Nitrogen Fixation by Non-leguminousPlants CharleneVan Raalte ALL STUDENTS OF BIOLOGY learn about legumes, ation in the non-legumes, I could find little information. Downloaded from http://online.ucpress.edu/abt/article-pdf/44/4/229/339852/4447478.pdf by guest on 03 October 2021 including such agriculturally essential plants as peas, My interest in these plants subsequently led me to several beans, and alfalfa that form a symbiosis with nitrogen- teams of biologists actively researching many aspects of fixing root nodule bacteria. But most biology students the biology and ecology of the non-leguminous nitrogen never learn about another abundant, widespread, and fixers. These scientists have recently made some impor- perhaps equally important group of plants that have tant discoveries of theoretical as well as immediate prac- nitrogen-fixingroot nodules quite different from those of tical interest. Some of these findings will be described the legumes. This group of non-leguminous nitrogen- here. fixing plants includes alder trees and shrubs (Alnus sp.), bayberry and sweet gale (Myrica sp.), and sweet-fern (Comptonia peregrina). These plants are rarely men- TABLE1. The BiologicalCharacteristics of NitrogenFixation tioned in basic biology or ecology texts, despite the fact The initialreaction N2 + 6H+-2NFL that their nitrogen-enrichingability makes them important FinalProducts components of their ecosystems and potentially very Aminoacids and proteins useful to farmers and foresters. OrganismsResponsible Onlybacteria (many types) The so-called nitrogen-fixing plants are of special in- EnzymeInvolved Nitrogenase(common to all N terest to me because I study vegetation tolerant of fixers) nutrient-poor soils. These plants have an advantage in Energetics Energyrequired to breakthe tripleN2 bond such soils since they associate with bacteriathat can con- vert or "fix" nitrogenous gas to ammonium (table 1). Importance Untilthe industrialfixation of N in the 1940s, mostof the N in the The plant harbors the bacteria in nodules that develop biospherehad been fixedby on its roots; the bacteria obtain respiratory carbon sub- bacteria. strates from the plant, and the plant obtains nitrogen containing amino acids from the bacteria. Most of the nitrogen-fixingplants growing in the habitats I have been This story is about plants, bacteria, nitrogen, and even studying-sand dunes, burned sites, and eroded soils sheep; perhaps more importantly,it is also a story about in the Northeast-are not legumes. But several years our ignorance until recent years of a basic biological ago when I attempted to learn more about nitrogen fix- phenomenon, recognition of that ignorance, and the rapid development of a new field in both basic and ap- Charlene Van Raalte (no photograph available) is an assistant professor plied research. of ecology at Hampshire College, Amherst, Massachusetts 01002. She has also taught at Dalhousie University and at the Marine Biological Non-leguminous Laboratory, Woods Hole, Massachusetts. She was awarded a B.A. Nitrogen-fixing degree with highest honors from Skidmore College and her Ph.D. Plants vs. Legumes degree from the Boston University Marine Program at the Marine Bio- logical Laboratory. She was the recipient of the Mellon Foundation Since the legumes are at least somewhat familiar to Faculty Development grant at Hampshire College in 1979 and 1980, most biologists, it is useful to compare the two types of among other fellowships and awards. Dr. Van Raalte is a member of the American Society of Limnologists and Oceanographers, American symbioses. Legumes have a worldwide distributionand, Women in Science, the New England Estuarine Research Society, and among the angiosperms, rank second or third in number AAAS. Her research has focused on nitrogen fixation in plants since of species contained. Most of these species are trees, 1975, and she is currently studying plant ecology of riverine marshes. She has published extensively in several publications, including ABT shrubs, or vines in inaccessible tropical habitats and are, [See ABT 39(5)]. therefore, little studied. -Vincent (1974) lists 12,880 NITROGEN FIXATION 229 species in the Leguminosae, of which only about 10% nodules or even in culture, look more like fungal fila- have been examined for presence of root nodules. Of ments than bacteria. Actinomycetes comprise a very course, the species which are cultivated for food, forage, common group of bacteria, members of which are pri- and pasture in temperate habitats have been much better marily responsible for decomposition of composts and studied. These plants include peas, beans, clovers, soy- man ures. beans, and lupins. The total global importance of legume- fixed nitrogen, clearly great, can only be estimated. TABLE2. Plantsin NorthAmerica Known to FormRoot According to Delwiche (1970), about 15-35 x 106 metric Noduleswith Actinomycete Bacteria* tons of nitrogen are fixed annually by the world's le- gumes. Reliable estimates exist for annual fixation rates Genus Family EcologicalSites by legumes in pastures or agriculturalfields. Soybeans, Alnus Betulaceae Poorsoils, glacial till, bogs, fix about 20-200 for example, can kg/ha (18-180 lbs./ gravel acre) of nitrogen annually-about equivalent to the Casuarina Casuarinaceae Sand dunes,salt marshes, amount of nitrogen a farmer would add as fertilizer. tropicalforests, deserts, In all legumes, the bacteria found in the root nodules (introducedto N. America) belong to one genus, Rhizobium. Rhizobia are gram- Ceanothus Rhamnaceae Dryforest, chaparral, sub- negative rods occurring singly or in pairs and usually alpine,disturbed sandy Downloaded from http://online.ucpress.edu/abt/article-pdf/44/4/229/339852/4447478.pdf by guest on 03 October 2021 motile when young. Inside the nodules these bacteria soils become deformed and are called "bacteroids."There are Comptonia Myricaceae Disturbedor gravellyareas many species of rhizobia;some only form the association Eleagnus Eleagnaceae Poorsoils with one species of legume, whereas others are more Myrica Myricaceae Acidicbogs, sand dunes, promiscuous in their affiliations.Rhizobium sp. were first mine wastes isolated in culture in the 1880s. They could not be shown Shepherdia Eleagnaceae Sandysoils, disturbed to fix N -nitrogen in culture and require media contain- sites ing either oxidized or reduced forms of nitrogen; there- * fore, microbiologists have long assumed that these bac- since Torrey (1978). teria could fix only in the presence of the host plant, requiring genetic input from the plant. However, Keister For many years, the Frankia could be easily seen in and Evans (1976) have recently demonstrated, under nodules of the non-leguminous fixers, but could not be stringent laboratory conditions, that these bacteria can fix isolated in pure culture in the laboratory.Since the micro- N2 and therefore must possess the so-called "nif" (nitro- biologists' main tool in the study of bacterialmorphology, gen-fixing) genes after all. metabolism, etc., is isolation and growth of the organism The process by which rhizobia invade the plant ex- in defined media, the lack of success with these actinomy- emplifies the complexities of such symbioses. The first cetes was very frustrating.Finally, three years ago Calla- discernible event-seen even with an ordinary stereo- ham, Del Tredici, and Torrey (1978) were able to isolate microscope-is "curling" of the host's root hairs. The the Frankia sp. from sweet-fern (Comptonia peregrina). bacteria enter the tip of the curled hair, become enclosed In addition to using careful techniques and the proper in a polysaccharide membrane or thread, and invade medium, these researchers were probably successful, the root cortical cells. Here they multiply and eventually in contrast to others, for one simple reason: they were fill the cells. Nodules result from the bacterial and root patient. Frankia sp. are extremely slow-growing organ- cell division. Inside the nodules, gaseous N2 is reduced to isms. Whereas most bacteria will cloud a nutrient broth the ammonium ion; the reduced nitrogen is then incor- culture overnight, the actinomycete nitrogen-fixer from porated into amino acids such as asparagine and trans- sweet-ferns requires some months to come to sizeable ported through the plant in the xylem. biomass. Quite possibly, past researchers had isolated The non-leguminous nitrogen-fixing plants are similar the organism but had thrown it away after a week or two! to the legumes in some ways and in many other ways Several species of Frankia have now been isolated are very different. The non-legumes do not belong to a from Alnus and Eleagnus sp.; thus, their growth patterns single family; they are a diverse group of woody dicoty- in culture can be examined, their morphology can be lendous angiosperms comprised of at least 160 species, studied with light and electron microscopes, and the spanning fifteen genera and seven families (Torrey1978). process of plant invasion can be compared with that of These plants typically grow in sites with poor soils- the legumes. The last, for example, is best done by grow- burned areas, dry forests, sandy habitats, and bogs (table ing the plants from seed axenically, i.e., in the absence of 2). Like the legumes, all of the few genera so far studied bacterial or other contaminants. The axenic condition harbor in their root nodules bacteria belonging to a single is important because all events occurring are then known genus. These bacteria are not in the genus Rhizobium, but to