Supernodulating Soybean Mutant Research Articles

Misexpression of miR482, miR1512, and miR1515 Increases Soybean Nodulation

MicroRNAs (miRNAs) are important regulators of plant growth and development. Previously, we identified a group of conserved and novel miRNA families from soybean (Glycine max) roots. Many of these miRNAs are specifically induced during soybean-Bradyrhizobium japonicum interactions. Here, we examined the gene expression levels of six families of novel miRNAs and investigated their functions in nodule development. We used northern-blot analyses to study the tissue specificity and time course of miRNA expression. Transgenic expression of miR482, miR1512, and miR1515 led to significant increases of nodule numbers, while root length, lateral root density, and the number of nodule primordia were not altered in all tested miRNA lines. We also found differential expression of these miRNAs in nonnodulating and supernodulating soybean mutants. The expression levels of 22 predicted target genes regulated by six novel miRNAs were studied by real-time polymerase chain reaction and quantitative real-time polymerase chain reaction. These results suggested that miRNAs play important roles in soybean nodule development.

Effect of N fertilizer top-dressing on N accumulation and N2 fixation of supernodulating soybean mutant

Increased application of nitrogen (N) fertilizer top-dressing during growth is an effective option for enhancing N supply to soybean plants. SS2-2 was characterized by the superior ability of symbiotic N2 fixation at the level of 30 kg N ha−1. But, the response of nitrogen fixation ability of supernodulating soybean mutant, SS2-2, to N fertilizer application rate remains unclear. The objective of this experiment was to compare the response of N fertilizer top-dressing on N accumulation and N2 fixation between supernodulating mutant, SS2-2, and wild-type, Sinpaldalkong 2. The effect of N fertilizer top-dressing (0.6 g N pot−1 top-dressing) on the nitrogen accumulation and redistribution were compared between SS2-2 and Sinpaldalkong 2. N fertilizer top-dressing at R1 stage increase in plant dry weight, relative growth rate (RGR), net assimilation rate (NAR), nitrogen harvest index (NHI), and N redistribution (NR). SS2-2 showed highest N concentration, 65.0 mg N g DW−1, followed by Sinpaldalkong 2 and En1282, and the N content per plant did not show a significant difference between SS2-2 and Sinpaldalkong 2. The N2 fixation rate was significantly reduced by N top-dressing, but the amount of N2 fixation was not changed due to an improved dry weight without changes of N concentration. In addition, SS2-2 showed higher NHI, NR and NRE than Sinpaldalkong 2. These results suggested that supernodulating soybean mutants, SS2-2, could be characterized by high N concentration and N2 fixation regardless of N fertilizer top-dressing due to a higher nitrate tolerance of supernodulating mutants than that of wild-type.

SNP discovery, linkage analysis and microsynteny in tentative consensus sequences derived from roots cDNA in a supernodulating soybean mutant

A soybean transcript map with genes mainly expressed in roots of a supernodulating mutant was constructed by mapping of single nucleotide polymorphism (SNP) markers into pre-existing SSR-based map, using a recombinant inbred line (RIL) population from Pureunkong and Jinpumkong 2. For SNP discovery, primer pairs were designed from 793 tentative consensus sequences (TCs) derived from the roots of a supernodulating soybean mutant. Among 520 loci showing high-quality sequence, more than 300 SNPs were identified in 116 loci between the parental genotypes. Average SNP frequency between the parental genotypes was one SNP per 1,010 bp. A total of 325 markers, including 98 SNPs, 208 SSRs, 15 AFLPs and four classical markers, were used to construct a genetic linkage map with 20 linkage groups (LGs) and a total length of 2,379 cM. Ten nonsynonymous changes were detected out of these SNP markers, indicating the possible usage of these markers as functional markers. With 40 of the SNP markers, putative synteny between soybean and Medicago truncatula (Mt) was revealed by identification of 19 short colinear blocks between the two genomes. This genetic map will provide the basis for the identification of gene in relation to nodulation and nitrogen fixation in partially sequenced genome like soybean by comparisons of the model genome (Mt). And, the integration of SNP markers and expression data with available genetic and phenotypic information would be helpful for clarifying function of cloned genes.

Nodulation and Growth of a Supernodulating Soybean Mutant SS2-2 Symbiotically Associated with Bradyrhizobium japonicum

<p class="p1">Mutan kedelai penghasil nodulsuper menunjukkan kelemahan dalam kontrol autoregulasi pada nodulasi dan perbedaan fenotip dibandingkan dengan tipe liarnya. Studi untuk mengevaluasi karakter pertumbuhan dan nodulasi dari kedelai penghasil nodulsuper dalam asosiasinya dengan <em>Bradyrhizobium japonicum </em>dilakukan dalam penelitian ini. Tiga genotip kedelai, yaitu mutan kedelai penghasil nodulsuper SS2-2, tipe liarnya Sinpaldalkong 2 dan kedelai kontrol Jangyeobkong diinokulasi dengan <em>B. japonicum </em>USDA 110, kemudian ditumbuhkan di rumah kaca dalam kondisi terkontrol. Karakter nodulasi, fiksasi nitrogen (Acetylene Reduction Activity/ARA), pertumbuhan tanaman, dan hasil biji ditentukan untuk mengevaluasi asosiasi simbiotik antara <em>B. japonicum </em>dan kedelai nodulsuper. Kedelai yang diinokulasi dengan <em>B. </em><em>japonicum </em>menunjukkan peningkatan jumlah dan berat kering nodul serta berat kering total tanaman dibandingkan dengan tanpa inokulasi. Tanaman SS2-2 yang diinokulasi menunjukkan jumlah nodul sekitar 20 kali lipat lebih tinggi daripada tipe liarnya. Inokulasi <em>B. japonicum </em>ternyata juga meningkatkan fiksasi nitrogen seiring dengan perkembangan nodul. Tanaman S2-2 lebih pendek dan menghasilkan fiksasi nitrogen (ARA) lebih tinggi, tetapi spesifik ARA dan hasil biji lebih rendah dibandingkan dengan tipe liarnya. Berdasarkan hasil evaluasi terhadap nodulasi dan pertumbuhannya, interaksi <em>Rhizobium </em>dan kedelai penghasil nodulsuper SS2-2 mempunyai respon asosiasi simbiotik lebih rendah dibandingkan kedelai penghasil nodul normal (kedelai yang tidak mendapat perlakuan mutasi).</p>

Differentially expressed genes related to symbiotic association in a supernodulating soybean mutant and its wild‐type

SUMMARY To understand the molecular basis of symbiotic association, a cDNA-AFLP technique was used to identify differentially expressed transcripts between a supernodulating soybean mutant, SS2-2, and its wild-type, Sinpaldalkong 2. As sources of cDNA-AFLP templates, trifoliates of 2-week-old plants were collected 1 week after Bradyrhizobium japonicum inoculation. A total of 147 bands out of 4000 amplicons were recognized as differentially expressed fragments, with 40 transcript-derived fragments (TDFs) in SS2-2 and 65 TDFs in Sinpaldalkong 2. Qualitative and quantitative real-time RT-PCR assays suggested that the expression patterns of genes in both genotypes were clearly differentiated. TDFs homologous to nodulin (65S2) and a putative senescence-associated protein (9S1) were up-regulated in SS2-2, whereas Sinpaldalkong 2 showed up-regulation of a receptor-like kinase (48sin1) and a kinase-like protein (17sin1). This indicates that different genes may be involved in regulation of the symbiotic programme that distinguishes SS2-2 from its wild-type. A TDF showing a change in a single base from A (Sinpaldalkong 2) to T (SS2-2) in this study was identified as a Glycine max nodule autoregulation receptor-like protein kinase precursor, previously identified by map-based cloning. These results demonstrate that cDNA-AFLP is a powerful technique to detect interesting genes without prior assumptions about the nature of the genes. The differentially expressed genes between Sinpaldalkong 2 and SS2-2 suggest that different signal transduction pathways for symbiosis may be involved in the two soybean genotypes.

Newly featured infection events in a supernodulating soybean mutant SS2-2 by Bradyrhizobium japonicum

Supernodulating soybean (Glycine max L. Merr.) mutant SS2-2 and its wild-type counterpart, Sinpaldalkong 2, were examined for the microstructural events associated with nodule formation and development. SS2-2 produced a substantially higher percentage of curled root hairs than the wild type, especially at 14 days after inoculation with Bradyrhizobium japonicum. In addition, there was new evidence that in SS2-2, B. japonicum also entered through fissures created by the emerging adventitious root primordia. Early steps of nodule ontogeny were faster in SS2-2, and continued development of initiated nodules was more frequent and occurred at a higher frequency than in the wild type. These data suggest that the early expression of autoregulation is facilitated by decreasing the speed of cortical cell development, leading to the subsequent termination of less-developed nodules. The nodules of SS2-2 developed into spherical nodules like those formed on the wild type. In both the wild type and supernodulating mutant, vascular bundles bifurcate from root stele and branch off in the nodule cortex to surround the central infected zone. These findings indicate that SS2-2 has complete endosymbiosis and forms completely developed nodule vascular bundles like the wild type, but that the speed of nodule ontogeny differs between the wild type and SS2-2. Thus, SS2-2 has a novel symbiotic phenotype with regard to nodule organogenesis.

Shoot-applied polyamines suppress nodule formation in soybean ( Glycine max)

In legumes, the number of root nodules is controlled by a mechanism called autoregulation. Recently, we found that the foliar brassinosteroid (BR), a plant growth-regulating hormone, systemically regulates the nodule number in soybean plants. In the present study we report that such down-regulation of root nodule formation by a BR may occur through a change of the polyamine contents, with the experimental evidence as follows. The foliar contents of both spermidine (Spd) and spermine (Spm) in the super-nodulating soybean mutant, En6500, were always lower than those in its parent line, Enrei. This lower Spd and Spm content accompanied a striking accumulation of putrescine (Put) in the former plant. This finding indicates that Spd and Spm biosynthesis from their precursor Put is repressed in En6500. The foliar treatments with Spd or Spm of En6500 led to a reduction of both nodule number and root growth. On the other hand, foliar treatment with MDL74038, a specific inhibitor of Spd biosynthesis, apparently increased the root nodule number in Enrei. Foliar application of brassinolide (BL) of En6500 increased the leaf Spd level and reduced the nodule number. These results suggested that BL-induced Spd synthesis in shoots might suppress the root nodule formation.

The nitrate-tolerant symbiosis of Glycine max (L.) Merr. nts382 and Bradyrhizobium japonicum is also tolerant of ammonium

Previously, we have shown that the Glycine max (L.) Merr. -Bradyrhizobium japonicum symbiosis is very sensitive to inhibition by NH4+. The current study addresses whether the supernodulating soybean mutant, nts382, which is known to be tolerant of NO3-, is also tolerant of NH4+. The nts382 mutant and its wild-type parent, Bragg, were grown in continuous-flow hydroponic culture in the presence of 0, 0.25, 0.5, or 1.0 mM 15N-enriched NH4+. Plants were harvested at 14, 21, and 28 days after inoculation. Both cultivars had the highest dry weight (DW) at each harvest date when grown on 0.25 mM NH4+. At 0.25 mM NH4+, whole plant DW increased by 5.3- and 3.2-fold in Bragg and nts382, respectively, compared with the 0.0 mM NH4+ control by the end of the experiment. As expected, whole-plant nodulation (nodules per plant), DW-specific nodulation (nodules per gram root dry weight), and nodule DW were severely inhibited in Bragg at all levels of NH4+. However, in nts382, whole-plant nodulation was not affected by NH4+ treatment, and nodule DW increased by as much as fivefold. Whereas DW-specific nodulation decreased by 94% in Bragg, this parameter decreased by only 52% in the nts382 mutant. Likewise, while the nitrogen derived from the atmosphere decreased by approximately 40% in NH4+-supplied Bragg, it increased 2.8-fold at 0.25 and 0.5 mM NH4+ in nts382. This study demonstrates that both nodulation and N2 fixation in nts382 are more tolerant of NH4+ than in the wild-type Bragg.

Growth Characteristics and Nitrogen Use Efficiency in a Supernodulating Soybean Mutant, En6500, Derived from the Variety of Enrei.

It was reported that a supernodulating soybean mutant, En6500 derived from the variety of Enrei, could produce many root nodules, but the growth rate of the plant was not necessarily high. The purpose of this paper, therefore, is to examine the characteristics of nodulation and also of plant growth in En6500 for elucidation of the mechanism of its low growth rate. The relative growth rate was low in En6500 compared with Enrei. The dry weight of the nodules per unit root mass was greater in En6500 than in Enrei, suggesting that the root of En6500 is supporting too many or an excess of nodules, which presumably leads to a reduction of plant growth rate. Reduced growth rate was associated with low net assimilation rate and further with low leaf photosynthesis. Moreover, the reduced leaf photosynthesis was associated with low partitioning of nitrogen to the leaves in En6500. Much allocation of nitrogen to the plant organs besides the leaves resulted in low photosynthesis, and also in the reduced nitrogen-use efficiency for dry matter production, which might be a potential mechanism for the low growth rate of En6500, a supernodulating soybean mutant.

Genetic and Allelism Analyses of Hypernodulation Soybean Mutants from Two Genetic Backgrounds

Several chemically mutagenized lines of soybean [Glycine max (L.) Merr.] have shown enhanced nodulation and partial tolerance to high NO−3 levels. Previous genetic analyses of hypernodulating or supernodulating soybean mutants indicated that they were genetically controlled by recessive monogenes following simple Mendelian principles. In the present study, we determined the genetic control of hypernodulating mutants selected from two soybean cultivars, Williams and Enrei, and analyzed the allelism of mutant genes controlling the hypernodulating phenotypes. Reciprocal crosses between four hypernodulating mutants (NOD1‐3, NOD2‐4, NOD4, and En6500) and two normally nodulated cultivars (Williams 82 and Harosoy 63) were made. The patterns of nodulation in F1s, F2s, and F2:3 progeny were visually evaluated at 14 d after planting in the greenhouse. Chi squares for each progeny and homogeneity tests for each cross were calculated. The results indicated that hypernodulation in the mutants studied was conditioned by single recessive genes. Three mutants (NOD1‐3, NOD4, and En6500) were controlled by a single recessive allele rj7, although they were isolated from distinctly different genetic materials. The allelism study indicated that another non‐allelic mutant gene was identified which was responsible for hypernodulation in mutant NOD2‐4. This new gene has been tentatively designated rj8. With respect to genetic interaction between the two mutant genes identified, the segregation ratio in the F2 did not fit a classical pattern and therefore we speculated that these two genes interact to modify expression in an unknown fashion. Hence, additional genetic analysis is needed to clarify the genetic interaction between rj7 and the proposed rj8 gene.

Characterization of nitrogen assimilation in a supernodulating soybean mutant En6500

Abstract A supernodulating and nitrate-tolerant soybean (Glycine max (L.) Merr.) mutant En6500 and its wild-type parent, cv. Enrei were cultured hydroponically at various nitrate (KNO3) levels (0.5, 5.0, and 15.0 mol m-3) to analyze the characteristics of nitrogen fixation, nitrate absorption, and associated characters in this mutant. The mutant produced a larger number of nodules, higher nodule dry weight, and a larger amount of fixed nitrogen per plant, thus depending more on fixed nitrogen at all nitrate levels than its parent. The mutant also exhibited a nitrate tolerance in nodule number and nodule dry matter, though the amount of fixed nitrogen per plant or on a nodule dry weight basis was reduced with increasing nitrate levels. The ability of nitrate absorption and plant growth in the mutant were inherently less active than those of the parent even in the absence of inoculation (non-nodulating conditions). Inoculation enhanced the low performance of nitrate absorption and growth in the mutant, whic...

Phosphorus requirement and sources of nitrogen in three soybean (Glycine max) genotypes, Bragg, nts 382 and Chippewa

The effect of different levels of soil phosphorus on nodulation, N2 fixation and growth of supernodulating soybean mutant, nts 382, its parent, Bragg, and soybean cultivar, Chippewa, were compared in a greenhouse study. The P rates were, 0, 30, 60 and 90 mg kg−1 soil (P0, P1, P2 and P3, respectively).

Shoot control of nodulation is modified by the root in the supernodulating soybean mutant En6500 and its wild-type parent cultivar Enrei

Abstract Based on grafting studies, both supernodulating (Carroll et al. 1985a, b) and hypernodulating (Gremaud and Harper 1989) soybean ([itGlycine max} L. Merr.) phenotypes were reported to be under the control of shoot factors (Delves et al. 1986, 1987; Cho and Harper 1991). Recently Akao and Kouchi (1992) have isolated a new supernodulating mutant (En6500) from ethyl methane sulfonate (EMS)-treated Enrei, a cultivar which is widely grown in the central districts of Japan. This mutant has been shown to produce several fold as many nodules as its wild-type parent cultivar when grown at a low concentration (0.5 mol m-3) of nitrate. Moreover, it exhibited a continuous increase in the nodule number with the increasing nitrate concentration, even at 15 mol m-3 (Francisco et al. 1992), in contrast to the similar mutant nts382 in which the nodulation decreased even at the relatively low nitrate level of 5.5 mol m-3 (Carroll et al. 1985a). In this study we conducted grafting experiments to determine which plant part controls the supernodulation of En6500.

Sealed-tube combustion for natural 15N abundance estimation of N2 fixation and application to supernodulating soybean mutants

Measurement of biological N2 fixation by the natural 15N abundance technique is based on the fact that soil N is usually enriched in 15N compared with atmospheric N2. The technique has been limited by the care required to prevent isotope fractionation in the Kjeldahl method. We describe a sealed-tube combustion technique that reduces isotope fractionation problems. Plant samples were combusted at 850 °C in Vycor tubes with Cu, CuO, CaO, and Ag foil, and N2 gas was admitted directly into the mass spectrometer. A δ15N value of 5.43 ± 0.01 (SE, n = 11) was obtained for a nonnodulating soybean seed sample. This method was used to estimate N2 fixation by soybean cv. Bragg and three supernodulating mutants, nts246, nts382, and nts1007, in the field. Average percent N derived from the atmosphere was 70 ± 4% in vegetative parts at physiological maturity and 72 ± 2% in the grain at harvest. No significant differences were found. The N harvest index averaged 77%; thus, despite the high percent N derived from the atmosphere, these soybean crops would have been net exporters of soil N. The natural 15N abundance method consistently gave estimates of N2 fixation that were 60–90 kg N ha−1 higher than the N difference method. Key words: sealed-tube combustion, natural 15N abundance, supernodulating soybean mutants, biological N2 fixation.

Short-Term Nitrate Effects on Hydroponically-Grown Soybean cv. Bragg and its Supernodulating Mutant

Fully symbiotic or nitrate treated (3 d, 4 0 mol m 3) soybean (Glycine max [L.] Merr.) cv. Bragg and a nitrate tolerant supernodulating soybean mutant nts 1007 were exposed to 13C enriched C02 for a period of 10 h. During this period and for the subsequent 24 h, continuous measurements of 13C02 and 12C02 evolution of their root systems were undertaken. Three harvests during the experiment allowed determinations of the distribution of recently fixed carbon in different plant organs. These measurements indicated higher dependence of N2 fixation in nts 1007 on recently fixed carbon (RFC) by showing elevated RFC concentrations in nodules as well as their augmented respiration. Root respiration of both genotypes was generally more reliant on stored carbon. Nitrate induced in all measured parameters a clear response in the mutant analogous to the wild type, but quantitative differences remained throughout. Nodule respiratory activity, the relative specific activity (RSA), and the utilization of RFC were substantially reduced, but remained higher in nts 1007 than in Bragg, while the demand of roots for RFC increased in Bragg more than in the supernodulator. The elevated carbon requirement of the nodule complement of the mutant and a high dependence on recently fixed carbon could be attributed to higher nodule growth and maintenance costs of the supernodulating genotype and were not associated with augmented nitrogen fixation activity. This less efficient utilization of carbon and the associated almost parasitic character of the nodule complement of nts 1007 is considered to be the cause of reduced growth of the mutant. No evidence was found for a physiologically based nitrate tolerance in terms of nitrogen fixation.

Efficiency of Nodule Initiation and Autoregulatory Responses in a Supernodulating Soybean Mutant

We compared the formation of nodules on the primary roots of a soybean cultivar (Glycine max (L.) Merr. cv. Bragg) and a supernodulating mutant derivative, nts382. Inoculation with Bradyrhizobium japonicum USDA 110 at different times after seed imbibition showed that the roots acquired full susceptibility to infection only between 3 and 4 days postgermination. When the plants were inoculated with serial dilutions of a bacterial suspension, the number of nodules formed in the initially susceptible region of the roots was linearly dependent on the logarithm of the inoculum dose until an optimum dose was reached. At least 10-fold-lower doses were required to induce half-maximal nodulation responses on nts382 than on the wild type. However, at optimal doses, about six times as many nodules formed in the initially susceptible region of the roots in nts382. Since there was no appreciable difference in the apparent rates of nodule emergence, the increased efficiency of nodule initiation in the supernodulating mutant could have resulted from a lower threshold of response to bacterial symbiotic signals. Two inoculations (24 h apart) of G. max cv. Bragg revealed that there was a host-mediated regulatory response that suppressed nodulation in younger portions of the primary roots, as reported previously for other soybean cultivar-Bradyrhizobium combinations. Similar experiments with nts382 revealed a comparable suppressive response, but this response was not as pronounced as it was in the wild type. This and other results suggest that there are additional control mechanisms for nodulation that are different from the systemic autoregulatory control of nodulation altered in supernodulating mutants.

Interactions between Irradiance Levels, Nodulation and Nitrogenase Activity of Soybean cv. Bragg and a Supernodulating Mutant

Summary A comparison between the supernodulating soybean mutant (nts382) and its parent cultivar (Bragg) under different levels of irradiance revealed a substantially decreased shoot and root (minus nodule) biomass production as a consequence of higher carbon demand of the greater nodule complement carried by nts382. At high irradiance this coincided with an increased specific nodule activity. At low light conditions, nodule activity decreased whereas carbon demand remained high due to the presence of a large number of small nodules with low contents of haem and soluble sugars. Diminished sugar levels in nodules indicated a restricted carbon flow from the shoot. In contrast, haem and sugar content of the wild type remained essentially unchanged in response to the treatments. Measurements of nitrogenase activity under elevated pO2 concurred with the concept of carbon limitation in the mutant under low light conditions in providing no evidence for O2 limitation of N2 fixation activity. The results emphasize the lack of nodulation control by the mutant, but not its parent cultivar, even under severely adverse conditions.

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.

Nitrogenase activity and ureide levels in a supernodulating soybean mutant: Effects of inoculum dose and nitrate treatment

The effects of nitrate on nitrogenase (EC 1.18.2.1) activity of soybean (Glycine max [L.] Merr) cv. Bragg and its supernodulating mutant derivative, nts382, were compared. A short‐term nitrate treatment was used to allow effects on nitrogenase activity to be studied in the absence of effects on nodule growth and a low inoculum dose, which prevented supernodulation of nts382, was employed to test for any interaction between supernodulation and the magnitude of the effect of nitrate on nitrogenase activity. At the usual inoculum dose, nitrogenase activity, per g nocule, of nts382 was lower than that of Bragg and was proportionally less affected by nitrate. Decreasing the inoculum dose increased nitrogenase activity of nts382 and also the proportional decline in response to nitrate. The decline in the ureide conentration in xylem exudate in response to nitrate was proportionally similar to the decline in nitrogenase activity per plant. However, although nitrogenase activity per plant of nts382 was several‐fold less than that of Bragg, the ureide flux rate (ureide concentration x xylem sap exudation rate), was not different. At the usual inodulum dose, the ureide content of the nocules, stems plus petioles and leaves of nts382 was greater than that of Bragg. Decreasing the inoculum dose reduced the ureide content of the nodules of nts382 but not of Bragg. Ureide degradative capacity of the leaves was the same for Bragg and nts382. Low activities of 5‐phosphoribosyl pyrophosphate amidotransferase (EC 2.4.2.14) and glutamine synthetase (EC 6.3.1.2) in the nodules reflected the low nitrogenase activity of nts382.

Nodule Physiology of a Supernodulating Soybean (Glyine max) Mutant

The nodule physiology of a supernodulating, nitrate tolerant symbiosis soybean (Glycine max (L.) Merr.) mutant (nts382) was compared to that of its wild-type parent, cv. Bragg. Nodule number and mass were greater in nts382 than cv. Bragg and individual nodule mass, bacteroid and haem content, and acetylene reduction activity per g nodule were less. Acetylene reduction activity expressed per mg bacteroid protein was the same in the two genotypes. In median sections, the ratio of infected to total nodule area was smaller in nts382, infected cell size was smaller and there were fewer bacteroids per peribacteroid envelope. When inoculum dose was decreased from 109 to 103 viable cells per pot, nodule number on nts382 decreased approximately to that on cv. Bragg; nodule size, bacteroid and haem contents increased as did nodule acetylene reduction activity. Application of moderate levels of nitrate, which did not significantly affect symbiotic parameters of cv. Bragg or high inoculum nts382, stimulated nodule growth and nitrogenase activity of low inoculum nts382. A combination of nitrate and low inoculum levels enhanced nodule parameters of nts382 to the level usually seen with cv. Bragg. When supernodulated, plant dry weight of nts382 was less than that of Bragg; decreasing inoculum dose had no significant effect but nitrate application increased plant growth; nitrate plus low inoculum induced similar plant growth to that of cv. Bragg. Nodule carbohydrate content was similar in both genotypes but nodule and xylem sap ureide contents were higher in nts382. In general, nodules of supernodulated nts382 resembled under-developed cv. Bragg nodules; when supernodulation was avoided by using low inoculum doses, ,nts382 nodules resembled those of cv. Bragg. Nitrogen metabolism in the mutant seems to be disturbed, resulting in ureide accumulation.