Ann Oaks

Compartmentation of Nitrogen Assimilation in Higher Plants

Publisher Summary This chapter describes important reactions of nitrogen assimilation and initiatives leading to new insights in the roles of several reactions involved in nitrogen metabolism. The chapter discusses the localization of these reactions and the advantage of such compartmentation to an organism and emphasizes leaf metabolism and the roles of chloroplasts, peroxisomes, and mitochondria in utilizing carbon and nitrogen intermediates involved in photorespiration. NO 3 - and NH 4 + are the common forms of nitrogen added to the soil. NO 3 - taken up from the soil is either converted to NH 4 + in the roots by the action of nitrate (NR) and nitrite (NiR) reductases or is transported to the shoot before assimilation. The conversion of NO 3 - to NH 4 + involves three proteins that are induced by NO 3 - : (1) a permease that permits the selective uptake of NO 3 - from the medium (soil), (2) NR reductase, and (3) NiR reductase. Depending on the level of NO 3 - administered to a system, NO 3 - can be stored in the root, transferred to the leaf to be stored in the vacuole, or be reduced in the roots or leaves. End products of NO 3 - assimilation—NH 4 + and amino acids— inhibit the induction of NR in Neurospora crassa.

Enzymes of nitrogen assimilation in maize roots

The enzymes nitrate reductase (NR), glutamate dehydrogenase (GDH), glutamate synthase (GOGAT), glutamine synthetase (GS) and asparagine synthetase (AS) have been assayed in various regions along the seedling root ofZea mays L. In the intact attached root and calculated on a protein basis NR, GOGAT, and GS are found to have slightly higher specific activities in the apical 5 mm than in more mature regions of the root. GDH and AS, on the other hand, are much more active in extracts prepared from mature regions of the root than in the apical region. In excised root tips incubated in the presence of NH4+ and NO3− there was a marked increase in GDH and AS, and a slight decrease in GOGAT and GS. Additions of NO3− are required for NR activity but neither NO3− nor NH4+ additions altered the activity levels of the other four enzymes. Additions of glucose to the medium inhibited the development of AS and GDH activities and resulted in higher activity levels of NR, GS and GOGAT. Glucose additions also enhanced the incorporation of acetate-14C and leucine-14C into protein. Additions of cycloheximide inhibit the development of NR, AS and GDH activities and also the incorporation of acetate-14C and leucine into protein.

Efficiency of Nitrogen Utilization in C3 and C4 Cereals

Addition of nitrogen leads to increased dry matter accumulation in vegetative plant parts and to increased final yields in cereal crops (Hageman and Lambert, 1988). The efficiency with which nitrogen is used varies with plant species and with environmental conditions. For example, plants that possess a C4 pattem of photosynthesis have, in addition to a superior method for trapping COz from the atmosphere, a greater nitrogen use efficiency (g dry matter gain per mg nitrogen utilized) than do C3 plants (Brown, 1978). Although there are many differences in the metabolism of C3 and C4 plants, the major difference between these two pattems of photosynthesis is the contribution of photorespiration to both carbon and nitrogen metabolism. When photorespiration is reduced in C3 plants either by increasing ambient levels of COz or reducing levels of 02, both the yield (vegetative dry matter) and nitrogen use efficiency are enhanced (Evans, 1989). As indicated in Table I, this effect is apparent in wheat, a C3 cereal, but not in maize, a C4 cereal (Hocking and Meyer, 1991).

Regulation of the Accumulation and Reduction of Nitrate by Nitrogen and Carbon Metabolites in Maize Seedlings

The accumulation and reduction of nitrate in the presence of the nitrogen metabolites asparagine (Asn) and glutamine (Gln) and the carbon metabolite sucrose (Suc) were examined in maize (Zea mays L.) seedlings in an attempt to separate their effects on the nitrate uptake system and the nitrate reduction system. After 8 h of exposure to nitrate in the presence of 1 mM Asn, tissue nitrate accumulation was reduced at 250 [mu]M external nitrate, but not at 5 mM Asn. The induction of nitrate reductase (NR) activity was reduced at both external nitrate concentrations. In the presence of 1 mM Gln or 1% Suc, tissue nitrate concentration was not significantly altered, but the induction of root NR activity was reduced or enhanced, respectively. The induction of root nitrite reductase (NiR) activity was also reduced in the presence of Asn or Gln and enhanced in the presence of Suc. Transcript levels of NR and NiR in roots were reduced in the presence of the amides and enhanced in the presence of Suc. When Suc was present in combination with either amide, there was complete relief from the inhibition of NiR transcription observed in the presence of amide alone. In the case of NR, however, this relief from inhibition was negligible. The inhibition of the induction of NR and NiR activities in the presence of Gln and Asn is a direct effect and is not the result of altered nitrate uptake in the presence of these metabolites.

Intracellular distribution of enzymes associated with nitrogen assimilation in roots

The cellular distribution of enzymes involved in nitrogen assimilation: nitrate reductase (EC 1.6.6.2), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) has been studied in the roots of five plants: maize (Zea mays L. hybrid W 64A x W 182E), rice (Oryza sativa L. cv. Delta), bean (Phaseolus vulgaris L. cv. Contender), pea (Pisum sativum L. cv. Demi-nain), and barley (Hordeum vulgare L.). Initially, cell organelles were separated from soluble proteins by differential centrifugation. Cell organelles were also subjected to sucrose density gradients. The results obtained by these two methods indicate that nitrite reductase and glutamate synthase are localized in plastids, nitrate reductase and glutamine synthetase are present in the cytosol, and glutamate dehydrogenase is a mitochondrial enzyme.

Metabolic Regulation of Ammonium uptake and Assimilation

Plants take up inorganic nitrogen as NO3 or NH4 ions, or as N2 fixed by symbiotic bacteria. The subjects of nitrate uptake as well as symbiotic N2 fixation will be discussed in chapter 1 and 4, respectively. N-containing molecules as amino acids, proteins, chlorophyll and nucleic acids, are all products of NH4 assimilation. Aspects of the synthesis and regulation of amino acids and amides (Lam et al. 1996), the interaction of sucrose, amides and NO3 with the assimilation of NH4 (Sivasankar and Oaks 1996) and the role of NO3 as a signal regulating metabolism and growth (Crawford and Glass 1998, Stitt et al. 2002) have already been discussed in detail, but will be considered in the light of current developments in our understanding of NH4 assimilation. This chapter will focus on NH4 uptake, its assimilation into glutamine and glutamate and its metabolic regulation. Recent progress in molecular biology, gene manipulation, and post-genomic research on NH4 metabolism will be described.

Regulation of nitrate reductase during early seedling growth : a role for asparagine and glutamine

Growth systems that either permit (wet system) or prevent (dry system) the hydrolysis of endosperm reserves in maize (Zea mays) seedlings were developed to study the effect of endosperm reserves on the acquisition of external nitrogen. Three-day-old seedlings treated with 5 mM KNO3 for 24 h had higher levels of nitrate reductase (NR) activity and protein in shoot and root tissues in the dry relative to the wet system. This suggests that the induction of NR is sensitive to products of hydrolysis of endosperm reserves. Asparagine (1 mM) or glutamine (1 mM), potential products of that hydrolysis, inhibited the induction of NADH-dependent root NR in the dry system by about 70%. The inhibition of the induction of NR activity in the wet system was only about 35%, suggesting that the enzyme in the wet system was already partially repressed at 3 d. At 5 d, when asparagine and glutamine levels in the plant tissue had decreased, the induction of root NR activity was inhibited to a similar extent in the two growth systems by amide additions. The shoot enzyme was less sensitive to amide additions, and 10 mM concentrations of either amide was required for a 65% inhibition.

Development of Endopeptidase Activities in Maize (Zea mays L.) Endosperms

An activity stain was used after native polyacrylamide gel electrophoresis, and at least 17 different endopeptidase activities were detected in maize (Zea mays L.) endosperm extracts prepared during the first 6 d after imbibition. The enzymes detected were classified into four groups based on their time of appearance and on their mobility in polyacrylamide gels. The first group, which included two enzymes present in dry endosperms, disappeared soon after imbibition. The second group, comprising five activity bands, appeared during the first 2 to 3 d after imbibition and then disappeared. The third set of enzymes increased continuously throughout the experimental period. The fourth group appeared after d 3 and remained at a constant level after that time. The endopeptidase activities were characterized by the effect of specific inhibitors on their activities. The two enzymes of the first group are metalloendopeptidases based on their sensitivity to ethylenediaminetetracetate (EDTA). Enzymes of the second, third, and fourth groups are sulfhydryl-endopeptidases as judged by their sensitivity to antipain, chymostatin, leupeptin, and E-64 and by their requirement for 2-mercaptoethanol. Pepstatin, phenylmethylsulfonyl fluoride, or EDTA had no effect on these enzymes. Many of the second, third, and fourth group enzymes cleaved [alpha]-zein-rich proteins as well as such easily obtained proteins as gelatin (used in our standard assay) and hemoglobin. The second group had a high affinity for [gamma]-zein, whereas none of the bands in the fourth group of enzymes cleaved this type of zein. The two metalloenzymes of the first group cleaved neither [alpha]- nor [gamma]-zeins.

The regulation of nitrate reductase in suspension cultures of soybean cells.

Abstract 1. 1. When soybean cells (Glycine max L. variety Mandarin) are transferred to fresh media increases in nitrate reductase (NADH:nitrate oxidoreductase, EC 1.6.6.1) occur within 3 h. A peak in activity is reached between 60–80 h. 2. 2. Cells grown in ammonium citrate or glutamine and transferred to fresh media do not develop a nitrate reductase activity when NO3− is added to the system.

Enzymes of asparagine synthesis in maize roots.

An asparagine synthetase which is active with either glutamine or NH4+has been found in maize (Zea mays L.) roots. Unlike the enzyme obtained from legume cotyledons, the maize-root enzyme is only slightly more efficient with glutamine (Km, 1.0 mM) than with NH4+(Km, 2.0–3.0 mM). The activity of this enzyme is higher in the mature root than in the root-tip region, i.e. root cells develop a capacity to make asparagine from glutamine or NH4+as they mature. β-Cyanoalanine synthetase is also present in maize roots. The apparent Km for cysteine is 2.6 mM and for cyanide is 0.57 mM. The enzyme is more active in the root tip than in mature root tissue. Thus, if asparagine were made in the root tip, the cyanide pathway could represent the mechanism of synthesis. It is our contention, however, that this potential is not realized under normal conditions because 14C-experiments performed previously have indicated a limited availability of both CN and cysteine in the maize root.