The cytosolic glutamine synthetase GLN1;2 plays a role in the control of plant growth and ammonium homeostasis in Arabidopsis rosettes when nitrate supply is not limiting

Glutamine synthetase (EC 6.3.1.2) is a key enzyme of ammonium assimilation and recycling in plants where it catalyses the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, five GLN1 genes encode GS1 isoforms. GLN1;2 is the most highly expressed in leaves and is over-expressed in roots by ammonium supply and in rosettes by ample nitrate supply compared with limiting nitrate supply. It is shown here that the GLN1;2 promoter is mainly active in the minor veins of leaves and flowers and, to a lower extent, in the parenchyma of mature leaves. Cytoimmunochemistry reveals that the GLN1;2 protein is present in the companion cells. The role of GLN1;2 was determined by examining the physiology of gln1;2 knockout mutants. Mutants displayed lower glutamine synthetase activity, higher ammonium concentration, and reduced rosette biomass compared with the wild type (WT) under ample nitrate supply only. No difference between mutant and WT can be detected under limiting nitrate conditions. Despite total amino acid concentration was increased in the old leaves of mutants at high nitrate, no significant difference in nitrogen remobilization can be detected using (15)N tracing. Growing plants in vitro with ammonium or nitrate as the sole nitrogen source allowed us to confirm that GLN1;2 is induced by ammonium in roots and to observe that gln1;2 mutants displayed, under such conditions, longer root hair and smaller rosette phenotypes in ammonium. Altogether the results suggest that GLN1;2 is essential for nitrogen assimilation under ample nitrate supply and for ammonium detoxification.     

NADH‐dependent glutamate synthase plays a crucial role in assimilating ammonium in the Arabidopsis root

Plant roots under nitrogen deficient conditions with access to both ammonium and nitrate ions, will take up ammonium first. This preference for ammonium rather than nitrate emphasizes the importance of ammonium assimilation machinery in roots. Glutamine synthetase (GS) and glutamate synthase (GOGAT) catalyze the conversion of ammonium and 2‐oxoglutarate to glutamine and glutamate. Higher plants have two GOGAT species, ferredoxin‐dependent glutamate synthase (Fd‐GOGAT) and nicotinamide adenine dinucleotide (NADH)‐GOGAT. While Fd‐GOGAT participates in the assimilation of ammonium, which is derived from photorespiration in leaves, NADH‐GOGAT is highly expressed in roots and its importance needs to be elucidated. While ammonium as a minor nitrogen form in most soils is directly taken up, nitrate as the major nitrogen source needs to be converted to ammonium prior to uptake. The aim of this study was to investigate and quantify the contribution of NADH‐GOGAT to the ammonium assimilation in Arabidopsis (Arabidopsis thaliana Columbia) roots. Quantitative real‐time polymerase chain reaction (PCR) and protein gel blot analysis showed an accumulation of NADH‐GOGAT in response to ammonium supplied to the roots. In addition the localization of NADH‐GOGAT and Fd‐GOGAT did not fully overlap. Promoter–β‐glucuronidase (GUS) fusion analysis and immunohistochemistry showed that NADH‐GOGAT was highly accumulated in non‐green tissue like vascular bundles, shoot apical meristem, pollen, stigma and roots. Reverse genetic approaches suggested a reduction in glutamate production and biomass accumulation in NADH‐GOGAT transfer DNA (T‐DNA) insertion lines under normal CO₂ condition. The data emphasize the importance of NADH‐GOGAT in the ammonium assimilation in Arabidopsis roots.     

Nitrogen Assimilation in Barley (Hordeum vulgare L. cv. Mazurka) in Response to Nitrate and Ammonium Nutrition

Barley plants (Hordeum vulgare L. cv. Mazurka) were grown inaerated solution cultures with 2 mM or 8 mM inorganic nitrogensupplied as nitrate alone, ammonium alone or 1:1 nitrate+ammonium.Activities of the principal inorganic nitrogen assimilatoryenzymes and nitrogen transport were measured. Activities ofnitrate and nitrite reductases, glutamine synthetase and glutamatesynthase were greater in leaves than in roots but glutamatedehydrogenase was most active in roots. Only nitrate and nitritereductases changed notably (4–10 times) in response tothe different nitrogen treatments. Nitrate reductase appearedto be rate-limiting for nitrate assimilation to glutamate inroots and also in leaves, where its total in vitro activitywas closely related to nitrate flux in the xylem sap and wasslightly in excess of that needed to reduce the transportednitrate. Xylem nitrate concentration was 13 times greater thanthat in the nutrient solution. Ammonium nitrogen was assimilatedalmost completely in the roots and the small amount releasedinto the xylem sap was similar for the nitrate and the ammoniumtreatments. The presence of ammonium in the nutrient decreasedboth export of nitrate to the xylem and its accumulation inleaves and roots. Nitrate was stored in stem bases and was releasedto the xylem and thence to the leaves during nitrogen starvation.In these experiments, ammonium was assimilated principally inthe roots and nitrate in the leaves. Any advantage of this divisionof function may depend partly on total conversion of inorganicnitrogen to amino acids when nitrate and ammonium are givenin optimal concentrations. Hordeum vulgare L., barley, nitrate, ammonium, nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, glutamate dehydrogenase, nitrogen transport     

Glutamine transport and feedback regulation of nitrate reductase activity in barley roots leads to changes in cytosolic nitrate pools

The size of tissue amino acid pools in plants may indicate nitrogen status and provide a signal that can regulate nitrate uptake and assimilation. The effects of treating barley roots with glutamine have been examined, first to identify the transport system for the uptake of the amino acid and then to measure root NR activity and cellular pools of nitrate. Treating N replete roots with glutamine elicited a change in the cell membrane potential and the size of this response was concentration dependent. In addition, the size of the electrical change depended on the previous exposures of the root to glutamine and was lost after a few cycles of treatment. Whole root tissue pools of glutamine and phenylalanine increased when roots were incubated in a nutrient solution containing 10 mM nitrate and 1 mM glutamine. Treating roots with 1 mM glutamine increased cytosolic nitrate activity from 3 mM to 7 mM and this change peaked after 2 h of treatment. Parallel measurements of root nitrate reductase activity during treatment with 1 mM glutamine showed a decrease. These measurements provide evidence for feedback regulation on NR activity that result in changes in cytosolic nitrate activity. After 6 h in glutamine both root NR activity and cytosolic nitrate activity returned to pretreatment values, while tissue concentrations of glutamine and phenylalanine remained elevated. The data are discussed in terms of the mechanisms that are most likely to be responsible for the changes in cytosolic nitrate.     

Tobacco plants that lack expression of functional nitrate reductase in roots show changes in growth rates and metabolite accumulation.

When tobacco is provided with a high nitrate supply, only a small amount of the nitrate taken up by the roots is immediately assimilated inside the roots, while the majority is transported to the leaves where it is reduced to ammonium. To elucidate the importance of root nitrate assimilation, tobacco plants have been engineered that showed no detectable nitrate reductase activity in the roots. These plants expressed the nitrate reductase structural gene nia2 under control of the leaf-specific potato promoter ST-LS1 in the nitrate reductase-mutant Nia30 of Nicotiana tabacum. Homozygous T2-transformants grown in sand or hydroponics with 5.1 mM nitrate had approximately 55-70% of wild-type nitrate reductase acivity in leaves, but lacked nitrate reductase acivity in roots. These plants showed a retarded growth as compared with wild-type plants. The activation state of nitrate reductase was unchanged; however, diurnal variation of nitrate reductase acivity was not as pronounced as in wild-type plants. The transformants had higher levels of nitrate in the leaves and reduced amounts of glutamine both in leaves and roots, while roots showed higher levels of hexoses (3-fold) and sucrose (10-fold). It may be concluded that the loss of nitrate reductase acivity in the roots changes the allocation of reduced nitrogen compounds and sugars in the plant. These plants will be a useful tool for laboratories studying nitrate assimilation and its interactions with carbon metabolism.     

Nitrogen uptake and metabolism in Populus x canescens as affected by salinity

External salinization can affect different steps of nitrogen (N) metabolism (ion uptake, N assimilation, and amino acid and protein synthesis) depending on the inorganic N source. Here, we assessed the net uptake of N supplied as nitrate or ammonium and N assimilation (combining metabolite analyses with molecular biological approaches) in grey poplar (Populus x canescens) plants grown under saline (75 mM NaCl) and control conditions. The specific (micromol N g(-1) dry weight fine roots h(-1)) and total plant (micromol N per plant h(-1)) N net uptake rates, total plant N content, total plant biomass and total leaf protein concentration were reduced under saline conditions when plants were supplied with ammonium. In both nutritional groups, salt treatment caused pronounced accumulation of soluble N compounds in the leaves. The mRNAs of genes coding for enzymes catalyzing rate-limiting steps of both proline synthesis and degradation (delta-1-pyrroline-5-carboxylate synthase and proline dehydrogenase) as well as for NADH-dependent glutamate synthase were accumulated under saline conditions. Whereas under control conditions the plant N status seemed to be superior when ammonium was supplied, the N balance of ammonium-fed plants was more severely affected by salt stress than that of plants supplied with nitrate. Possible metabolic implications of stress-related accumulation of particular amino acids are discussed.     

Diurnal changes in nitrogen assimilation of tobacco roots.

To gain an insight into the diurnal changes of nitrogen assimilation in roots the in vitro activities of cytosolic and plasma membrane-bound nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.7.7.1) and cytosolic and plastidic glutamine synthetase (EC 6.3.1.2) were studied. Simultaneously, changes in the contents of total protein, nitrate, nitrite, and ammonium were followed. Roots of intact tobacco plants (Nicotiana tabacum cv. Samsun) were extracted every 3 h during a diurnal cycle. Nitrate reductase, nitrite reductase and glutamine synthetase were active throughout the day-night cycle. Two temporarily distinct peaks of nitrate reductase were detected: during the day a peak of soluble nitrate reductase in the cytosol, in the dark phase a peak of plasma membrane-bound nitrate reductase in the apoplast. The total activities of nitrate reduction were similar by day and night. High activities of nitrite reductase prevented the accumulation of toxic amounts of nitrite throughout the entire diurnal cycle. The resulting ammonium was assimilated by cytosolic glutamine synthetase whose two activity peaks, one in the light period and one in the dark, closely followed those of nitrate reductase. The contribution of plastidic glutamine synthetase was negligible. These results strongly indicate that nitrate assimilation in roots takes place at similar rates day and night and is thus differently regulated from that in leaves.     

Changes in the activities of nitrogen assimilation enzymes ofLolium perenne L. during regrowth after cutting

The activities of key enzymes involved in N assimilation were investigated after defoliation of 6-week-old ryegrass plants grown in water culture conditions. In a first experiment, nitrate reductase, glutamine synthetase and glutamate dehydrogenase activities were measured in roots, stubble and leaves on the day of cutting and at 7-day intervals over the following 5-week period of regrowth. Ammonia assimilation enzymes showed little change whereas the nitrate reductase activity sharply decreased 2 weeks after clipping. In a second experiment, the nitrate reductase activity was measured at 2- or 3-day intervals 1 week before and 3 weeks after clipping.In vivo andin vitro assays both showed an increasing activity in leaves up to 8 days after cutting while root activity decreased. The opposite changes then occurred and both organs recovered their initial nitrate reductase activity levels after 12–14 days of regrowth. These fluctuations in nitrate reductase activity were considered to be related to the capacity for C assimilation and the nitrate availability.     

Sink control of nitrogen uptake and assimilation during regrowth after-cutting of ryegrass (Lolium perenne L.)

Abstract. The ¹⁵N isotope was used to compare the uptake and the assimilation of NH₄⁺ and NO₃ nitrogen in ryegrass ( Lolium perenne L.) during regrowth after cutting. Uptake of nitrate-N, expressed per plant, was at all times greater than ammonium-N uptake and assimilation decreased in roots and stubble while its assimilation was maintained at a high level in leaves. It has been suggested that ammonium assimilation is directly related to the availability of carbohydrates in the sink organ (leaves) resulting from their remobilization from the source organs (roots and stubble). Nitrate reduction decreased in all organs, while the uptake of NO₃ was still high. After this first period of regrowth, nitrogen assimilation both from nitrate and ammonium increased in all the plants. Nitrate reduction capacity (expressed in μg NO₃-N reduced per g D.W. per d) is 7.5 and 22.5 times greater in leaves than in stubble and roots, respectively. Therefore, nitrogen assimilation in stubble and particularly in roots was mainly dependent on ammonium nitrogen.     

Effect of the gln-1b mutation on nitrogen metabolite repression in Neurospora crassa.

In Neurospora crassa, synthesis of the enzymes of nitrate assimilation, nitrate reductase and nitrite reductase, was repressed by the presence of ammonium, glutamate, or glutamine. This phenomenon was a manifestation of the regulatory process termed nitrogen metabolite repression whereby alternative pathways of nitrogen acquisition are not expressed in cells enjoying nitrogen sufficiency. However, the glutamine synthetase mutant gln-1b had derepressed levels of the nitrate assimilation enzymes. The inability of glutamine to achieve nitrogen metabolite repression in this mutant militated against its potential role as the direct effector of this regulation.     

Nitrate reductase,nitrite reductase and glutamate dehydrogenase levels in roots and leaves of maize seedlings

Total activities of nitrate and nitrite reductases were higher in 4 to 20 day old maize plants in the leaves than in the roots. The ratio of activities found in the leaves and in the roots respectively was much higher in the case of nitrate reductase than in the case of nitrite reductase. On the other hand higher glutamate dehydrogenase activity in the roots than in the leaves clearly indicates that the roots play a more important role in the assimilation of ammonium than in the assimilation of nitrate. When comparing the distribution of seminal and nodal adventitious roots of maize seedlings with the assimilation of inorganic nitrogen on the basis of enzyme levels, it could be deduced that during the first 20 days of seedling growth seminal roots were more involved in the assimilation of nitrate whereas nodal adventitious roots were more active in ammonium assimilation.     

An amino-acid-grown maize cell line for use in investigating nitrate assimilation

Summary Studies on uptake and assimilation of nitrate in plants are confounded by differences in cell function associated with anatomical features of roots as well as by problems inherent with growing plants without nitrate. To circumvent these problems, a Zea mays L. embryo cell line was grown in suspension culture using an amino-acid-based medium consisting of a Murashige and Skoog medium in which ammonium and nitrate were replaced by aspartic acid (100 mg/l), glycine (100 mg/l), arginine (150 mg/l), and glutamine (1 g/l). The growth, cellular characteristics, and physical appearance of the amino-acid-grown cells were similar to cells grown in the presence of nitrate. The amino-acid-grown cells exhibited the expected induction pattern and inhibitor sensitivity of nitrate uptake. This cell line should facilitate research on the induction of nitrate uptake and the regulation of nitrate assimilation into proteins.