Light-Independent Nitrogen Assimilation in Plant Leaves: Nitrate Incorporation into Glutamine, Glutamate, Aspartate, and Asparagine Traced by 15N

Although the nitrate assimilation into amino acids in photosynthetic leaf tissues is active under the light, the studies during 1950s and 1970s in the dark nitrate assimilation provided fragmental and variable activities, and the mechanism of reductant supply to nitrate assimilation in darkness remained unclear. 15N tracing experiments unraveled the assimilatory mechanism of nitrogen from nitrate into amino acids in the light and in darkness by the reactions of nitrate and nitrite reductases, glutamine synthetase, glutamate synthase, aspartate aminotransferase, and asparagine synthetase. Nitrogen assimilation in illuminated leaves and non-photosynthetic roots occurs either in the redundant way or in the specific manner regarding the isoforms of nitrogen assimilatory enzymes in their cellular compartments. The electron supplying systems necessary to the enzymatic reactions share in part a similar electron donor system at the expense of carbohydrates in both leaves and roots, but also distinct reducing systems regarding the reactions of Fd-nitrite reductase and Fd-glutamate synthase in the photosynthetic and non-photosynthetic organs.


Introduction
Plants use inorganic nitrogen present in the soil for their growth mainly in the form of nitrate (NO 3 − ). Following the absorption through the roots, the most oxidized form of NO 3 − (+6) is reduced to organic forms (-2) such as amino acids prior to their incorporation into proteins, nucleotides, and chlorophylls. Although plants can assimilate NO 3 − to amino acids in both the photosynthetic leaves and non-photosynthetic roots, the major sites of nitrate assimilation are green shoots where energy (ATP), reductant (electrons) and organic skeletons are produced by photosynthetic reactions using solar energy. In addition, the nitrate assimilation in the leaves takes place in the night, i.e., by using storage carbohydrates [1]. However, the activities of nitrate assimilation in darkness measured from the 1950s to 1970s were fragmental and variable. Here, we present physiological aspects of light-independent nitrate-to-asparagine assimilation in the leaves by referring to the light-independent nitrate assimilation in the non-photosynthetic roots at the expense of carbohydrates, supplied by transport from the shoots, as reviewed recently [2].

15 N Tracing Analysis of Dark Nitrate Assimilation into Amino Acids in Leaves
Direct evidence of dark nitrate assimilation into amino acids in the leaves was obtained by incubating the leaf tissues with 15 N-nitrate in darkness. Delwiche [3] fed detached immature leaves of tobacco (Nicotiana tabacum) with 15 N-KNO 3 solution (15 atom % excess 15 N) for 24 h via petioles in darkness, and detected 15 N-labelling in ammonia plus amide-N, and in ammonia fraction by 2.89 and 0.61 atom % excess 15 N, respectively. Mendel and Visser [4] conducted a short incubation (30 min) of tomato leaf discs with 15 N-KNO 3 (14 atom % excess 15 N) in the dark, and detected 15 N-labelling in free ammonia fraction in duplicated samples by 2.13 and 2.42 atom % excess 15 N. Canvin and Atkins [5] incubated leaf segments of wheat (Triticum aestivum L.) and corn (Zea mays L.) with 15 N-NaNO 3 solution (95 atom % 15 N) for 15 and 30 min in darkness. After 15 min, they detected little 15 N enrichment in the soluble amino acid fraction in darkness compared with the high 15 N labelled amino acids in the light and concluded that the nitrate assimilation was strictly dependent on the light.
Shortly after the findings of 15 N-labelled amino acids in the roots of rice (Oryza sativa L.) seedlings by feeding with 15 N-ammonium [6] and 15 Table 1. In these studies, 15 N-labelling of individual amino acids was determined by a combination of their separation on two-dimension thin-layer chromatography and 15 N enrichment by emission optical spectrometry, as first described by Yoneyama and Kumazawa [6]. Dark 15 N-labelling in the amino-N of glutamine, glutamate, and aspartate was less than that in the light, while the dark 15 N-labelling of the amide-N of glutamine was higher than that in the light irrespective of the feeding with 15  In the 1970s, a simple in vivo assay of nitrate reductase activity was widely employed using leaf segments without extraction of enzymes. In this technique, it was assumed that nitrite, nitrate reductase reaction product, was barely assimilated when the assay was carried out in the dark [10][11][12]. In vivo nitrate reductase assays were conducted using 15 N-NaNO 3 to measure its reduction to nitrite and the assimilation into amino acids under aerobic or anaerobic conditions [13]. Nitrite production was active under anaerobic conditions while 15 N incorporation into amino acids was intensive under aerobic conditions than under anaerobic conditions ( Table 2). The 15 N-labelling experiments were carried out using green and white chlorophyll-less leaves of albino mutant seedlings of rice, which were produced by chemical mutation. It was shown that the white leaves fed with 15 NO 2 − in darkness [14] had lower 15 N-enrichments (atom % excess 15 N) in glutamine, glutamate, and aspartate than green leaves, but a large 15 N accumulation occurred in glutamine and particularly in asparagine in the white leaves (Table 3). In vitro activities of both nitrate reductase and nitrite reductase were detected in the leaf extracts of normal and chlorophyll-less leaves of albino mutant seedlings of barley (Hordeum vulgare L.), although their specific activities were less in the chlorophyll-less leaves than in the normal leaves [15].

Nitrate Reduction to Ammonia
Nitrate is not only an essential nutrient but also a signaling molecule of cellular events in response to its fluctuating availability in both space and time. Nitrate is taken up into the roots by the nitrate transporters on the plasma membrane and regulates lateral root developments [16]. Transported in the xylem, nitrate is distributed within the plant by the nitrate transporters located in the shoots, leaves, flowers and seeds, and triggers expression of nitrate-responding genes, leaf development, and seed germination [17][18][19]. Nitrate reductase in the cytosol (Equation (1)) and ferredoxin (Fd)-dependent nitrite reductase in the chloroplasts/plastids (Equation (2)) catalyze the sequential reactions of nitrate reduction to nitrite and nitrite to ammonia, respectively ( Figure 1).

Glutamine Synthesis and Metabolism to Glutamate and Asparagine in Leaves
Up to 1974, it was generally accepted that ammonia assimilation is catalyzed by ammoniainducible glutamate dehydrogenase (GDH, EC 1.4.1.2), which catalyzes a reversible amination of 2oxoglutarate by ammonia generating L-glutamate and its conversion to ammonia and 2-oxoglutarate. Under conditions of ammonia excess, ammonia was assimilated into glutamine by glutamine synthetase (GS or L-glutamate:ammonia ligase (ADP), EC 6.3.1.2, Equation (3)), and under more Ferredoxin-dependent NiR (Fd-NiR), localized in the chloroplasts/plastids [25,[27][28][29], utilizes six electrons from photo-reduced ferredoxin (Fd) as the electron donor to reduce nitrite to ammonia. Fd-NiR has two prosthetic groups: a siroheme and an iron-sulfur cluster, linked by one of four cysteine residues of the iron-sulfur cluster [26,30]. The high NiR activity, assayed by the disappearance of nitrite or formation of ammonia, was detected in the presence of strong reducing dyes methyl viologen or benzyl viologen reduced chemically by dithionite [31,32]. NiR activity in vitro was found in the presence of Fd reduced by ferredoxin: NADP + oxidoreductase (FNR, EC 1.18.1.2) depending on NADPH, which was generated by a diaphorase containing glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) [33]. The in vitro titration analysis showed that the NiR may make a complex close to 1:1 with reduced Fd for the efficient electron transfer [34].

Glutamine Synthesis and Metabolism to Glutamate and Asparagine in Leaves
Up to 1974, it was generally accepted that ammonia assimilation is catalyzed by ammonia-inducible glutamate dehydrogenase (GDH, EC 1.4.1.2), which catalyzes a reversible amination of 2-oxoglutarate by ammonia generating L-glutamate and its conversion to ammonia and 2-oxoglutarate. Under conditions of ammonia excess, ammonia was assimilated into glutamine by glutamine synthetase (GS or L-glutamate:ammonia ligase (ADP), EC 6.3.1.2, Equation (3)), and under more excessive levels of ammonia and glutamine, asparagine served as an storage compound of nitrogen via the catalysis by glutamine-dependent asparagine synthetase (AS, EC 6.3.5.4).
Glutamine synthetase (GS, EC 6.3.1.2) Fd-glutamate synthase (Fd-GOGAT, EC 1.4.7.1) 2e 15 N tracing studies confirmed an in vivo operation of GS2/Fd-GOGAT cycle for the nitrogen assimilation from nitrate, nitrite, and ammonia into amino acids in the light or in darkness (see Table 1). The GS occurs in two forms, cytosolic GS1 and plastidial GS2, in both leaves and roots with different ratio according to plants [43]. The cytosolic GS1 in the senescent leaves may function to assimilate a high level of ammonia during nitrogen remobilization [44,45]. Two forms of GOGAT, Fd-GOGAT and NADH-GOGAT (EC 1.4.1.14), are distinguished in leaves and roots of different plant species. The Fd-GOGAT in vitro activity was found active in the light-grown mature leaves, and the isolated chloroplasts showed a high activity of Fd-GOGAT in the light and low activity in darkness [46,47]. The enhancement of Fd-GOGAT activity and the Fd-GOGAT protein level during the greening of the etiolated plants [48] via a reversible red/far-red reaction provided evidence for a regulation mediated by the phytochromes [39,49]. Two molecules of glutamate are formed from glutamine and 2-OG through the intramolecular reactions of NH 2 -releasing glutaminase and 2-OG transamidation with -NH 2 using reduced Fd (Equation (4)) [50][51][52].
In the cytosol of leaf cells, the amide-N of glutamine is transferred to aspartic acid to form asparagine by asparagine synthetase utilizing ATP (AS, Equation (5)). AS could use both ammonia and glutamine-amide while glutamine is a preferred amide donor. Km for glutamine (0.04-1.0 mM) was 40-fold lower than for ammonium ion [53]. The accumulation of asparagine [54] and AS-mRNA of Class I ASN genes [55][56][57] was enhanced in darkness. This is consistent with a carbon supply by an anaplerotic reaction of cytosolic phosphoenolpyruvate carboxylase [58,59]. Oxaloacetate thus formed is transaminated with glutamate by aspartate aminotransferase to aspartate, substrate of AS (AspAT, Equation (6)).
Asparagine synthetase (AS, EC 6.3.5.4) L-Glutamine + L-Aspartate + ATP → L-Asparagine + L-Glutamate + AMP + PPi Aspartate aminotransferase (AspAT, EC 2.6.1.1) L-Glutamate + Oxaloacetic acid ↔ L-Aspartate + 2-Oxoglutarate (6) AspAT in plants exists as isoforms, which are located in different subcellular compartments. The ASP2 mRNA for cytosolic AspAT2 in Arabidopsis was most abundantly expressed in root tissue and accumulated at higher levels in illuminated leaves and dark-adapted leaves [60], indicating that AspAT2 may be involved in synthesizing aspartate pool for asparagine synthesis by AS2 in dark-adapted plants. Figure 1 depicts the scheme of nitrate assimilation in darkness in the leaf cells. Nitrate delivered from the xylem is reduced to NO 2 − by the cytosolic NR using NADH from glycolysis. Nitrite is diffused into the chloroplasts [61] and reduced to ammonia by Fd-NiR. The ammonium is assimilated to glutamine by chloroplast-localized GS2 using ATP imported [62] and then to glutamate by Fd-GOGAT using 2-OG produced via cytosolic NADP-dependent isocitrate dehydrogenase (NADPH-ICDH, EC 1.  [66]. Glutamate and glutamine in the chloroplasts are released to the cytosol and glutamate is metabolized to aspartate by cytosolic AspAT2. Aspartate thus produced is combined with the amide of glutamine, forming asparagine by leaf cytosolic AS2. The nitrogen assimilation pathway from nitrate to asparagine catalyzed by Fd-NiR, GS, Fd-GOGAT, AspAT and AS in darkness in the green leaves was in line with 15 NO 2 − tracing data shown in Table 3. In chlorophyll-less white leaves, which contained proplastids [14], the activity of AS2 (asparagine formation) might be higher than the Fd-GOGAT activity (glutamate formation), suggesting a low level of Fd-GOGAT without light [48].
The previous investigations of 15 NO 3 − and 15 NO 2 − reduction and assimilation into amino acids in leaf segments in darkness showed high magnitudes than in the light in tobacco [3], tomato [4], sunflower [8], and rice [14], while negligible in wheat and maize leaves [5]. Such difference in magnitudes may be derived from an availability of electron in NADPH-FNR-Fd systems under changing electron donating system in darkness (Figure 2).  Under anaerobic condition, the nitrite accumulation was large and glutamine quantity became small ( Table 2). These results were caused by the elevated level of NO3 − -reducing NADH and the shortage of NADPH [12,86]: The NADPH deficiency may reduce nitrite-to-ammonia reduction and induce the glutaminase activity of GOGAT, causing disappearance of glutamine [85]. Expression studies determined the level of Fd and FNR in the plants deprived of light by continuous or prolonged darkness. This dark stress declined the photosynthetic FNR subforms (LFNRI and LFNRII) at both mRNA and protein levels at the base section of wheat leaf in the presence of nitrate [87], and leaf-type Fds (Fd I and Fd II) in maize leaves [68], suggesting a less efficient contribution of NADPH-FNR-Fd system necessary to the reactions of Fd-NiR and Fd-GOGAT. Exposure of Arabidopsis to environmental stress such as extended high light (120 h at approximately 500 micromole photons m 2 sec 1 ) resulted in a gradual decrease of AtFd2 (At1g60950) in its mRNA (to 10% of the WT level) and protein (to 13%) [88]. Down-regulation or mutation of Fd in Arabidopsis [89] and potato (Solanum Under anaerobic condition, the nitrite accumulation was large and glutamine quantity became small (Table 2). These results were caused by the elevated level of NO 3 − -reducing NADH and the shortage of NADPH [12,86]: The NADPH deficiency may reduce nitrite-to-ammonia reduction and induce the glutaminase activity of GOGAT, causing disappearance of glutamine [85]. Expression studies determined the level of Fd and FNR in the plants deprived of light by continuous or prolonged darkness.
This dark stress declined the photosynthetic FNR subforms (LFNRI and LFNRII) at both mRNA and protein levels at the base section of wheat leaf in the presence of nitrate [87], and leaf-type Fds (Fd I and Fd II) in maize leaves [68], suggesting a less efficient contribution of NADPH-FNR-Fd system necessary to the reactions of Fd-NiR and Fd-GOGAT. Exposure of Arabidopsis to environmental stress such as extended high light (120 h at approximately 500 micromole photons m 2 sec 1 ) resulted in a gradual decrease of AtFd2 (At1g60950) in its mRNA (to 10% of the WT level) and protein (to 13%) [88]. Down-regulation or mutation of Fd in Arabidopsis [89] and potato (Solanum tuberosum) [90] caused an inactivated photosynthesis and inhibited plant growth.

Reductant Supply to the Fd-Dependent GOGAT
Ammonia produced by NiR in the plastids is assimilated into glutamine by GS2 using energy (ATP) from mitochondria [62,79]. The glutamine amide-N is transferred to 2-OG, yielding two molecules of glutamate takes place in the chloroplasts/plastids by Fd-GOGAT [36,52,91]. Fd-GOGAT is a flavin and iron-sulfur-containing protein. The isopotential of these chromophore and cluster were reported to have Em of −225 ± 10 mV in the enzyme from spinach leaves [92]. NADPH, generated by the oxidative pentose phosphate pathway, was also shown to be a primary electron donor for the reactions of Fd-GOGAT in darkness [80].
A 15 N-tracing study in sunflower leaf discs (Table 1) showed that the activity of glutamine amide-N transfer to 2-oxoglutamate forming glutamate in darkness was less active than in the light, where PSI supplied electrons to Fd (see Figure 2). The activity of Fd-GOGAT in vegetable leaves was the major regulating step of nitrate assimilation in the whole plant [1].

Conclusions
Leaves represent a major site of primary nitrogen assimilation in concert with roots, photorespiratory NH 4 + re-fixation, and translocation of nitrogen within the plant. 15 N-tracing studies with leaves demonstrated that nitrate was reduced to ammonia and assimilated into glutamine, glutamate, aspartate, and asparagine in the light and in darkness. In the present review, we examined that the reductive incorporation of nitrate into amino acids occurs in darkness in the leaves through the isoforms of NR, NiR, GS, Fd-GOGAT, AspAT, and AS. To provide reducing equivalents to the NiR and Fd-GOGAT reactions in the dark, single leaf contains the photosynthetic form of FNR and Fd and heterotrophic form of FNR and Fd, indicating inter-connected electron supply systems in the light and in darkness. It remains to dissect the operation mechanism of electron donation systems in distinct types of photosynthetic cells and heterotrophic cells of a leaf.