Bacteria-plant interactions in symbiotic nitrogen fixation

In the inter- and intracellular N₂-fixing symbioses between plants and micro-symbionts, the development of an endophytic form of the micro-symbiont is essential. This development includes a series of steps consisting of plant-bacteria interactions. Considerable progress in the elucidation of these steps has been made by applications of the methods of molecular genetics. Several genes with a role during infection and nodulation have been indicated in Rhizobium and Bradyrhizobium like the common nod genes A, B, C, I and J, and the host-specific genes nod E, F and H. The nod D gene is the only constitutive gene, and its product is essential for activity of all other nod genes, provided some flavonoids from the root exudate are present as well. Mutants in these genes show phenotypic effects, in which the products of the genes must be involved. Far more difficult is the biochemical and physiological study of these products and their direct effects. The difficulties involved in such biochemical-physiological studies is illustrated by a short discussion of the controversies around the possible role of plant lectins. While in Rhizobium the nod genes are present on a large sym-plasmid, other essential genes must be present on the bacterial chromosome and on other plasmids. Induction of plant genes is evident from the formation of nodule-specific proteins, the nodulins. Though many different plant and bacterial genes are involved in the series of steps in the development of an effective root nodule, there are indications that regulation is affected by a smaller number of essential regulatory genes. This is illustrated by the effect of the regulatory nod D gene during infection and nodulation, and of ntrA and nifA genes for the formation and activation of the nitrogen-fixing systems. Moreover, every step, once initiated, may lead to cascade effects on subsequent reactions. Finally, some further consequences of the endophytic way of life are discussed, which affect either the metabolic and transport activities of the endophytes or their viability. This is illustrated by the possible role of membrane integrity as evident during the isolation of Frankia from its endophytic form.     

Genomics insights into symbiotic nitrogen fixation

Following an interaction with rhizobial soil bacteria, legume plants are able to form a novel organ, termed the root nodule. This organ houses the rhizobial microsymbionts, which perform the biological nitrogen fixation process resulting in the incorporation of ammonia into plant organic molecules. Recent advances in genomics have opened exciting new perspectives in this field by providing the complete gene inventory of two rhizobial microsymbionts. The complete genome sequences of Mesorhizobium loti, the symbiont of several Lotus species, and Sinorhizobium meliloti, the symbiont of alfalfa, were determined and annotated in detail. For legume macrosymbionts, expressed sequence tag projects and expression analyses using DNA arrays in conjunction with proteomics approaches have identified numerous genes involved in root nodule formation and nitrogen fixation. The isolation of legume genes by tagging or positional cloning recently allowed the identification of genes that control the very early steps of root nodule organogenesis.     

Methods for enhancing symbiotic nitrogen fixation

Biological nitrogen fixation of leguminous crops is becoming increasingly important in attempts to develop sustainable agricultural production. However, these crops are quite variable in their effectiveness in fixing nitrogen. By the use of the ¹⁵N isotope dilution method some species have been found to fix large proportions of their nitrogen, while others like common bean have been considered rather inefficient. Methods for increasing N₂ fixation are therefore of great importance in any legume work. Attempts to enhance nitrogen fixation of grain legumes has been mainly the domain of microbiologists who have selected rhizobial strains with superior effectiveness or competitive ability. Few projects have focused on the plant symbiont with the objective of improving N₂ fixation as done in the FAO/IAEA Co-ordinated Research Programme which is being reported in this volume. The objective of the present paper is to discuss some possibilities available for scientists interested in enhancing symbiotic nitrogen fixation in grain legumes. Examples will be presented on work performed using agronomic methods, as well as work on the plant and microbial symbionts. There are several methods available to scientists working on enhancement of N₂ fixation. No one approach is better than the others; rather work on the legume/Rhizobium symbiosis combining experience from various disciplines in inter-disciplinary research programmes should be pursued.     

Control of symbiotic nitrogen fixation in Rhizobia regulation of NH4 assimilation

The metabolic fate of gaseous nitrogen (¹⁵N₂) fixed by free-living cultures of Rhizobia (root nodule bacteria) induced for their N₂-fixation system was followed. A majority of the fixed ¹⁵N₂ was found to be exported into the cell supernatant. For example, as much as 94% of the ¹⁵N₂ fixed by Rhizobium japonicum (soybean symbiont) was recovered as ¹⁵NH₄⁺ from the cell supernatant following alkaline diffusion. Several species of root nodule bacteria also exported large quantities of NH₄⁺ from l-histidine. Evidence is presented that overproduction and export of NH₄⁺ by free-living Rhizobia may be closely linked to the control of several key enzymes of NH₄⁺ assimilation. For instance, NH₄⁺ was found to repress glutamine synthetase whereas l-glutamate repressed glutamate synthase. Assimilation of NH₄⁺ as nitrogen source for growth of Rhizobia was inhibited by glutamate. The mechanism of regulation of NH₄⁺ production by root nodule bacteria is discussed.     

Nitrate reduction and nitrogen fixation in symbiotic association Rhizobium-legumes

The inhibitory effect of nitrate on nitrogenase activity in root nodules of legume plants has been known for a long time. The major factor inducing changes in nitrogenase activity is the concentration of free oxygen inside nodules. Oxygen availability in the infected zone of nodule is limited, among others, by the gas diffusion resistance in nodule cortex. The presence of nitrate may cause changes in the resistance to O2 diffusion. The aim of this paper is to review literature data concerning the effect of nitrate on the symbiotic association between rhizobia and legume plants, with special emphasis on nitrogenase activity. Recent advances indicate that symbiotic associations of Rhizobium strains characterized by a high nitrate reductase activity are less susceptible to inhibition by nitrate. A thesis may be put forward that dissimilatory nitrate reduction, catalyzed by bacteroid nitrate reductase, significantly facilitates the symbiotic function of bacteroids.     

Localized and internal effect of nitrate on symbiotic dinitrogen fixation

Alfalfa (Medicago sativa L.) is a deeply rooted perennial legume which, under field conditions, may be exposed to varying NO₃^? concentrations with depth. Our objective was to characterize the effect of localized (deep vs shallow) exposure of alfalfa root systems to NO₃^? on symbiotic N₂ fixation and NO₃^?-N uptake. Cuttings of a single alfalfa plant were grown in vertical split root systems in a controlled environment chamber. The split root system was a rigid acrylic tube (5 cm diam. by 60 cm long) filled with silica sand and divided into upper and lower sections at the 30-cm depth by a 5-mm-thick wax layer. Roots penetrated the wax layer, but mixing of nutrient solutions between the sections was prevented. Nodulation was restricted to the upper section. The plants were subjected for 10 days to the following treatments: both sections of the split root system received nutrient solution containing either 0.5, 5.25, or 10 mM NO₃^?; the upper section received 0.5 mM NO₃^? while the lower section received 10 mM NO₃^?; or the upper section received 10 mM NO₃^? while the lower section received 0.5 mM NO₃^?. Increasing supply of NO₃^? in the nutrient solution to both sections resulted in higher NO₃^?-N uptake, lower nodule mass and lower specific nitrogenase activity. Although NO₃^?-N uptake did not differ, plants exposed to 10 mM NO₃^? for 10 days in the upper, nodulated section of the root system had a 20% lower nodule mass than plants exposed to the same NO₃^? concentration in the lower, non-nodulated section of the root system. Specific nitrogenase activity was not different between these two treatments. Therefore, we conclude that: (1) nodule mass was dependent on two factors, the amount of NO₃^?-N taken up and the concentration of NO₃^? within the nodulated root zone; and (2) specific nitrogenase activity was little affected by the concentration of NO₃^? surrounding the nodules, but was strongly inhibited by NO₃^?-N taken up.     

Molecular genetics of Rhizobium Meliloti symbiotic nitrogen fixation

The application of recombinant DNA techniques to the study of symbiotic nitrogen fixation has yielded a growing list of Rhizobium meliloti genes involved in the processes of nodulation, infection thread formation and nitrogenase activity in nodules on the roots of the host plant, Medicago sativa (alfalfa). Interaction with the plant is initiated by genes encoding sensing and motility systems by which the bacteria recognizes and approaches the root. Signal molecules, such as flavonoids, mediate a complex interplay of bacterial and plant nodulation genes leading to entry of the bacteria through a root hair. As the nodule develops, the bacteria proceed inward towards the cortex within infection threads, the formation of which depends on bacterial genes involved in polysaccharide synthesis. Within the cortex, the bacteria enter host cells and differentiate into forms known as bacteroids. Genes which encode and regulate nitrogenase enzyme are expressed in the mature nodule, together with other genes required for import and metabolism of carbon and energy sources offered by the plant.     

Ontogenetic interactions between photosynthesis and symbiotic nitrogen fixation in pigeonpea

Total nodule nitrogenase activity (TNA, μmols ethylene plant^-1 h^-1) in pigeonpea (Cajanus cajari) increased with plant growth to reach maximum at flowering (75 days after sowing), decreasing thereafter until maturity (120 days after sowing). However, specific nodule nitrogenase activity (SNA, μmols ethylene g^-1 nodule fresh wt h^-1) reached its maximum earlier (45 days after sowing). The rate of photosynthesis and shoot and nodule respiration followed a similar pattern to TNA. However, higest rates of root respiration were observed at flowering and again immediately before final harvest. ¹⁴CO₂ feeding studies showed that assimilates produced in leaves before flowering were retained in the vegetative parts. Assimilates produced after flowering were exported to the reproductive structure at the expense of the nodules. It is suggested that the decreased availability of photosynthate to nodules decreased nitrogen fixation.     

Rhizobium japonicum mutants defective in symbiotic nitrogen fixation.

Rhizobium japonicum strains 3I1b110 and 61A76 were mutagenized to obtain 25 independently derived mutants that produced soybean nodules defective in nitrogen fixation, as assayed by acetylene reduction. The proteins of both the bacterial and the plant portions of the nodules were analyzed by two-dimensional polyacrylamide gel electrophoresis. All of the mutants had lower-than-normal levels of the nitrogenase components, and all but four contained a prominent bacteroid protein not observed in wild-type bacteroids. Experiments with bacteria grown ex planta suggested that this protein was derepressed by the absence of ammonia. Nitrogenase component II of one mutant was altered in isoelectric point. The soluble plant fraction of the nodules of seven mutants had very low levels of heme, yet the nodules of five of these seven mutants contained the polypeptide of leghemoglobin. Thus, the synthesis of the globin may not be coupled to the content of available heme in soybean nodules. The nodules of the other two of these seven mutants lacked not only leghemoglobin but most of the other normal plant and bacteroid proteins. Ultrastructural examination of nodules formed by these two mutants indicated normal ramification of infection threads but suggested a problem in subsequent survival of the bacteria and their release from the infection threads.