RWTH Aachen, Okologie des Bodens, Germany
The symbiosis between Rhizobiaceae and leguminous plants is agronomically the most important biological mechanism for adding nitrogen to the soil/plant system. The interaction between micro- (the bacterium) and macro- (the plant) symbiont results in the formation of a specialized, highly differentiated plant organ, the root nodule, in which the microsymbiont converts atmospheric nitrogen to ammonia, which is translocated to the host plant. Agricultural important crops that can benefit from this symbiosis include grain legumes such as peas, beans and soybeans and forage legumes like alfalfa and clover. The symbiotic process involves a complex and sequential exchange of molecular signals between the two partners (for review see 2,4,11) which is highly specific and results in a strict host-specificity, i.e., that only certain rhizobial species (and biovars) can interact with certain leguminous plants.
Rhizobia able to nodulate plants of the genera Pisum, Vicia, Lathyrus and Lens are called Rhizobium leguminosarum biovar viciae. Phenolic compounds exudated from the plant root (mainly flavones and flavanones) induce the expression of certain bacterial genes, the nodulation (nod) genes, which consequently leads to the production and excretion of specific lipo-chitooligosaccharides. The backbone of these so-called nodulation or nod factors consists of four to five b-1,4-linked N-acetylglucosamine residues with a fatty acid moiety at the non-reducing end. This basic structure is identical in all rhizobia and is produced through the action of so-called common nod genes. The host specificity of the nodulation factor is mediated by specific modifications which are the product of nodulation genes not conserved between different Rhizobium species (host-specific nodulation or hsn genes). For example, the Nod factors produced by R.leguminosarum bv. viciae are characterized by a 6-0-acetyl group at the non-reducing sugar and a C18 fatty acid with four double bonds, whereas the nodulation factor synthesized by Sinorhizobium meliloti is sulfated and carries a C16 unsaturated fatty acid at the non-reducing end (9,10).
The nodulation factors in turn act as signal molecules to induce specific developmental processes in compatible host plants. For example, they trigger the de-differentiation of root cortical cells which restart cell division to establish a nodule primordium that ultimately results in the formation of the nodule. Bacteria invade these primordia through infection threads from which they are endocytosed into the plant cytoplasm and enveloped by a plant derived membrane, the peribacteroid membrane.
The actual reduction of atmospheric dinitrogen gas (N2) to ammonia (NH3) which is the fixed nitrogen that can be assimilated into amino acids by biological systems, is mediated by an enzyme complex, the nitrogenase, which is physically and functionally conserved in diverse nitrogen-fixing organisms. It is composed of two components, the iron-molybdenum protein (MoFe protein) and the iron protein (Fe protein). The MoFe protein (also called dinitrogenase) is a tetrameric (a2b2) protein containing a molybdenum-iron cofactor (FeMoCo), which comprises the catalytic site for N2 reduction. The Fe protein (also called dinitrogenase reductase) is a homodimer and its function is to transfer electrons to the dinitrogenase enzyme. Both components are highly oxygen sensitive and are irreversibly inactivated in the presence of oxygen (1). A precondition for the synthesis of nitrogenase is the irreversible differentiation of the bacteria into so-called bacteroids. This process includes physiological and morphological alterations as well as changes in the pattern of gene expression, which is triggered by the low oxygen tension present in the nodule. Among the genes expressed under these conditions are the structural genes for the nitrogenase complex (nif genes) as well as accessory genes (fix genes), for example fixNOQP encoding a membrane bound cytochrome oxidase which most probably is part of a bacteroid-specific respiratory chain functioning under low oxygen conditions (for a review see 3).
A number of oxygen-regulatory proteins has been identified in various rhizobia (for a review see 3). For example, in Rhizobium leguminosarum bv. viciae, we found NifA, a central, and in diazotrophs highly conserved, oxygen-responding transcriptional activator which mediates transcription of the structural nif genes (5), FixL, a heme binding protein resembling the sensor moiety of classical two-component-regulatory systems (6), and two Fnr-like proteins, one of which (FnrN) also responds to oxygen at the protein level and activates e.g., the fixNOQP operon (7,8). Similar or identical elements have been found in other rhizobia, but interestingly, their interrelation and significance for nitrogen fixation differs considerably between different species and even biovars.
The knowledge that we have on the processes and mechanisms occurring in the bacterial partner, is the result of about two decades of molecular and genetic research. On the plant side, far less is known. Although various genes have been identified in different legumes, their biochemical functions are still unknown. Despite its drawbacks for molecular genetics, pea is certainly still a genetic model system for studying bacteria-plant interactions. It will be exciting to follow the research and progress in this field.