Ammonia assimilation pathways

There is thus good evidence from the inhibitor studies that glutamine synthetase plays a key role in the process of photorespiration. Possible explanations for the rapid fall in photosynthetic rates will be discussed in the following section, which describes the properties of mutants lacking enzymes of the ammonia assimilation pathway. 

In 1980, Miflin and Lea pointed out that much of the information on biochemical pathways has arisen from the use of mutants of bacteria and yeast larking key enzymes. They complained at the time that no mutants of higher plants were available in the ammonia assimilation pathway. A glance at Table I clearly indicates that a number of such mutants are now available. In addition mutants of maize with low (but not zero) levels of glutamate dehydrogenase have also been studied (Rhodes et ai, 1989). 

GS in plants is the key enzyme of the GS/GOGAT (glutamine synthetase/g1utamine 2-oxyg1utarate aminotransferase) pathway and thus plays a crucial role in ammonia assimilation/reassimilation (117. 118). GS inhibition by phosphinothricin causes accumulation of toxic levels of ammonia. Since ammonia production is increased by photorespiration and conversion of nitrite to ammonia (also light-dependent), nitrogen fertilizers and light act to promote phosphinothricin efficacy (119). 

Photosynthetic inhibition caused by NOx may be due to competition for NADPH between the processes of nitrite reduction and carbon assimilation in chloroplasts. N02 has been shown to cause swelling of chloroplast membranes (Wellburn et al. 1972). Biochemical and membrane injury may be caused by ammonia produced from N03, if it is not utilized soon after its formation. Plants can metabolize the dissolved NOx through their N02 assimilation pathway ... 

As implied in the introduction, various types of evidence can be used to resolve the question as to which pathway of ammonia assimilation is operating. In this section we wish to critically appraise the nature of such evidence. In doing this it must be borne in mind that, as in all scientific proofs, we are trying to reject invalid hypotheses rather thanprove the validity of any given one the best that we can hope to do is to show that all the available evidence is compatible with only one hypothesis. 

Thus, although the general conclusion that ammonia assimilation in fungi can occur via GDH remains valid, it would appear that several fungi have the capability to use the alternate pathway which may provide an adaptive advantage under certain environmental conditions (see Section V). 

In broad outline, the reduction and assimilation of inorganic sulfate and nitrate in plants have several features in common. Both processes entail 8 c reductions to inorganic forms (sulfide and ammonia, respectively) in energy-requiring reactions prior to incorporation into appropriate acceptor molecules. With the exception of the partial reduction of nitrate to nitrite in the cytoplasm, assimilation of sulfate and nitrite occurs in chloroplasts in reactions which are dependent on light for a supply of Fdred and ATP. However, the processes differ in many aspects of detail. For example ATP is required for activation of sulfate prior to reduction but in the nitrate assimilation pathway ATP is required after reduction for the incorporation of ammonia into glutamine. In addition, sulfate activation has no counterpart in nitrate reduction and, whereas sulfate remains bound to a carrier during reduction, the intermediates of nitrate remain free. 

This hypothesis is interesting from the point of view of the role of mitochondrial GDH enzymes in higher plant tissues, which are considered to assimilate ammonia via the GS/GOGAT pathway (Miflin and Lea, 1976). Thus the late evolution of the higher plant enzyme suggests that it could have a role in the mitochondrion and that it is not simply an obsolete ancestral enzyme of ammonia assimilation. 

Seeds of pea (Thurman et al., 1965) and Medicago sativa (Hartmann et al., 1973) possess 7 GDH isoenzymes (pattern 1) which decrease in number during germination, while a new pattern of isoenzymes (pattern 2) becomes visible on gel electrophoresis. Pattern 1 is characteristic of the seed, pattern 2 is characteristic of the root, while the shoot possesses some pattern 1 and some pattern 2 isoenzymes. Prior to the discovery of the GS/GOGAT pathway in higher plant tissues, it was postulated that pattern 1 enzymes were involved in deamination reactions, while pattern 2 enzymes were involved in ammonia assimilation. 

The chloroplast NADP linked GDH of Lactuca has a for ammonia of 5.2 mM, and the observation that ammonia uncouples chloroplasts at concentrations of 2 mM makes a role for this enzyme in ammonia assimilation unlikely (Miflin and Lea, 1976). It is, however, possible that the values of GDH enzymes within the cell may be lower than the values determined in vitro, or that high concentrations of ammonia may be localized at the site of the enzyme in the cell. Alternatively, GDH may function primarily in deamination reactions in higher plants, and alternative pathways for ammonia assimilation have been proposed (Miflin and Lea 1976 and this volume. Chapter 4). 

An alternative approach to estimating the metabolic capabilities of chloroplasts entails measurement of the light-dependent metabolism of radioactive tracers. Using isolated pea chloroplasts. Mills and Wilson (1978a) found that lysine, methionine, threonine, and isoleucine were synthesized from [ C]aspartate. Further evidence that aspartate was being metabolized via the anticipated pathways was provided by the demonstration that the synthesis of homoserine was inhibited by lysine and threonine (Lea et al., 1979). These results, combined with those relating to enzyme localization, lead to the concept that chloroplasts contain a complete functional sequence of enzymes which can facilitate the synthesis of the aspartate family and at least some of the branched-chain amino acids. This is consistent with the importance of chloroplasts in ammonia assimilation (Miflin and Lea, this volume. Chapter 4) and with the evidence that protein can be synthesized from CO2 in isolated plastids (Shepard and Leven, 1972 Huberer al., 1977). The actual fraction of [ ]02 which is utilized for amino acid biosynthesis in isolated plastids is usually quite small. Thus, reactions which normally occur outside of chloroplasts are considered to be of major importance in the synthesis of carbon skeletons such as oxaloacetate or pyruvate (Kirk and Leech, 1972 Leech and Murphy, 1976). 

However, models which assume that ammonia is assimilated solely via GDH cannot account in quantitative terms for the observed rates of NH/ incorporation (Rhodes et al, 1980 Fentem et ai, 1983a). Rhodes et ai, (1989) modeled the kinetics of NH4 incorporation in tobacco cell cultures and suggest that up to 30% of the ammonia assimilated may enter via the GDH reaction. However, the relative sizes of the glutamate and glutamine pools made it difficult to distinguish between the simultaneous operation of alternative pathways and Rhodes et al. were unable to verify the model when inhibitors of GS and GOGAT were used to block the glutamate synthase cycle. 

Ammonia is oxidized in nature to nitrate via several intermediates in the process of nitrification. Nitrate may be reduced to nitrite by either a dissimilatory or an assimilatory process. Nitrite may be assimilated into the cell via reduction to ammonia, or it may be reduced by microorganisms to N20 and N2 in denitrification. A major part of the total nitrogen in this pathway is lost to the atmosphere. However, in turn, atmospheric dinitrogen is converted to ammonia by various bacteria in nitrogen fixation. 

HaUatn, S. J., Mincer, T. J., Schleper, C., Preston, C. M., Roberts, K., Richardson, P. M., and DeLong, E. F. (2006). Pathways of carbon assimilation and ammonia oxidation suggested by environmental genomic analyses of marine Crenarchaeota. Plos Biology 4, 520-536. 

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