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CHAIR'S COMMENTS: NODULE METABOLISM: OVERVIEW
Department of Natural Resource Sciences, McGill University Ste-Anne-de-Bellevue, Québec, Canada
Nutrient exchange is at the heart of the symbiosis between legumes and rhizobia. The basics of this exchange are fairly well understood: in exchange for an energy source from the plant, rhizobia fix nitrogen within nodules and supply the host with nitrogenous compounds that are readily assimilated. As with signaling and nodulation however, there appear to be differences in nutrient metabolism and exchange between the different rhizobium-legume symbioses. There are still many unanswered questions such as; which compounds are being exchanged, how are the metabolic pathways regulated, what is the role of storage compounds and nutrient supply during infection, and what are the metabolic changes that occur during the transition from infecting rhizobia to bacteroids. In addition, there are questions regarding the role of metabolism in bacterial survival and competition, and how legume yield may be improved through engineered changes to rhizobial and plant metabolic processes. The metabolism and transport of carbon and nitrogen in root nodules has been the subject of many reviews over the years (for example: Dunn 1998; McDermott el al. 1989; Poole, Allaway 2000; Tajima, Kouchi 1996; Udvardi, Day 1997; Vance, Heichel 1991). This overview is very brief, and focused mainly around the topics covered by the session speakers (CP Vance, TC Charles, PS Poole, C Atkins).
C4-dicarboxylic acids (C4-DCA) such as malate are found in high concentrations in root nodule cells, and are the principal source of energy supplied by the plant to N2-fixing bacteroids. The role of malate in symbiosis has been reviewed by Vance and Heichel (1991). There is evidence that some legume enzymes in the pathway which convert photosynthate to malate are highly expressed in nodule tissue, such as the nodule-enhanced malate dehydrogenase (MDH) of alfalfa (Miller et al. 1998).
Bacteroids rely on the oxidation of C4-DCA via the tricarboxylic acid (TCA) cycle to support the energy requirements of the nitrogenase enzyme. In S. meliloti bacteroids, malate appears to be channeled through two pathways (i) MDH, and (ii) NAD+ malic enzyme and pyruvate dehydrogenase, to produce the oxaloacetate and acetyl-CoA, respectively, required for synthesis of citrate (Cabanes et al. 2000; Driscoll, Finan 1993). There is a large body of evidence indicating that C4-DCA are the principal source of carbon supplied by the plant to N2-fixing bacteroids, and that they are metabolized by bacteroids to generate the energy necessary to support N2-fixation. Nitrogenase activity in isolated bacteroids is highly stimulated by C4-DCA, but not by sugars (Bergersen, Turner 1967; Miller et al. 1988). Transport of C4-DCA via the bacterial C4-DCA transport (dct) system is essential in N2-fixing bacteroids, as Dct" mutants induce root nodules, but are unable to fix N2 (Ronson et al. 1981, and many others). Transport of C4-DCA across the peribacteroid membrane has also been demonstrated (see Udvardi, Day 1997 for review).
C4-DCA are metabolized directly via the TCA cycle. All of the TCA cycle enzymes have been detected in Rhizobium species, and they appear to be highly active in bacteroids (see reviews by Dunn 1998; Poole, Allaway 2000). While most mutants that completely lack a TCA cycle enzyme form nodules that fail to fix nitrogen, B. japonicum sueA mutants are not completely Fix", possibly due to a metabolic bypass of 2-oxoglutarate dehydrogenase via succinic semialdehyde (Green et al. 2000). Poole et al. (1999) showed that the R. leguminosarum mdh is in an operon with sucCD, and possibly with sucAB. Their results indicate that arabinose stimulates expression of an mdh::lacZ gene fusion, and that the very high expression of this fusion in a sucD mutant background may be the result of the accumulation of a metabolic intermediate, perhaps arabinose itself. In
S. meliloti, the mdh gene is in an operon with sucCD, while sucAB and sdhCDAB appear to constitute separate operons even though the former are directly downstream of mdh sucCD (S.I. Dymov et al., unpublished).
While it has long been recognized that legumes assimilate nitrogen either via the amide or the ureide pathway, it has been recently shown that bacteroids of different rhizobia may export different forms of nitrogen. B. japonicum bacteroids export alanine rather than ammonium, as previously believed (Waters et al. 1998). R. leguminosarum bacteroids appear to supply both ammonium and alanine, and while pea plants inoculated with alanine dehydrogenase mutants are Fix+, they show reduced dry weight values, indicating that both compounds contribute to plant growth (Allaway et al. 2000). In tropical legumes such as cowpea and soybean, enzymatic (Shelp et al. 1983) and gene expression studies (Smith et al. 1998) indicate that nitrogen assimilation is via purine synthesis. The mode by which amino groups are transferred from alanine to this pathway is not yet clear, however.
The emergence of complete genome sequences, such as that of S. meliloti (Galibert et al. 2001), will enhance the ongoing goal to understand nodule metabolism, making the tedious process of completely sequencing metabolic genes an unnecessary step. The post genomics era will return the focus to the genetic and biochemical characterization of biochemical processes, and allow a better comparison of the differences observed between the various symbioses. There are many metabolic processes that occur in nodules that we do not understand well enough yet, such as the processes that allow legumes to balance the supply of energy to bacteroids with the supply of carbon to the rest of the plant, bacterial growth in the infection thread, the transition from bacteria to bacteroids, how bacteroids generate energy under nodule conditions (with a highly controlled 02 supply), and the assimilation of alanine and/or ammonium. Interesting questions outside of the nodule also exist, such as the role of bacterial storage compounds like polyhydroxybutyrate (Aneja, Charles 1999) in the soil rhizosphere, and how these compounds affect survival and competition for nodulation.
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