energy costs between the two types of transport compounds (Sprent and Sprent, 1990). There are also differences in solubility, ureides being less soluble than amides in solution, but this does not appear to present problems for ureide-exporting plants grown at low temperatures (Neves and Hungria, 1987).
Detoxification of ammonia
Teleologically speaking, GS serves two roles. The first is to assimilate ammonia and thus to provide useable N for general metabolism. The second results from the observation that free ammonia is actually toxic, and thus the action of GS can also be viewed as a detoxification mechanism. In animals, excess N is generally excreted, e.g. as a component of urea. In plants, however, where N is often a limiting factor, the potentially toxic NH4+ is sequestered in the form of storage compounds that are non-toxic and are also available for later provision of N. When N is required from this reservoir, it is generally released by catabolic breakdown of the storage compound followed by reassimilation of the ammonia so released (Schubert, 1986).
The storage of high amounts of N in legumes makes them especially valuable as fodder with a high protein content in pasture systems (Chapter 10).
Inhibition by fixed N in free-living N2-fixing organisms
As discussed above, at least 16 ATP molecules are needed to reduce one molecule of dinitrogen. The net result is generally provision of an amide group on glutamate.
Thus, if nitrogen is supplied in this or any other form of combined N, as certainly happens in many laboratory-grown cultures, fixation of N2would be a waste of ATP, and it is not surprising that all free-living N2-fixing organisms studied to date appear not to show nitrogenase activity if sufficient combined nitrogen is already available in the cell.
Regulation in response to combined nitrogen has been most intensively studied inK. pneumoniae. It has been shown that the signal that indicates the intracellular availability of combined nitrogen is the ratio between glutamine and 2-oxoglutarate.
If it is low, this indicates a low cellular concentration of NH4+(the logic behind this is clear from the GS/GOGAT pathway of ammonia assimilation; Fig. 3.4), and leads, through a complex cascade of genetic regulation, to synthesis of a series of enzymes involved in N metabolism. Some of these enzymes are involved in catabolism of nitrogen-rich amino acids (histidine, proline and arginine). The other enzyme of nitrogen metabolism that is regulated in this manner is nitrogenase. By this means nitrogenase is only synthesized if nitrogen is sufficiently limiting to warrant fixation of N2. It should be noted that a deficiency of combined nitrogen is not the only factor regulating induction of nitrogenase activity inK. pneumoniae. A system of genetic control also exists to prevent synthesis of nitrogenase if the concentration of O2is too high (see above).
Another form of regulation of nitrogenase activity that has been identified in some free-living N2-fixing organisms is temporary inactivation of the enzyme by covalent modification in response to availability of ammonia. It is mediated by two enzymes, one of which, Fe protein ADP-ribosyl transferase (DRAT), covalently modifies Fe protein by addition of an ADP molecule in the presence of ammonium.
This temporarily inactivates nitrogenase. When the ammonium concentration falls, the ADP-ribosylation is reversed by a second enzyme called dinitrogenase reductase activating glycohydrolase (DRAG) and nitrogenase activity is restored. This phenomenon was originally observed in R. rubrum and has since been found in other organisms, includingR. capsulatus,Azospirillumspp. and, most probably,A.
caulinodans(Robertset al., 1990). However, it is by no means universal amongst N2-fixers, not being found inAzotobacterspp. or inK. pneumoniae.
Regulation of nodulation and N2-fixation in symbioses with plants In the case of legume symbioses, regulation of N2-fixation in response to available fixed N is mediated by the host legume rather than by the bacterial symbiont. It has long been known that a large concentration of nitrate will inhibit the development of root nodules (Fred and Graul, 1916). However, the exact significance of this is not entirely clear, as the different energy requirements of nitrate assimilation and N2-fixation plus associated costs are not so great.
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The effect of nitrate on a number of stages of the nodulation process has been examined, and all, from induction of rhizobial nodulation genes through root hair development and penetration and infection thread formation, are inhibited to a greater or lesser extent by the presence of nitrate (Carroll and Mathews, 1990). The end result is that the actual number of nodules formed on legume roots is reduced, leading eventually to complete suppression of nodulation if concentrations of nitrate exceed a certain threshold value. This may vary for different species, but is in the range of 2–20 mM (Harper and Gibson, 1984). At intermediate concentrations of nitrate, partial inhibition of nodulation will occur and this observation is used in the calibration of the ureide method for measurement of N2-fixation (Chapter 4). At such intermediate concentrations the effect may be manifested in the developing nodules being smaller, such that the nodule mass per plant is reduced while the total number of nodules remains almost unaltered (Streeter, 1988). It should be pointed out that low concentrations of nitrate (1–2 mM) actually promote nodulation by ensuring early, rapid growth of the plant and development of a healthy root system able to nodulate profusely. This is termed the ‘starter effect’.
A third effect of nitrate is actual inhibition of fixation in active nodules. This has been demonstrated both in greenhouse (e.g. Streeter, 1985; Minchinet al., 1989) and in field-grown plants (Eardlyet al., 1984). Many of these results were obtained using ARA as a measure of nitrogenase activity, and it has since been shown that acetylene itself induces major changes in nodule nitrogenase activity (Chapter 4).
Nevertheless, the phenomenon of nitrate inhibition of symbiotic N2-fixation is a real one (Streeter, 1988) that appears to occur in three stages. In the first stage, nitrate is restricted to the cytoplasm of nodule cells and the immediate decrease in nitrogenase activity is due to an increase in the resistance of the O2 diffusion barrier. This is then followed by a reduction in C metabolism (Arrese-Igoret al., 1997). Irreversible nodule senescence follows, perhaps encouraged by toxic effects of nitrite on nitro- genase and leghaemoglobin (Becana and Sprent, 1987; Escuredoet al., 1996).
A phenomenon that is almost certainly related to the above observations is autoregulation. This means that a legume root system will develop only a certain number of active nodules, and then no further new infection occurs. It appears that early infection events in the root produce a signal that is translocated to the shoot and the shoot in turn produces a signal that is transported to all parts of the root, thus suppressing further development of nodules (Carroll and Mathews, 1990). However, this autoregulation must somehow be sensing the N2-fixing activity of the nodules, since if the nodules are induced by an ineffective rhizobial strain, new nodules continue to be formed until the nitrogen requirements of the plant are met.
Parsonset al. (1993b) suggested that further regulation of nodule growth and activity is triggered by the concentration of reduced N compounds in the phloem.
This N feedback mechanism is supported by an array of direct and indirect evidence (Hartwig, 1998; Serraj et al., 1999), and the effects of nitrate on nodulation and N2-fixation described above (including autoregulation) may be mediated through signals from the shoot. The N feedback mechanism provides an elegant explanation of how the plant host may regulate both the degree of infection and the activity of
N2-fixation in nodules, which probably also extends to actinorhizal symbioses (Baker et al., 1997) andParasponiaandGunnerasymbioses.
Conclusions
N2is reduced by the enzyme nitrogenase to ammonia. In most N2-fixing organisms there appears to be one form of nitrogenase, the Mo nitrogenase, but two other forms of nitrogenase have been identified in Azotobacter. The Mo nitrogenase reduces two protons to one molecule of H2 for every molecule of N2reduced. In some species there is an uptake hydrogenase that oxidizes this hydrogen to water, thereby generating ATP and salvaging some of the energy ‘wasted’ in the evolution of H2by nitrogenase.
The ammonia generated by nitrogenase is assimilated as the NH4+ ion into glutamate. In both bacteria and plants this occurs via the joint action of the enzymes glutamine synthetase (GS) and glutamine-2-oxoglutarate-amino-transferase (GOGAT). In legumes the fixed nitrogen is further assimilated and transported predominantly as the amino acids asparagine and glutamine in amide-exporters, or as the ureides allantoin and allantoic acid in the ureide-exporters.
N2-fixation is inhibited by a supply of combined N in all systems. In free-living diazotrophs, this occurs through inhibition of nitrogenase synthesis or by temporary inactivation of nitrogenase through covalent modification of the enzyme. In legume–rhizobial symbioses, inhibition by combined nitrogen can occur at three levels: nodulation may be suppressed completely; the total nodule mass may be reduced; or the nitrogenase activity of mature nodules may be inhibited. Legume symbioses also show autoregulation whereby further nodule formation is inhibited once a sufficient number of effective nodules have developed.
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Measuring N2-fixation Chapter 4