Root Nodules (Rhizobium, Legumes)

Nicholas J Brewin, John Innes Centre, Norwich, UK

Legume root nodules provide a built-in supply of nitrogen fertilizer. Nodules arise from a symbiotic association with soil bacteria termed rhizobia that have the capacity to convert atmospheric nitrogen into a form that is available for plant growth.

Introductory article
Article Contents
. Introduction . Biochemistry and Physiology . Bacterial and Plant Genetics . Taxonomy and Evolution

Introduction
Nitrogen is an essential nutrient because it is a major component of proteins and nucleic acids. Although 80% of the planets atmosphere is nitrogen, dinitrogen gas is chemically inert and unavailable to higher plants and animals. The global nitrogen cycle depends on the capacity of certain bacteria and cyanobacteria to reduce atmospheric nitrogen into ammonia, a nitrogenous form that can be assimilated by all other organisms. During the course of human evolution, it is curious that primitive agriculturalists selected and cultivated a large number of plants belonging to the family Leguminosae (Fabaceae). Examples of grain legumes include beans, peas and lentils, all of which are particularly nutritious because of the high protein content of their seed. Examples of fodder legumes include clover, alfalfa and vetch, which contribute to the fertility of pasture and hence to the nourishment of ruminant animals. Some woody legumes are important sources of timber (e.g. Acacia and Leucaena), while others (e.g. gorse and broom) are colonizers of poor and sandy soils where nitrogen (nitrate) is at a premium. In all of these situations, legumes have a competitive advantage because they are able to establish a root nodule symbiosis that allows them to be independent of the nitrogen status of the soil. Legumes have root nodules that harbour soil bacteria collectively termed rhizobia. These symbiotic bacteria have the capacity to establish a new organ (the root nodule); to colonize host cells; and to develop the capacity for nitrogen xation inside plant tissues. The legume root nodule is a fully dierentiated tissue with many distinct cell types showing morphological and functional specialization. Its physiology is fully integrated into the growth and development of the whole plant.

ammonia requires large amounts of energy and reductant. At least 16 molecules of energy-carrying ATP (adenosine triphosphate) are consumed during the reduction of each molecule of nitrogen. Hydrogen is always evolved as a byproduct (eqn [I]).
N2 +8H+ +8e 2NH3 +H2 [I]

The nitrogenase enzymes are highly sensitive to oxygen damage, yet rhizobia require oxygen for growth and respiration. In general, rhizobia do not x nitrogen in the soil but only within root nodules where the internal oxygen concentration is regulated to be about 1000 times below ambient (330 nmol L 1). Despite the low oxygen concentration, the intracellular rhizobia sustain a vigorous level of oxidative respiration. Oxygen diusion is facilitated through intercellular air spaces and through an oxygen scavenging haemoprotein (leghaemoglobin) that represents a major component of the cytoplasm in nodule cells. The dierentiated nitrogen-xing forms of rhizobia are termed bacteroids. Bacteroids are enclosed within a hostderived membrane (the peribacteroid membrane) that regulates metabolic exchanges between the bacterium and its intracellular environment. Organic acids (e.g. malate), are apparently the principal carbon source transported to bacteroids. Ammonia, the primary product of nitrogen xation, is exported to the host cell cytoplasm and is subsequently assimilated by the action of glutamine synthase. In eect, the peribacteroid unit operates like a prokaryote organelle concerned with nitrogen xation: it is often referred to as a symbiosome. Symbiosis is based on metabolic exchange for mutual benet. The plant supplies carbon from photosynthesis and the root nodule provides a low-oxygen niche for rhizobium. In exchange, rhizobium provides the plant with its own source of xed nitrogen.

Biochemistry and Physiology

Nitrogen xation depends on a complex of bacterial enzymes termed nitrogenase. At the heart of the catalytic process is a highly complex cofactor comprising iron, molybdenum and sulfur. The conversion of dinitrogen to

Bacterial and Plant Genetics

A number of rhizobium genes are critical for host-specic nodulation on legumes, but are not necessary for survival as free-living soil bacteria. The products of these nod genes
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Root Nodules (Rhizobium, Legumes)

are involved in a molecular dialogue that results in the initiation of nodule development on the appropriate host legume. Each legume host exudes a characteristic set of organic compounds (avonoids) that are sensed specically by the nodD gene product(s) of an infective rhizobium strain. Following this recognition event, transcription of a further set of rhizobium nod genes takes place. This culminates in the synthesis and secretion of specic signal molecules, termed Nod factors that specically activate host cells of the appropriate legume. Structurally, Nod factors are chemically decorated oligomers of N-acetylglucosamine (the constituent of chitin) and, characteristically, they each carry an unsaturated acyl chain on the nonreducing sugar. By a process that is still not understood at the molecular level, Nod factors initiate nodule initiation by inducing cortical cell divisions. Nod factors also promote host cell colonization by provoking a reorganization of cell wall growth in epidermal cells and root hairs.

independently. However, the predisposition to form root nodules is not restricted to the Leguminosae but rather to the Rosid clade I, which includes the Leguminosae as just one of its families. Unlike all other groups of higher plants, this clade contains several instances of the evolution of non-legume root nodule symbioses. In these cases the nodule endophyte is not a rhizobial strain (as currently dened) but is instead an actinomycete, Frankia, a lamentous Gram-positive bacterium. Examples of actinorhizal plants include Alnus, Caeanothus and Casuarina. There is one example of a non-legume root nodule symbiosis in which the endophyte is unquestionably a rhizobium strain: this involves the ulmaceous shrub Parasponia, which also belongs to Rosid clade I.

Further Reading
Brewin NJ (1991) Development of the legume root nodule. Annual Review of Cell Biology 7: 191226. Cook DR (1999) Medicago truncatula a model in the making! Current Opinion in Plant Biology 2: 301304. Crespi M and Galvez S (2000) Molecular mechanisms in root nodule development. Journal of Plant Growth Regulation 19: 155166. Downie JA and Brewin NJ (1999) Plantmicroorganism symbiosis. In: Russo VEA et al. (eds) Development Genetics, Epigenetics and Environmental Regulation, pp. 211232. Berlin: Springer-Verlag. Gualtieri G and Bisseling T (2000) The evolution of nodulation. Plant Molecular Biology 42: 181194. Parniske M (2000) Intracellular accommodation of microbes by plants: a common developmental program for symbiosis and disease? Current Opinion in Plant Biology 3: 320328. Schultze M and Kondorosi A (1998) Regulation of symbiotic root nodule development. Annual Review of Genetics 32: 3357. Spaink HP (2000) Root nodulation and infection factors produced by rhizobial bacteria. Annual Review of Microbiology 54: 257288. Sprent JI (2001) Nodulation in Legumes. London: Cromwell Press. Stougaard J (2000) Regulators and regulation of legume root nodule development. Plant Physiology 124: 531540.

Taxonomy and Evolution

When rhizobial strains are analysed by standard taxonomic criteria based on gene sequence comparisons, it is clear that these bacteria do not form part of a homogeneous taxonomic group. The Rhizobiaceae as a distinct bacterial family does not exist. The clear implication is that nod genes, nitrogenase genes and other genes concerned with intracellular symbiosis have been transferred horizontally by gene exchange between various groups of soil bacteria to create new genetic combinations that are optimally adapted both for survival in the soil and for the nodulation of some particular legume host. On the plant side, the Leguminosae constitute a very large family (18 000 species), within which the capacity to develop root nodules appears to have evolved several times

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