Plants assimilate inorganic nitrogen into these N-transport amino acids: glutamate, glutamine, aspartate and asparagine. These compounds are used to transfer nitrogen in the phloem from source organs to sink tissues and to build up reserves during periods of nitrogen availability for subsequent use in growth, defence and reproductive processes (Lam et al., 1996; Coruzzi and Zhou, 2001). The concentration of these transported amino acids are not static but are modulated by factors such as light. The most prominent enzymes that synthesise N-transport amino acids are glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH) aspartate aminotransferase (AspAT) and asparagine synthetase (AS). These enzymes are involved in the primary assimilation of inorganic nitrogen from the soil as well as in the re-assimilation (secondary assimilation) of free ammonium within the plant. Nitrogen assimilated into glutamate and glutamine is readily disseminated into plant metabolism, because these amino acids donate nitrogen in the biosynthesis of amino acids, nucleic acids and other N-containing compounds. Because of the high affinity of GS for ammonia, nearly all plant nitrogen is first assimilated into organic form as glutamine. GS acts in concert with glutamate synthase (GOGAT) in what is generally referred to as the glutamate or GS/GOGAT cycle (Figure 27.1). The GS/GOGAT cycle is most likely the principal route of ammonium assimilation in plants. The net effect of this is the amination of 2-oxoglutarate to glutamate. A similar result is achieved by glutamate dehydrogenase (GDH), which also produces glutamate from 2-oxoglutarate and ammonia, but it is generally believed that this enzyme is primarily involved in glutamate oxidation, and contributes little to ammonia assimilation. GS isoforms can be separated into two classes by ion-exchange chromatography—one localised in the cytosol (GS1), the other in the chloroplast (GS2). Plants examined thus far appear to possess a single nuclear gene encoding GS2 and multiple (up to four) nuclear genes encoding GS1 subunits. The plant GS holoenzyme functions as octamer and GS1 polypeptides can assemble into homo- or heterotetramers. Although both GS isoforms do not significantly differ in their biochemical properties, they display distinct in vivo functions. The GS2 holoenzyme is predominant in leaves and very probably involved in primary ammonia assimilation and re-assimilation of respiratory ammonia. GS1 isoenzymes are present at low concentrations in leaves and at higher concentrations in roots suggesting that this enzyme has a role in primary assimilation in roots.Asparagine synthetase (AS) catalyses the amidation of aspartate to asparagine, using ammonia or glutamine as amino donor. In most cases, it appears that the in vivo substrate for this enzyme is glutamine, not ammonia, and hence AS does not usually constitute a route for ammonium assimilation.
Asparagine synthesis is particularly important in the root nodules of legumes, where much of the nitrogen fixed by the bacteria is rapidly transferred to asparagine through the joint activities of GS and AS. Thus, a lot of nodule-derived nitrogen transported in the xylem is in the form of asparagine. Asparagine levels in plant tissues vary diurnally, and often increase under stress conditions, such as nutrient deficiencies, salt stress or drought. The significance of such increases has not been established but may be a means of storing nitrogen when protein synthesis is limited by the stress that the plant is experiencing.
Proline and Arginine
Glutamate is the precursor of glutamine, arginine and proline (Buchanan et al., 2000). Glutamine synthesis has been handled earlier (in the section on ‘Nitrogen Assimilation and Reduction’ in this chapter and in the chapter on nitrogen fixation of this handbook). The first step of proline synthesis, is the activation of glutamate to an energy rich glutamyl-5′-phosphate consuming ATP and its consecutive reduction to glutamyl-5′-semialdehyde (Aral and Kamoun, 1997). In plants, different from e.g. bacteria, the kinase and dehydrogenase are synthesised as a bifunctional fusion protein, ΔΔ1-pyrroline-5-carboxylate synthetase (P5CS). The semialdehyde spontaneously cyclises to give pyrroline 5′-carboxylic acid, which is then reduced to the imino acid proline. Due to its rigid ring structure proline acts as a chain breaker when inserted into proteins, disrupting regular folding patterns of αα-helices. Physiological features of proline will be discussed in more detail when addressing the biotechnological perspectives.
Arginine biosynthesis resembles very much proline biosynthesis as glutamate is first δδ-phosphorylated by a kinase forming N-acetyl-glutamyl-5′-phosphate and then reduced to N-acetyl- glutamyl-5′-semialdehyde. However, initial N-acetylation of the amino group of glutamate prevents cyclisation. After transamidation of the 5′-semialdehyde with another glutamate as amino group donor the resulting N-acetyl-ornithine is converted in a series of reactions, similar to the animal urea cycle, to the non-protein amino acids ornithine, citrulline and finally arginine. Arginine plays a major role as a basic protein constituent often participating in active centre reactions, e.g. in the substrate-binding site of lactate dehydrogenase where it probably helps to orientate the substrate while a histidine residue acts in the conversion of lactate to pyruvate.
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