Nitrate assimilation pathway

Fig. 1. The nitrate assimilation pathway in higher plants. The pathway of nitrate assimilation in the tobacco leaf is illustrated. In some other species an additional cytosolic GS is found in the leaf. The pathway in plant roots is more poorly documented and more variable GS in roots is mostly cytosolic, and some enzymes such as GOGAT are found as isoforms utilising alternate reducing substrates. T, expected nitrate carrier NR, nitrate reductase NiR, nitrite reductase GS, glutamine synthetase GOGAT, glutamate synthase Fd, ferredoxin Gin, glutamine Glu, glutamate.

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. 

Baebprasert, W., Jantaro, S., Khetkom, W., Lindblad, P., Incharoensakdi, A., 2011. Increased H2 production in the cyanobacterium Synechocystis sp. strain PCC 6803 by redirecting the electron supply via genetic engineering of the nitrate assimilation pathway. Metabolic Engineering 13, 610—616. 

Nitrate reductase, the first enzyme in the nitrate assimilation pathway, catalyzes the reduction of nitrate to nitrite. This requires two electrons which are donated by either NADH or NADPH in the eukaryotic NRs. The NADH-spe-cific NRs (EC 1.6.6.1) are found in most higher plants and numerous eukaryotic algae while the NADPH-specific NRs (EC 1.6.6.3) are found in the fungi. The NAD(P)H-bispecific NRs (EC 1.6.6.2) occur in some higher plant and algae species. The prokaryotic NRs which utilize a variety of electron donors, including ferredoxin, reduced pyridine nucleotides, and respiratory intermediates, will not be considered in this article (see Stewart, 1988). 

Fig. 3. Regulation of the bound pathway for the assimilation of sulfate into cysteine and associated processes. Carrier refers to an endogenous thiol of uncertain identity in higher plants. Enzymes associated with the sulfate assimilation pathway and the synthesis of O-acetylseiine are (1) high-ailinity sulfate uptake mechanism, (2) ATP-sulfurylase, (3) adenosine S -phosphosulfate (APS) sulfotransferase, (4) organic thiosulfate reductase, (5) cysteine synthase, and (6) serine transacetylase. Cysteine sulfhydrase (7), an enzyme of cysteine catabolism, and nitrate reductase (8), the first enzyme of the nitrate assimilation pathway, are also shown. Inhibitory control of the pathways is shown by discontinuous lines (----) and enhancement by continuous lines (------).

The principles of the coordinated control of sulfate and nitrate assimilation were formulated by Reuveny and Filner (1977) following their pioneering work on the effect of nitrate and sulfate stress on the levels of ATP-sulfiirylase and nitrate reductase in cultured tobacco cells. Coordinated control involves independent internal control of the sulfate and nitrate assimilation pathways by their own substrates and products (such as discussed for the enzymes of sulfate assimilation in the previous section) and stimulation of the pathways of sulfate and nitrate assimilation by products of the other pathway. Thus for the sulfate assimilation pathway this involves stimulation by a reduced form of nitrogen while for the nitrate assimilation pathway it involves stimulation by a reduced form of sulfur. 

The internal regulation of the sulfate assimilation pathway and its coordination with the nitrate assimilation pathway are summarized in Fig. 3. It shows that cysteine is a negative effector of serine transacetylase and that it also controls the level of APS sulfotransferase. The inhibitory effects of HjS on the level of APS sulfotransferase are probably mediated via cysteine, though HjS itself at high concentrations inhibits cysteine synthase. 

In tobacco cell cultures the extractable levels of ATP-sulfurylase and cysteine synthase are very low when the cells are subject to nitrogen stress but increase rapidly upon alleviation of the stress, suggesting that a product of nitrogen assimilation derepresses the levels of these enzymes. In Lemna (and possibly in cultured Rosa cells) it appears that this role is fulfilled by APS sulfotransferase and that ATP-sulfurylase and cysteine play unimportant roles in coordinating the sulfate assimilation pathway with the nitrate assimilation pathway. A further regulatory mechanism known to occur in cultured tobacco cells is that excessively high concentrations of cysteine induce the synthesis of cysteine desulfiiydrase (see Section VI). 

Burger, G., Tilbur, J. Scazzocchio, C. (1991). Molecular cloning and functional characterization of the pathway-specific regulatory gene nir A, which controls nitrate assimilation in Aspergillus nidulans. Molecular and Cellular Biology 11, 795-802. 

The findings that in tobacco cells sulfur starvation prevents induction of nitrate reductase (P. Filner, unpublished observation cited in Reuveny and Filner, 1977), and that repression of nitrate reductase by a number of nonsulfur amino acids is antagonized by cysteine (Filner, 1966) led Reuveny and Filner (1977) to suggest that a product of the sulfate assimilation pathway may also play a positive role in control of nitrate assimilation. If so, a reciprocal relationship would exist between the nitrogen and sulfur assimilation pathways such that a product of each of these pathways is required as positive effector for the other. 

Nitrate is a major source of inorganic nitrogen for plants, fungi, and many species of bacteria. Animals cannot assimilate nitrate. Assimilation involves three pathway-specific steps uptake followed by reduction to nitrite, and further reduction to ammonia which is then metabolized into central pathways. 

Regardless of whether the process of nitrate reduction is located in photosynthetic or nonphotosynthetic tissues, it still involves a cytoplasmically located nitrate reductase [reaction (6)] and a nitrite reductase complex [reaction (7)], which is located in plastids. Possible sources of reductant for these reactions have been discussed in several reviews (e.g., Lee, 1980 Abrol et al., 1983 Smirnoff and Stewart, 1985) and the conclusion reached that in heterotrophic (nonphotosynthetic) nitrate assimilation the NADH required by nitrate reductase might be derived from glycolysis, from the oxidative pentose phosphate pathway, or even from mitochondrial dehydrogenases (see I e, 1980), whereas the pentose phosphate pathway may be of singular significance in supplying NADPH for nitrite assimilation. As indicated for root tissue by Ernes et al. 

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. 

Because NTR links the reduced and oxidized sides of the N cycle, it can be considered a central process that provides substrate to microbes that employ nitrate or nitrite as oxidant (see Chapter 5 by Ward, this volume Fig. 19.1, arrow 4). Like NH4, the products of NTR, N02, and NOs , may experience one of several possible fates, including (1) flux from the sediment, (2) assimilation within the sediment or at the sediment—water interface, or (3) reduction by one of three possible dissimilatory pathways DNF, dissimilatory nitrate reduction to ammonium (DNRA), or ANAM (Fig. 19.1, arrows 5, 6, and 7 Fig. 19.2). Uptake of NO by... 

---