Thomas A. LaRue

How Much Nitrogen do Legumes Fix

Publisher Summary This chapter presents the estimates of nitrogen fixation by legume crops and discusses the methods of estimating fixation by crops. The pulses are sources of good-quality protein. The forages have a long-documented history of supporting livestock on poor soil. Both these factors are attributed in part to the ability of legumes, in symbiosis with rhizobia, to obtain nitrogen from the air. Legumes require more phosphate fertilizer than cereals, and many are more demanding of water. There is not a single legume crop for which valid estimates of the nitrogen fixed in agriculture is available. There are good estimates for soybean grown in representative locations in experimental plots. Dry beans are the important legume crop for human consumption in Latin America. There is no good evidence that any legume crop satisfies all its nitrogen requirements by fixation. The highest estimates are typical of low-fertility soil or soils artificially made N-poor by admixture or carbon amendment. The simplest estimate of nitrogen fixation is by total nitrogen accumulation of the crop. This is based on the intuitive assumption that the crop derives all its nitrogen via symbiotic fixation.

Induced symbiosis mutants of pea (Pisum sativum) and sweetclover (Melilotus alba annua)

Abstract Treatment of Pisum sativum (L.) cv. ‘Sparkle’ with ethylmethane sulfonic acid or γ or neutron radiation induced stable mutants which do not form nodules, or form few nodules. Six mutants were at the previously described sym 5 locus. Non-nodulating mutants of Melilotus alba annua (Desr.) cv. U389 were obtained by treatment with ethylmethane sulfonic acid (EMS) or neutron radiation, but not by γ radiation or azide.

Pisum sativum mutants insensitive to nodulation are also insensitive to invasion in vitro by the mycorrhizal fungus, Gigaspora margarita

Abstract Transformed root cultures were established using Agrobacterium rhizigenes inoculation of pea mutants, altered in their interaction with Rhizobium . They were tested in an in vitro model for sensitivity to the vesicular-arbuscular mycorrhizal (VAM) fungus, gigaspora margarita . VAM development was assessed using light and electron microscopy. Two non-nodulating, non-nitrogen fixing (Nod − , Fix − ) pea mutants were resistant to VAM colonization in vitro: Mycelium developed on the root surface but failed to colonize the interior. A nodulating (Nod + ) genotype, which was unable to fix nitrogen (Fix − ) in association with Rhizobium and a parental line, Lincoln (Nod + , Fix + ), interacted normally with the fungus, showing extensive internal colonization. These results confirm, under axenic conditions, previous reports showing that defective nodulation is correlated with defective mycorrhization. We propose using this in vitro model to identify factors necessary to initiating and maintaining the VAM/plant symbiosis.

Genetic analysis of sym genes and other nodule-related genes in Pisum sativum

The formation of nodules on the roots of legumes is a complex process, involving genes on both the host’s and the bacterial genome. We use the garden pea (Pisum sativum L.) as our model system, concentrating on the genes of the host plant that affect nodulation. Two classes of genes are being analyzed: mutants that produce phenotypic changes in nodule number or physiology (referred to as sym mutants) and genes encoding nodulins or other proteins with important roles in nodule biochemistry. Our approach has been to carefully characterize the sym mutants, thereby defining as accurately as possible their role in nodule formation and to map each of the sym and nodulin genes on the pea linkage map. Functionally related genes do not generally form clusters in the genomes of higher eukaryotes, and we expected the genes involved in nodule formation to be widely distributed on the pea linkage map. Correspondance between the map positions of sym and nodulin genes might indicate that the two are genetically related. If the two are coded by the same locus the precise metabolic defect affecting nodule formation in the mutant and, conversely, the mechanism through which a nodulin influences nodulation would be revealed.

Carbon Metabolism in the Legume Nodule

The nodule uses photosynthate for its growth and maintenance, to provide energy and reductant for nitrogenase, and for the incorporation and transport of newly fixed nitrogen to the shoot. There is ample evidence that symbiotic fixation is demanding of energy, and imposes a respiratory burden on the legume plant. An increase in nitrogen fixation by crops might be achieved by increasing the supply of photosynthate to nodules, or by a more efficient use of carbon compounds within the nodule. This will require more knowledge than we now have on how carbon compounds are used by the symbionts.

Investigation of Four Classes of Non-nodulating White Sweetclover (Melilotus alba annua Desr.) Mutants and Their Responses to Arbuscular-Mycorrhizal Fungi.

Abstract The nitrogen-fixing symbiosis between Rhizobiaceae and legumes is one of the best-studied interactions established between prokaryotes and eukaryotes. The plant develops root nodules in which the bacteria are housed, and atmospheric nitrogen is fixed into ammonia by the rhizobia and made available to the plant in exchange for carbon compounds. It has been hypothesized that this symbiosis evolved from the more ancient arbuscular mycorrhizal (AM) symbiosis, in which the fungus associates with roots and aids the plant in the absorption of mineral nutrients, particularly phosphate. Support comes from several fronts: 1) legume mutants where Nod− and Myc− co-segregate, and 2) the fact that various early nodulin (ENOD) genes are expressed in legume AM. Both strongly argue for the idea that the signal transduction pathways between the two symbioses are conserved. We have analyzed the responses of four classes of non-nodulating Melilotus alba (white sweetclover) mutants to Glomus intraradices (the mycorrhizal symbiont) to investigate how Nod− mutations affect the establishment of this symbiosis. We also re-examined the root hair responses of the non-nodulating mutants to Sinorhizobium meliloti (the nitrogen-fixing symbiont). Of the four classes, several sweetclover sym mutants are both Nod− and Myc−. In an attempt to decipher the relationship between nodulation and mycorrhiza formation, we also performed co-inoculation experiments with mutant rhizobia and Glomus intraradices on Medicago sativa, a close relative of M. alba. Even though sulfated Nod factor was supplied by some of the bacterial mutants, the fungus did not complement symbiotically defective rhizobia for nodulation.

Simple estimate of ureides in soybean tissue

Abstract Extracts of soybean tissue are treated with an acidic cation-exchange resin to remove amino acids. The sample is treated with hypochlorite, pH 4.0, to convert the amide from allantoic acid or allantoin to products that react with alkaline phenol to form indophenol. The procedure is inexpensive, sensitive, and rapid. The method may be adapted for automated analysis with the Technicon autoanalyzer.

An altered constitutive peptide in sym 5 mutants of Pisum sativum L.

Mutational analysis of Pisum sativum L. was used to search for constitutive proteins that might function in nodule formation. The sym 5 locus is a mutational hot spot, represented by seven independently derived mutant lines with decreased nodulation. Comparison of two-dimensional polyacrylamide gels of in vitro-translated root RNA showed a consistent difference in the migrational pattern of one peptide. In the nodulating parental cultivar ‘Sparkle’, a 66 kDa peptide had a pI of 5.9. In four of the five tested sym 5 mutants, the 66 kDa peptide had a more acidic pI of 5.8. This 66 kDa peptide is found in lateral root, tap root, and shoot. Its expression was independent of rhizobial inoculation, root temperature, or light.

Ethylene and Nodulation

Ethylene is probably a regulator in many aspects of plant growth and development, response to stress, and senescence. Several factors such as species, tissue type, and stage of development affect the plant response to ethylene, but the concentrations of exogenous ethylene that produce visible symptoms are usually reported in the range of 0.1 to 1.0 μL/L.

EMS Derived Mutant of Pisum Sativum Resistant to Nodulation

A non-nodulated pea plant was obtained by screening M2 progeny from plants arising from EMS-treated seeds. The nodulation resistant character is stable, and has been maintained to the M8 generation. The parent cultivar ‘Sparkle’ and field pea cultivar ‘Trapper’ are well nodulated while the mutant is resistant to nodulation by 27 strains of Rhizobium leguminosarum tested, including 4 which nodulate the nod-resistant pea variety ‘Afghanistan’ (1,2). Occasionally 1–5 effective nodules are formed. Rhizobia isolated from these nodules are not infective when retested on the mutant. Segregation for nodulation in the F1 and F2 progeny of reciprocal crosses between the mutant and ‘Sparkle’ or ‘Trapper’ show that nodulation resistasnce is conditioned by a single pair of recessive alleles. This was confirmed by analysis of test crosses between F1 plants and the mutant line. Test crosses and F2 progeny of crosses between the mutant and ‘Afghanistan’ were scored for nodulation by R. leguminosarum strain TOM, which infects ‘Afghanistan,’ and by R. leguminosarum 128C53 which does not infect ‘Afghanistan.’ Segregation for nodulation indicates that there are at least two different loci controlling nodulation resistance in ‘Afghanistan’ (sym-2,sym-2) and the EMS derived mutant (sym-5,sym-5).