated withFrankia strain ArI3 and/or VAM (asGlomus intra-radices) under three phosphorus levels (10, 50 and 100 ppm). After 120 days differences in growth were observed at the 50 ppm P level between nodulated and non-nodulated plants; either if inoculated withFrankia+VAM or just with VAM. Interaction betweenFrankia and VAM was positive on nodule weight at 50 ppm P level. Differences in acetylene reduction, per gram of fresh nodule, were observed between and within both groups:Frankia inoculated andFrankia+VAM inoculated seedlings. InFrankia inoculated seedlings differences were observed between seedlings receiving 50 ppm P which showed higher nitrogenase activity than seedlings treated with 100 ppm P. Plants inoculated withFrankia andGlomus intra-radices at low P level (10 ppm) showed the highest acetylene reduction. It was 150% higher than the mean of the other treatments within the group, and 87% higher than the general mean of the onlyFrankia inoculated plants.

Rapid Dinitrogen Fixation During Soybean Pod Fill Enhances Net Photosynthetic Output and Seed Yield: A New Perspective

AbstractNitrogen is frequently the limiting nutrient in plant growth and reproduction. Nevertheless, it is widely reported that the seed yield of the nodulated soybean [Glycine max (L.) Merr.] grown on fertile soil is not N‐limited. This paper, based on new experimental evidence obtained under carefully controlled laboratory conditions, seeks to review and reevaluate this conclusion. To test this claim, inoculated soybean plants were grown hydroponically in the presence of suboptimal urea or with an excess of either nitrate or nitrate plus urea. Acetylene reduction activities (i.e., N2 fixation rates) of plants grown on suboptimal urea were approximately tenfold greater than those of plants grown on excess nitrate. At maturity, plants that had fixed N2 at a rapid rate during pod fill had a greater biomass (i.e., a greater net photosynthetic output), a higher N content, and produced larger seeds than uninoculated or poorly nodulated plants grown on excess nitrate. Likewise, published reports show that plant biomass, seed N, and mean seed weight (i.e., g/100 seeds) of nodulated plants grown in the field without added fertilizer N were significantly greater than those of nonnodulated plants grown on adjacent field plots and given a modest level of fertilizer N. Calculations show that mean seed weight of both hydroponically grown and fieldgrown plants correlated significantly (r ≥ 0.93) with seed yield. Because of this strong positive correlation and because rapid N2 fixation during pod fill significantly increases mean seed weight without lowering the number of seeds, it is concluded that elevated N2 fixation by field grown plants would increase soybean yield significantly. An increase in both total plant N and net photosynthetic output is thought to promote this increase in seed yield.

A non-nodulating alfalfa mutant displays neither root hair curling nor early cell division in response to Rhizobium meliloti.

The early events in the alfalfa-Rhizobium meliloti symbiosis include deformation of epidermal root hairs and the approximately concurrent stimulation of cell dedifferentiation and cell division in the root inner cortex. These early steps have been studied previously by analysis of R. meliloti mutants. Bacterial strains mutated in nodABC, for example, fail to stimulate either root hair curling or cell division events in the plant host, whereas exopolysaccharide (exo) mutants of R. meliloti stimulate host cell division but the resulting nodules are uninfected. As a further approach to understanding early symbiotic interactions, we have investigated the phenotype of a non-nodulating alfalfa mutant, MnNC-1008 (NN) (referred to as MN-1008). Nodulating and non-nodulating plants were inoculated with wild-type R. meliloti and scored for root hair curling and cell divisions. MN-1008 was found to be defective in both responses. Mutant plants inoculated with Exo- bacteria also showed no cell division response. Therefore, the genetic function mutated in MN-1008 is required for both root hair curling and cell division, as is true for the R. meliloti nodABC genes. These observations support the model that the distinct cellular processes of root hair curling and cell division are triggered by related mechanisms or components, or are causally linked.

Biological Characterization of Root Exudates and Extracts from Nonnodulating and Supernodulating Soybean Mutants

The root exudates and extracts of soybean nonnodulation (nod¯) mutants nod49, nod139, and nod772 were compared with those of the wild-type parent cultivar Bragg and supernodulation (nitrate tolerant symbiotic) mutant nts382 to assess their effects on the early stages of nodulation by Bradyrhizobium japonicum strain USDA110. When nod¯ mutants were cocultured in proximity with wild-type or mutant nts382, nodulation was as expected for the individual genotypes. Coculturing nts382 with wild-type or nonnodulating plants decreased nodule number on the supernodulating mutant when compared to similar plant density of nts382 alone. Pretreatment of B. japonicumwith collected root exudates of the soybean mutants did not affect nodulation efficiency or total nodule number per plant. Analysis of in vivo labeled proteins in root exudates of wild type, nts382, and nod49 showed similar patterns both on one- and two-dimensional gels. Radioactively (35S) labeled root exudates were subjected to immunoprecipitation with the antibody to soybean lectin (SBL) and similar amounts were found in the nod49, nts382, and wild-type soybean. Seedling root extracts of nts382, nod49, and Bragg were tested for their ability to induce a nodC::lacZ fusion in B. japonicum strain USDA110. Similar levels of induction were found in all cases, indicating that uninoculated seedlings of cultivar Bragg, nod49, and nts382 contained similar stimulating compounds. Based on the observation that mutants nod49, nod139, and nod772 lack root hair curling, we suggest that the nonnodulation phenotype may be caused by an inability to respond efficiently to bacterial factors designed to stimulate plant cell divisions and concomitant root hair curling.

Organ regulated expression of Parasponia andersonii haemoglobin gene in transgenic tobacco plants.

Plant haemoglobin genes are known to occur in legume and non-legume families and in both nodulating (e.g., Parasponia andersonii) and non-nodulating species (e.g., Trema tomentosa). Their presence in non-nondulating plants raises the possibility that haemoglobins might serve a function in non-symbiotic tissues distinct from their role in the nitrogen-fixing root nodules induced by micro-organisms. We report here that a P. andersonii haemoglobin promoter can regulate expression of either the P. andersonii haemoglobin gene, or a hybrid construct with the bacterial chloramphenicol acetyltransferase gene (cat), in the non-symbiotic plant, Nicotiana tabacum. Expression is predominantly in the roots, implying that haemoglobins might have a function in roots of non-nodulated plants. We have also observed a low level of haemoglobin protein in non-nodulated P. andersonii roots, but not leaves, supporting this assertion. The expression in transgenic plants will allow further characterization of the promoter sequences essential for the organ-specific expression of haemoglobins in non-symbiotic tissues.

Non‐Nodulating Mutants in Common Bean

Estimates of nitrogen fixation by common bean (Phaseolus vulgaris L.) have been made using non‐nodulating soybean or other nonfixing plant species to measure soil nitrogen because non‐nodulating bean lines are not available. Such lines are also needed for studies on the genetic control of nodulation. The objective of this study was to obtain (using induced matagenesis) nodulation mutants of the bean lines ‘RIZ 30’ and ‘RIZ 36’. One hundred and twelve nonnodulated plants were identified in the M2 generation following treatment of imbibed seeds with 0.08 M ethyl methane sulfonate for 4 h. Screening was carried out in perlite with a nitrogen‐free nutrient solution, and seeds were inoculated with a mixture of Rhizobium strains CIAT 632 and CIAT 899 before emergence. Plants were uprooted 3 wk after planting, and those without nodules were transferred to soil for seed production. The absence of nodulation by eight lines was confirmed in the M3 and M4 generations following inoculation with several other strains of Rhizobium, and in the M5 generation two mutant lines were evaluated in soil. Seed set in these lines is poor, though improved by applying nitrogen.

Biology of theParasponia-Bradyrhizobium symbiosis

Parasponia remains the only non-legume known to nodulate withRhizobium/Bradyrhizobium. It is a pioneer plant that is capable of rapid growth and fixing large quantities of nitrogen. In addition to its high agronomic potential, the symbiosis offers the scientist the unique opportunity of studying differences at the molecular level of both partners, and to investigate any possible extension of the symbiosis to other non-legumes of importance. Haemoglobin has been found in the nodule tissue ofParasponia and other nodulated non-legumes and the gene for it has been found and expressed in non-nodulating plants such asTrema tomentosa andCeltis australis. Bradyrhizobium strains isolated from species ofParasponia growing in Papua New Guinea form a group that are more specific in their host requirements thanBradyrhizobium strains from tropical legumes from the same area. They do not effectively nodulate (except CP283) tropical legumes, andParasponia is not readily nodulated withRhizobium andBradyrhizobium strains from legumes. The effectiveness of the symbiosis is influenced by host species, theBradyrhizobium strain and the environment.Parasponia andersonii forms a more effective symbiosis than the other species tested. In competition studies with strains from legumes, isolates fromParasponia always dominate in nodules onParasponia.

Functioning haemoglobin genes in non-nodulating plants

Haemoglobin has previously been recorded in plants only in the nitrogen-fixing nodules formed by symbiotic association between Rhizobium or Frankia and legume or non-legume hosts. Structural similarities amongst these and animal haemoglobins at the protein and gene level suggested a common evolutionary origin. This suggests that haemoglobin genes, inherited from an ancestor common to plants and animals, might be present in all plants. We report here the isolation of a haemoglobin gene from Trema tomentosa, a non-nodulating relative of Parasponia (Ulmaceae). The gene has three introns located at positions identical to those in the haemoglobin genes of nodulating plant species, strengthening the case for a common origin of all plant haemoglobin genes. The data argue strongly against horizontal haemoglobin gene transfer from animals to plants. The Trema gene has a tissue-specific pattern of transcription and translation, producing monomeric haemoglobin in Trema roots. We have also found that the Parasponia haemoglobin gene is transcribed in roots of non-nodulated plants. These results suggest that haemoglobin has a role in the respiratory metabolism of root cells of all plant species. We propose that its special role in nitrogen-fixing nodules has required adaptation of the haemoglobin-gene regulation pathway, to give high expression in the specialized environment of the nodule.

Uptake of soil nitrogen by soybean as influenced by symbiotic N 2-fixation or fertilizer nitrogen supply

Nodulated and N 2-fixing soybean plants ( Glycine max. L.) grown in a pot experiment took up significantly more soil N (labelled with 15N) than non-nodulated control plants. The organic matter in the experimental soil was labelled with 15N during a previous incubation, and the pool of labelled inorganic N originated mainly from mineralization of organic matter. Addition of non-labelled ammonium or nitrate-N to non-nodulated plants did not increase their uptake of labelled soil N. Plants grown with the various N-sources exhausted the soil for KCl-extractable N to almost the same low concentration. Where non-nodulated plants were grown, 60–75% of the inorganic N initially present could be accounted for in plants and KCl-extracts at harvest. An amount corresponding to 98% of the KCl-extractable N initially present was found in nodulated plants and the pool of KCl-extractable N.

Further Studies on Genetics of Nonnodulation in Peanut

In the peanut (Arachis hypogaea L.) nonnodulation has been reported to be controlled by both duplicate recessive genes and a single recessive gene. In the present study genetics of peanut nonnodulation was investigated in four crosses from data on F2 populations and F3 progenies from nonnodulating F2 plants. The F2 data of all the four crosses studied showed a better fit for a trigenic ratio of 61 nodulating to three nonnodulating plants compared to the previously reported duplicate digenic ratio of 15 nodulating to one nonnodulating F2, plants. Further, the segregation of F3 progenies from nonnodulating F2 plants indicated the inadequacy of the duplicate factor model. A new genetic model involving three genes in the inheritance of nonnodulation is proposed. Two genes produce nodulation while the third gene inhibits nodulation only when it is dominant and the former two genes are in recessive homozygous condition. By assuming differential heterozygosity at the three loci of the parents involved in various crosses, the duplicate factor and monogenic ratios reported by previous workers on nonnodulation could be explained by the new model. Although the occurrence or non‐occurrence of nodules is governed by a few major genes, the intensity of nodulation appears to be controlled quantitatively.

Glycine-Glomus-Rhizobium Symbiosis

Soybean (Glycine max [L.] Merr. cv Hobbit) plants were grown in a growth chamber for 56 days in a phosphorus- and nitrogen-deficient soil and were colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd) Gerd. and Trappe and Rhizobium japonicum strain USDA 136, or by either organism alone, or by neither. Non-VAM plants received supplemental phosphorus and nonnodulated plants supplemental nitrogen to achieve the same rate of growth in all treatments. Plants of all four treatments had the same (P > 0.05) dry weights at harvest, but VAM plants had higher rates of CO(2) exchange (CER, P < 0.05) and lower leaf P concentrations (P < 0.01). Leaf nitrogen concentrations were lower in nodulated than in nitrogen-supplemented plants (P < 0.01) while starch concentrations were higher (P < 0.01). There was a significant negative relationship between nitrogen and starch (r = -0.989). Statistical evaluation of the data showed that some parameters (CER, leaf area and phosphorus content) were associated with phosphorus nutrition (or the presence of the VAM fungus), others (leaf fresh weight and root dry weight) with nitrogen nutrition (or the presence of Rhizobium), and some (leaf nitrogen and starch content) by both factors. The development of microsymbiont structures and nodule activity were significantly lower in the tripartite association than in plants colonized by one endophyte only. The findings suggest that endophyte effects go beyond those of simple nutrition and associated source-sink relationships.

Benefits of mycorrhizae to soybeans grown on various regimes of nitrogen nutrition1

Abstract Nodulating and nonnodulating isolines of soybean (Glycine max Merr ‘Clay') were grown in sand culture in a greenhouse. The plants were cultured with or without mycorrhizal (Glomus mosseae) infection, and nodulating plants were inoculated with Rhizobium iaponicum. Phosphorous was supplied as hydroxyapatite or dicalcium phosphate with N nutrition from nitrate or as combinations of nitrate and ammonium or nitrate and urea. Best growth of the nodulating isoline was with urea nutrition. Best growth with the nonnodulating isoline was with ammonium nutrition. Urea‐treated nodulating plants showed increased growth due to mycorrhizae. Urea‐treated or ammonium‐treated nonnodulating plants showed growth increases due to mycorrhizae. Nitrate‐treated plants did not show increased growth due to mycorrhizae. Mycorrhizal infection was greatest with urea nutrition, and the infection increased the tissue N content of these plants relative to nonmycorrhizal plants. Enhancement of tissue P accumulation through mycor...

Effect of nitrogen on nodulation and yield of soya beans under two systems of production in Sudan

SummaryTwo experiments were run over a 3-year period in the central rainlands of Sudan under two systems of production, rainfed and irrigated, to assess the effects of system of production, inoculation and nitrogen fertilizers on plant and nodule development and grain yield of soya beans. Nodulated plants could fix more than 80 kg N/ha under irrigation whereas under rainfed conditions nodulation was neither effective nor efficient. Soya bean was responsive to nitrogen fertilizers under both systems of production giving significant increments in grain yields. Non-nodulating plants with added nitrogen fertilizers produced more total dry matter than nodulating plants during the vegetative phase until flowering time. At 2 weeks after flowering total dry-matter production for both types was equal and from then on to maturity nodulating plants outyielded non-nodulating ones in total dry-matter production. In 1979 and 1980 yield of irrigated nodulating soya-bean grain was 0·53 and 1·54 t/ha higher than rainfed yields whereas the difference in grain yields of the non-nodulating soya beans was 0·21 t/ha and zero during the same two seasons, respectively. There was a contrasting inverse relation between the number of nodules and dry weights under the two systems of production. Fewer and heavier nodules were produced under irrigation whereas under rainfed conditions nodulation was profuse and nodules were light. On the evidence available 1–4 g/m length of the granular form of soil implant inoculant (Nitragin), i.e. 16·6·66.4 kg/ha, is to be recommended for irrigated soya-bean production in Sudan.

Total and CO-reactive heme content of actinorhizal nodules and the roots of some non-nodulated plants

The concentration of total and CO-reactive heme was measured in actinorhizal nodules from six different genera. This gave the upper limit to hemoglobin concentration in these nodules. Quantitative extraction of CO-reactive heme was achieved under anaerobic conditions in a buffer equilibrated with CO and containing Triton X-100. The concentration of CO-reactive heme in nodules of Casuarina and Myrica was approximately half of that found in legume nodules, whereas in Comptonia, Alnus and Ceanothus the concentrations of heme were about 10 times lower than in legume nodules. There was no detectable CO-reactive heme in Datisca nodules, but low concentrations were detected in roots of all non-nodulating plants examined, includingZea mays. Difference spectra of CO treated minus dithionite-reduced extracts displayed similar wavelengths of maximal and minimal light absorption for all extracts, and were consistent with those of a hemoglobin. The concentration of CO-reactive heme was not correlated to the degree to which CO inhibited nitrogenase activity nor was it affected by reducing the oxygen concentration in the rooting zone. However, there was a positive correlation between heme concentration and suberization or lignification of the walls of infected host cells. These observations demonstrate that, unlike legume nodules, high concentrations of heme or hemoglobin are not needed for active nitrogen fixation in most actinorhizal nodules. Nonetheless, a significant amount of CO-reactive heme is found in the nodules of Alnus, Comptonia, and Ceanothus, and in the roots ofZea mays. The identity and function of this heme is unknown.

Comparisons of ureide and acetylene reduction methods for estimating biological nitrogen fixation by glasshouse-grown cowpea

In dry field conditions, obtaining estimates of biological nitrogen fixation using either the acetylene reduction assay or measurements of ureide concentrations in xylem sap is limited by the difficulties involved in digging out root-nodule systems or obtaining xylem sap. This study was conducted to determine whether ureide concentrations in extracts from stem tissues are as reliable for estimating rates of biological nitrogen fixation by cowpea [ Vigna unguiculata (L.) Walp.] as acetylene reduction assays or ureide concentrations in xylem sap. Glasshouse-grown cowpeas were subjected to different levels of NO 3 − (0, 2, 10 mM) in a basal nutrient solution to achieve different levels of nitrogen fixation. Both levels of added nitrogen (2 and 10 mM) reduced nodulation and nitrogen fixation as measured by the ureide methods and acetylene reduction assay. All methods used to estimate rates of nitrogen fixation exhibited similar seasonal trends but some differences were apparent among treatments. Hot-water extraction of ureides from dried stem tissue appears promising as a rapid technique that could be effective under dry field conditions where the other techniques are not effective. Relative ureide abundance, expressed as 100 × [ureide (N)]/[ureide (N) + NO 3 − (N) + NH 4 + (N)], in either xylem sap or stem tissue, provided approximate estimates of the extent to which cowpeas rely on biological nitrogen fixation. Values of 85–95% were obtained for plants relying totally on nitrogen fixation, whereas non-nodulated plants grown on NO 3 − had values of 3–12%.

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.