Nitrogen-Fixing Symbioses

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1 Research for Tomorrow Pathway to Stable Products of Photosynthetic Energy Conversion. CHOH CHOH CH2OPO3" CH2OPO3 2 CHOH COOH CH2OPO3 COO Photosynthetic COo Fixation CH2OPO3 *^ Respiration j With Loss CHOH ofc02 COOH of CO2 as shown in the lower pathway, and the result is a lowering of CO2 fixation and a decrease in productivity. This undesirable alternative often occurs in crop plants. Presently we are learning the precise structure and the chemical details of functioning of this enzyme. There is a good possibility that the powerful tools of biotechnology molecular biology and genetic engineering will allow us to regulate the choice between these two alternative reactions and so improve crop productivity at its most fundamental level. Nitrogen Fixation in Nonleguminous Plants Iris F. Martin, associate program manager, Competitive Research Grants Office, Office of Grants and Programs Tremendous advances have been made in the last 10 years toward understanding nitrogen fixation at the molecular level. The transfer to plants of nitrogen-fixation genes is an intriguing consideration. Let us first look at the process of biological nitrogen fixation and then at the more recent developments in molecular genetics that are providing information that may make such transfer of genes possible in future years. Process of Biological Nitrogen Fixation Nitrogen gas (N2) makes up 79 percent of the earth's atmosphere. But before plants can use this molecular nitrogen for the synthesis of amino acids and proteins, it must be converted to combined or "fixed" nitrogen compounds. Plants do not have this abifity. Making atmospheric nitrogen available to the food chain is restricted to certain prokaryotes (cellular organisms without a distinct nucleus), such as bacteria and cyanobacteria (blue-green algae), which contain an enzyme called nitrogenase. Nitrogenase is composed of two proteins iron and molybdenumiron and catalyzes the reduction of gaseous nitrogen (N2) to ammonia (NH3). This process requires large 112 BIOTECHNOLOGY: ITS APPLICATION TO PLANTS

2 Research for Tomorroiv amounts of energy (ATP) and reducing equivalents (electrons). The sun is the ultimate source of the energy for nitrogen fixation with the ATP being derived from carbon compounds such as sugars manufactured by the plants through photosynthesis. The reducing equivalents pass from an electron donor, a protein such as ferredoxin or flavodoxin, to the iron protein and then on to the molybdenum-iron protein where the conversion of N2 to NH3 occurs. Since both proteins of the enzyme are inactivated by oxygen, some bacteria fix nitrogen only when they are growing in the absence of oxygen. Others have evolved mechanisms and anatomical structures which protect the enzyme from oxygen. Nitrogen-Fixing Symbioses Some bacteria fix nitrogen in the free-living state and others only when living in a symbiotic relationship with a plant. Dissimilar organisms which live together in a mutually beneficial relationship are said to be in symbiosis. The smaller member is the symbiont. Legumes such as soybeans, peas, and alfalfa are well-known plants which enter into nitrogen-fixing symbioses when their roots are infected by specific bacteria called Rhizobia. The plant forms nodules in which the bacterium reduces N2 to ammonia. More complex compounds of nitrogen are synthesized from the ammonia and transported to other parts of the plant. The plant provides the bacterium with carbon compounds that are metabolized to obtain the energy for the reduction of the nitrogen. Some plants that are not legumes also enter into nitrogen-fixing relationships and contribute significant quantities of fixed nitrogen to their environments. One example is a small water-fern, Azolla, which harbors a When Anabaena filaments grow in ttie absence of fixed nitrogen, some cells may differentiate into tieterocysts (indicated by arrows). cyanobacterium Anabaena azollae, as a symbiont. It is used as a source of nitrogen in the cultivation of rice. Another group includes certain trees and Nitrogen Fixation in Nonteguminous Plants 113

3 Research for Tomorrow As an Azolla leaf develops at the apex of ttie stem, a cavity forms and becomes inoculated with Anabaena filaments. Klebsiella Pneumoniae Nitrogen Fixation Genes "o Electron Uptake Activatioti Repressoft Transpon B A LFM 01 I I 11 I (ZU [ZZl V s U X N I E Y K 1 I II H C Electrwi Transpon Low Potential ^ > Reductant (e-) ^J Molybdenum Iron Protein ^9^^'' MgADP 2H+ H, shrubs, whose roots become infected with the bacterium Frankia, for example, the alder tree. Study of the partners of these symbioses and their interactions are Ukely to provide new insights and unique opportunities to obtain genetic information that wul have parallels in the legumes. Azoffa-Anabaena Symbiosis. Nitrogen is frequently a limiting element in rice paddies, and the cost of nitrogen fertilizer is often prohibitive especially in the poorer countries. The water-fern, Azolla, can serve as a source of biologically fixed nitrogen for lice. The Anabaena symbiont can meet the complete nitrogen requirement of the fern, and decomposition of the fern supplies nitrogen to the rice plant. Azolla can be used as a green manure or grown as a companion crop with rice plants increasing rice yields by as much as 100 percent over unfertilized controls. Cyanobacteria such as Anabaena are not only among the few organisms which can fix nitrogen while growing under aerobic conditions (that is, with oxygen present) but are the only oxygen-evolving organisms with this capability. Free-living Anabaena grows in the presence of combined nitrogen as a filament of vege- 114 BIOTECHNOLOGY: ITS APPLICATION TO PLANTS

4 Research for Tomorroiir tative cells. In the absence of a combined nitrogen source, some cells along the filament differentiate into specialized cells called heterocysts. They develop an outer envelope, lose their oxygen-evolving capacity, and provide an environment in which nitrogenase can function. When certain strains oí Anabaena live in symbiosis with Azolla, they become less dependent on their own photosynthetic capacity as an energy supply. More vegetative cells differentiate into heterocysts, more nitrogen can therefore be fixed, and the nitrogen requirements of the host can be met. In Azolla the Anabaena reside in a cavity of the leaf This symbiosis exhibits synchronous development. As a leaf develops at the apex of the stem, the forming leaf cavity becomes inoculated with Anabaena. As leaves age, an increasing percentage, up to 40 percent, of the Anabaena cells become differentiated into heterocysts, and parallel adaptations occur in the fern tissues. Actinorhizal Symbioses» Strains of the bacterium, Frankia, infect a large array of nonleguminous trees and shrubs. These plants (actinorhizal) fix significant nitrogen in forests, especially in poor soils, and a number of them can grow under dry or acid conditions. These attributes make them excellent primary colonizers of post-glacial and post-mining soils. Although nodules have been observed on actinorhizal plants for over 150 yeais, Frankia was not isolated in pure culture until Many different strains of the bacterium have since been isolated. Some have recently been grown on a scale adequate for inoculant production. Frankia behave very much like the bacteria that infect legumes but considerably less is known about the actinorhizal symbioses. The bacterium multiplies rapidly in the area of the Nitrogen Fixation in Nonleguminous Plants root and there is evidence of recognition between the Frankia and the plant. The bacterium invades the root, and the plant responds to this invasion by the formation of a nodule. Unlike a soybean nodule, in which the concentration of oxygen is quite low, actinorhizal nodules contain oxygen at approximately atmospheric levels. Within the nodule of most host plants, Frankia develop vesicles, or pouches, which apparently protect nitrogenase from oxygen and in which nitrogen fixation occurs. A compound resembling hemoglobin, capable of readily binding and releasing oxygen, has recently been identified in nodules of some actinorhizal plants. It may prove to have a function comparable to that of leghemoglobin in legumes maintenance of oxygen flux at low concentration. Molecular Genetics Actinorhizal symbioses, cyanobacterial symbioses, and fi:ee-li\1ng cyanobacteria can be of agronomic benefit through their capacity to use the sun's energy for photosynthesis and through the conversion of nitrogen from the air to more usable forms. Recently developed probes of molecular genetics are providing significant new insights about nitrogen-fixing organisms, but they are dependent upon physiological and biochemical advances for an understanding of which genes should be modified and the function of the gene products. The following nitrogen-fixing bacteria have been studied at some length. KlBbsiella Pneumoniae, The nitrogen fixation (nif) genes have been most fully characterized in the free-hving bacterium, Klebsiella pneumoniae. The genes were first cloned, mapped, and subjected to nucleotide sequencing in the late 1970's. At least 17 adjacent genes code for the struc 1Í5

5 Research for Tomorr&w ture and regulation of the enzymes required for nitrogen fixation. The structural genes, nif H and D, and K that code for the protein precursors of the two nitrogenase components, the iron protein and the molybdenumiron protein respectively, have been most thoroughly studied. Their nucleotide sequences have been determined and the amino acid sequences of the proteins deduced from these. Nif A controls the activation of the other nif genes, and nif L codes for a protein that acts to repress nitrogen fixation. The expression of nif A and L is in turn regulated by a central nitrogen assimilatory system. lîhîzowa. Significant information has been gained in the last few years on the nitrogen fixation genes in Rhizobia, which primarily infect legumes. Genes required for nodulation have been located in some strains and evidence for signaling between the plant and the bacterium is just emerging. ilffabaena. Gene structure and regulation have been studied extensively in one strain of free-living Anabaena. Only four nif genes have so far been identified, among them the three structural genes for nitrogenase. In the vegetative cells where one does not observe any nitrogen fixation, one of these genes is separated from the other two. A significant recent development is the discovery of gene rearrangement that occurs when the vegetative cell differentiates to a heterocyst. A section of DNA that separated the genes in the vegetative ceu is excised such that, under nitrogen-fixing conditions, the structural genes are present and are transcribed as a single unit. When growing in symbiosis with Azolla, Anabaena exports a large portion of the ammonia produced to the. host. Biochemical and genetic studies have revealed significant modifica- tions in the nitrogen assimilatory system of the Anabaena. A thorough understanding of the regulatory system could allow genetic manipulation of Anabaena in the free-living state for large-scale ammonia production. FrmnkiBm Among the least studied of the symbiotic nitrogen-fixing bacteria are Frankia. Initially, their slow growth rate in culture made molecular genetic studies difficult. However, culture methods have been improved, and genetic studies are now possible. Work is actively being pursued on the molecular biology of both the nitrogen-fixation genes and the interaction of the symbionts. Frankia are unusual in that they are capable of infecting a wider range of host plants than other known nitrogen-fixing bacteria. Biotechnology may be able to further extend that range. FututB of Transfer of Nitrogen Fixation Genes It has been suggested that nitrogenfixation genes might be transferred to plants that do not now have this nitrogen-fixation capacity. These genes would have to be in a form that could be incorporated into the plant genome, replicated, and expressed. The genes would have to be expressed in an environment amenable to nitrogen fixation where the enzyme, nitrogenase, could be protected from oxygen and where the enzyme system could tap into the sources of reductant and energy from the plant. The fuu complement of nitrogen fixation genes has been cloned from Klebsiella pneumoniae and transferred to and expressed in another bacterium, Escherichia coli. These genes also have been transferred to yeast, but they could not be expressed. Transfer to a plant is a much more complicated process involving many more genes and still remains highly speculative. Í1B BIOTECHNOLOGY: ITS APPLICATION TO PLANTS