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 plant: 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 ability. 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 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.

When Anabaena filaments grow in the absence of fixed nitrogen, some cells may differentiate into heterocysts (indicated by arrows).

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.

As an Azolla leaf develops at the apex of the stem, a cavity forms and becomes inoculated with Anabaena filaments.