Nitrogen Metabolism -- nitrate reduction, ammonia
Goal: A good working knowledge and understanding of the
reduction of oxidized forms of nitrogen commonly encountered by plants to
ammonia and the assimilation of the reduced ammonia into major nitrogenous
compounds of plant tissues.
- Nitrate uptake
- Nitrate reduction to ammonia -- nitrate reductase & nitrite
- Ammonia assimilation into glutamate & glutamine
Background Readings for the discussion on
nitrate reduction and ammonia assimilation :
1 - Chapter 16, sections 16.6 - 16.10 of the Biochemistry & Molecular Biology of Plants class text.
2 - Han, Y.-L., Song, H.-X., Liao, Q., Yu, Y., Jian, S.-F., Lepo, J.E., Liu, Q., Rong, X.-M., Tian, C., Zeng, J., et al. (2016). Nitrogen use efficiency is mediated by vacuolar nitrate sequestration capacity in roots of Brassica napus. Plant Physiology 170:1684-1698.
1 - Lee, J.-H., B.S. Evans, G. Li, N.L. Kelleher, and W.A. van der Donk. 2009. In Vitro Characterization of a Heterologously Expressed Nonribosomal Peptide Synthetase Involved in Phosphinothricin Tripeptide Biosynthesis. Biochemistry 48:5054-5056..
2 - Crawford,
Nigel M. 1995. Nitrate: Nutrient and signal for plant growth. The Plant Cell
3 - Scheible, W.-R., R. Morcuende, T. Czechowski, C. Fritz, D. Osuna, N. Palacios-Rojas, D.
Schindelasch, O. Thimm, M.K. Udvardi, and M. Stitt. 2004. Genome-Wide Reprogramming of Primary and Secondary Metabolism, Protein Synthesis, Cellular Growth Processes, and the Regulatory Infrastructure of Arabidopsis in Response
to Nitrogen. Plant Physiol. 136: 2483-2499.
4 - Forde, B.G. 2002. LOCAL AND LONG-RANGE SIGNALING PATHWAYS REGULATING PLANT RESPONSES TO NITRATE.
Annu. Rev. Plant Biol. 53:
5 - Lea, U.S., M.-T. Leydecker, I. Quillere,
C. Meyer, and C. Lillo. 2006. Posttranslational Regulation of Nitrate
Reductase Strongly Affects the Levels of Free Amino Acids and Nitrate, whereas
Transcriptional Regulation Has Only Minor Influence. Plant
Physiol. 140: 1085-1094.
6 - Okamoto, M., A. Kumar, W. Li, Y. Wang, M.Y.
Siddiqi, N.M. Crawford, and A.D.M. Glass. 2006. High-Affinity Nitrate
Transport in Roots of Arabidopsis Depends on Expression of the NAR2-Like Gene
T., P. Nacry, M. Pervent, T. Girin, P. Tillard, M. Lepetit, and A. Gojon.
2006. A Central Role for the Nitrate Transporter NRT2.1 in the Integrated
Morphological and Physiological Responses of the Root System to Nitrogen
Limitation in Arabidopsis. Plant
Remember from the previous class discussion, nitrogen (N2) must
first be fixed usually into a reduced form such as ammonia. Ammonia is
usually rapidly oxidized into nitrate by nitrifying bacteria in soils so
nitrate is the usual form of nitrogen available to most plants.
- Nitrate Uptake
- The nitrate uptake system in plant must be versatile and
- Plants have to transport sufficient nitrate to
satisfy the total demand for nitrogen in the face of external
nitrate concentrations that can vary by five orders of
- Plants must compete for N in the soil with abiotic
and biotic processes such as erosion, leaching and microbial
- To function efficiently and the face of such
environmental variation, plants have evolved 3 transport systems
The energy that drives nitrate uptake comes from the proton
gradient maintained across the plasma membrane by the H+
See Fig. 1 of Crawford (1995) The Plant Cell 7: 859-868 and Fig. 16.34 of
the class text.
- The H+-ATPase in the PM pumps protons out of
the cell producing pH and electrical () gradients.
- The nitrate transporters (Ntr) cotransport two or more
protons per nitrate into the cell.
- Nitrate can be transported across the tonoplast membrane and
stored in the vacuole.
- Nitrate in the cytosol is reduced to nitrite that enters the
plastid and is reduced to ammonia.
- Ammonia is fixed into glutamate (Glu) to produce glutamine
(Gln) by the action of glutamine synthetase (GS).
- Nitrate also acts as a signal to increase the expression of
nitrate reductase (NR), nitrite reductase (NiR) and Ntr genes.
The initial uptake of nitrate occurs across the plasma membrane of
epidermal and cortical cells of the root. Subsequent transport
across the tonoplast membrane and the PM of cells in the vascular system
and leaf distributes NO3- throughout leaf and shoot
tissue. Ultimately N can be stored in the seed or other storage
organ. Figure 1 of Crawford `95 and Fig. 16.34 of the class text shows a brief outline of the major
routes of metabolism of nitrogen within a plant:
Plant have distinct transport systems with different affinities for
nitrate (Vidmar et al., 2000; Ortiz-Lopez et al., 2000).
- Constitutive high-affinity transport system (CHATS)
- Responsible for nitrate uptake at low
concentrations (below ~1 mM).
- Saturation kinetics, with Km values below 300 µM.
Inducible high-affinity transport system (IHATS)
- Induced by NO3- taken up by CHATS.
- Increases overall NO3- uptake transiently.
Low affinity transport system (LATS)
- Uptake of nitrate at high concentration (> 0.2 mM).
- Like the high affinity systems, the low affinity
systems are electrogenic and involve proton cotransport.
- Show linear rather than saturation kinetics.
- Not saturated even at 50 mM NO3-.
Studies on nitrate uptake
Measuring NO3- uptake has been hampered by the
lack of a suitable radioactive tracer.
- 13N has a half-life of only 10 min.
- 15N is not radioactive.
[not covered in 2017]
- When NO3- is supplied to cells it is
metabolized and effluxed as equilibrium is established with
- An alternative substrate of the
NO3- uptake mechanism.
- 36Cl can be used as the radioactive isotope to
study nitrate uptake properties.
- ClO3- inhibits
NO3- uptake and vice versa.
- ClO3- is not assimilated.
- ClO3- uptake into roots is extremely
sensitive to the inhibitor FCCP, an uncoupler of energy-dependent ion
Utilizing ion-specific electrodes it has been suggested that there is
a cotransport of NO3-/H+ across the
plasmalemma of maize root cells and that the cytoplasmic level of nitrate
is kept relatively constant by storage of nitrate in the vacuole.
In NO3--starved roots, uptake is dominated by a
constitutively expressed, low activity, high affinity system for
NO3- uptake in barley and maize roots.
When N-starved plants are treated with NO3-,
the root develops a high rate of NO3- uptake with a
greater affinity for NO3-:
- 13NO3- influx displays
typical Michaelis-Menten kinetics
- The rate of uptake is depending upon the length of the
previous treatment with nitrate, thus suggesting that a nitrate
transport system is induced by nitrate.
The induction of mRNA of the IHATS transporter is longer and greater at
1 mM (a) than 10 mM (b) NO3-.
Induced by NO3- and somewhat less so by
NO2- but inhibited by NH4+ (all at
Not only is the nitrate uptake system induced, but also nitrate
induces nitrate and nitrite reductase activities by altering gene
expression mainly by enhancing transcription of the respective
genes. Nitrate, light and sugars are inducers; glutamate and
glutamine are repressors.
Why might this be?
Nitrate is a signal for developmental changes in the physiology of the
The primary responses include:
- Induction of genes for nitrate and nitrite reduction.
- Nitrate uptake and translocation systems.
- DNA regulatory proteins required for expression of the
secondary response gene system.
The secondary response include more complex phenomena such as
- Proliferation of the root system.
- Enhancement of respiration.
- Other changes in the physiology of the plant.
A constitutive 'NO3- sensor' protein would
detect the presence of environmental NO3-, then
NO3- induction regulatory protein(s) would be
activated which would act to initiate transcription of the primary
response genes by RNA polymerase, resulting in NR, NiR,
NO3- transporters, NO3-
translocaters, ammonia assimilation enzymes.
What might be expected to inhibit this?
The fate of NO3- taken up by a root epidermal
Once transported into an epidermal cell, NO3-
has one of four fates:
- It may undergo efflux to the apoplast and soil environment.
- It may enter the vacuole and by stored.
- It may be reduced to ammonium by the combined action of NR
- It may be translocated via the symplast to the xylem.
- Virtually all biologically important N-compounds contain N in a
- The principal inorganic forms of N in the environment are in
an oxidized state. Thus, the entry of N into organisms
depends on the reduction of oxidized organic forms (N2
and NO3-) to NH4+.
- The reactions involving inorganic N-compounds occur only in
microorganisms and green plants. Animals acquire their N
from the catabolism of organic N-compounds mainly proteins,
obtained in the diet.
- Nitrate is reduced to ammonia by a two-step process catalyzed
by the enzymes nitrate reductase (NR) and nitrite reductase (NiR)
[covered in 2017]
- Nitrate and nitrite reductase
- NO3- + 2H+ + 2e- NO2- +
- NO2- + 8H+ + 6e- NH4+ +
- 4-1. Nitrate reductase (NR)
- Located primarily in the cytosol of root epidermal and
cortical cells and shoot mesophyll cells.
- Transfers 2 e- from NAD(P)H to nitrate via three redox
centers composed of two prosthetic groups (FAD and heme). It also has a
molybdenum cofactor (MoCo), a complex of molybdate and pterin, which
catalyzes the actual nitrate reduction.
- See Fig. 2 of Crawford (1995) The Plant Cell 7: 859-868 and
Figs. 16.38 - 40 of the class text.
- NR is typically a homodimer or homotetramer with 100-to 115 kD
- flavin (FAD) domain
- heme (Fe) domain
- molybdenum cofactor (MoCo) domain
[not covered in 2017]
The NR catalyzed reduction of NO3- starts with
e- transport from NAD(P)H to the flavin domain, through heme
and finally onto NO3- via the molybdenum
cofactor. Cytochrome c can be an alternative e- acceptor
(Fig. 2, 16.38 above).
The FAD domain has been crystallized and found to contain 2
lobes. One lobe contains 6 parallel ß-strands. The
other lobe, which binds to the FAD molecule tethered by several
hydrogen bonds, also contains ß-strands, but is antiparallel
(Fig. 2B Crawford, 1995, Fig. 16.40 class text).
The central heme containing 75-80 amino acids is similar to heme of
cytochrome b5s. See illustration at the
Directory of P450-containing Systems web page.
[end not covered 2017]
Green tissues have >>> NR than NiR activity so NR is rate
limiting. What might be the advantages of this?
It is very important that plants regulate the process of nitrate
reduction because it is a very energy intensive process consuming ~20% of
the e- produced by photosynthetic electron transport.
Also, the intermediate product of nitrate reduction, nitrite, is
cytotoxic and mutagenic. As mentioned above NR genes can be
regulated by nitrate, light, sugars, glutamate and glutamine. Also
plant hormones and other factors such as Ca++ can affect NR transcription. NR
can be regulated post translationally by reversible protein
phosphorylation (see Fig. 16.42 text).
Nitrite Reduction via Nitrite Reductase (NiR)
- Reduction of nitrite to ammonia:
- NiR enzyme
- the holoenzyme is a monomer, 60-70 kD with two redox centers
- a siroheme center
- an iron-sulfur center; 4Fe-4S cluster
- a ferredoxin binding domain
- Carbamoyl phosphate synthetase I
NH4+ + HCO3- + 2ATP
H2N-CO-O-PO3-2 + 2ADP + Pi
- Glutamate dehydrogenase (GDH) reaction:
GDH has a significantly higher km for NH4+
than does glutamine synthetase (GS). Consequently in
organisms confronting N-limitation GDH is not effective and GS is
the only NH4+ assimilation reaction.
It also appears that GS is the sole port of entry of N into amino
- Glutamine synthetase (GS)
- ATP-dependent amination of the g-carboxyl group of
glutamate to form glutamine
- Very high affinity for ammonia (Km
= 3-5 µM)
- Glutamine is the major N-donor in the biosynthesis of
many organic N compounds such as
- purines, pyrimidines
- other amino acids
The reaction: Glutamate + ammonia + ATP glutamine + ADP + Pi
involves activation of the g-carboxyl group of Glu by ATP followed by amination by
- 5-2. The glutamate consumed by the GS reaction is replenished by an
alternative mode of glutamate synthesis
- Glutamate synthases
- (=GoGAT glutamate: oxoglutarate
- reductant + a-KG +
Gln 2Glu + oxidized
- Scheme: See Fig. 1 of Lam et al. (1995) The Plant Cell 7:887-898.
- Two equivalents of glutamate are formed
- from amination of α-KG
- from deamination of Gln
These Glu can now serve as ammonia accepts for
glutamine synthesis by GS
Different organisms use different reductants
- H+ + NADH: yeast, N.
- H+ + NADPH: E. coli
- H+ + reduced ferredoxin: plants (solely
The GS/GoGAT pathway of ammonium assimilation:
Summary: 2 NH4+ +α-KG + 2H+ + 2 Fdred + 2
ATP Glutamine + 2 Fdox + 2 ADP +
These reactions result in conversion of α-KG to glutamine at the expense of 2 ATP and 1 NADPH (equivalent).
The assimilation of ammonia in higher plants via the GS/GoGAT cycle:
- The photorespiratory N cycle also provides N as discussed previously
by Dr. Bob Houtz.
Scheible et al. (2004) examined the genome-wide
metabolic responses of Arabidopsis to N. Numerous overall metabolic
changes after transfer of N-deficient Arabidopsis seedlings to NO3-
sufficient medium after 30 min.:
and after 3h:
Background Readings for the discussion on
amino acid and other N compound metabolism
1 - Chapter
16, sections 16.11 - 16.18 of the Biochemistry & Molecular Biology
of Plants class text.
1 - Lam
et al. 1995. Use of Arabidopsis mutants and genes to study amide amino
acid biosynthesis. The Plant Cell 7, pp. 887-898.
2 - Guo, F.Q., M. Okamoto and N.M. Crawford. 2003. Identification of a plant Nitric oxide synthase gene involved
in hormone signaling. Science 302: 100-103.
3 - Shewry
et al. 1995. Seed storage proteins: structures and biosynthesis. The Plant
Cell 7, pp. 945-956.
4 - von
Wettstein et al. 1995. Chlorophyll biosynthesis. The Plant Cell 7, pp.
5 - Zeier, J., M. Delledonne, T. Mishina, E. Severi, M.
Sonoda, and C. Lamb. 2004. Genetic Elucidation of Nitric Oxide Signaling
in Incompatible Plant-Pathogen Interactions. Plant Physiol. 136:
6 - Jasid, S., M. Simontacchi, C.G. Bartoli, and S. Puntarulo. 2006. Chloroplasts as a Nitric Oxide Cellular Source. Effect of Reactive Nitrogen Species on Chloroplastic Lipids and Proteins. Plant Physiol. 142:1246-1255.
7 - Peng Pan, Eilika Woehl and Michael F. Dunn. 1997. Protein
architecture, dynamics and allostery in tryptophan synthase channeling.
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or comments regarding the class or the webpages.
This page was last modified Feb 7, 2017.