NITRATE UPTAKE, REDUCTION AND ASSIMILATION - nitrogen cycle. essential for all life science students.

1. NITRATE UPTAKE AND
ASSIMILATION

2. NITRATE UPTAKE AND ASSIMILATION
Schematic and simplified representation of the nitrate uptake, transport and assimilation in plants.

3.  Nitrate movements through cellular membranes are always mediated by specific transporters.
 The translocation of NO3 ions is active and involves both high and low affinity proton symporters.
 In roots, three distinct groups of nitrate transporters have been identified.
 Two of them have been classified as high affinity transporter system (HATS) and include the
inducible (iHATS) and constitutive (cHATS) transport systems.
 Another group is represented by a constitutive low affinity transporter system (LATS).
 All these transporters are up-regulated by nitrate availability.
 Nitrate influx into the root cells is proton coupled, as above mentioned, therefore nitrate
transport is dependent on the H+ -ATPase pumps and it requires energy.
 It has been proposed that in both symporter systems, for each NO3 - loaded, two H+ cross the
plasma membrane and that this mechanism is tightly regulated by cellular pH .
 Nitrate in plants can be reduced to nitrite both in roots and in shoots or loaded and stored inside
the vacuole. NO3 - reduction is catalysed by the enzyme Nitrate Reductase.

4.  NR in the leaf is a cytoplasmic enzyme and works in the presence of NAD(P)H,
 whereas in the root two distinct types of nitrate reductase are present:
 a cytoplasmic isoform (cNR) and
 a plasmamembrane-bound isoform (PMNR),
 the latter also able to oxidize succinate.
 In normal conditions, NR reduces nitrates to nitrites, which are then transferred to the chloroplast/plastid
where they are reduced to ammonium by nitrite reductase.
 At last, ammonium is organicated to glutamate by glutamine synthetase to produce glutamine.
 Therefore, nitrate is assimilated into amino acids via the GS-GOGAT pathway (GS1, GS2, GOGAT), resulting
in glutamine and glutamate as primary N organic compounds.
 Glutamine synthetase (GS) and glutamate synthase (GOGAT,) are the enzymes involved in ammonium
assimilation, either derived from nitrate reduction, photorespiration or, in case it is externally supplied, as
NH3 and or NH4 +.
 In roots, NO2 - is converted into NH4 + mainly by the cytoplasmic GS isoform (named GS1), whereas the
plastidial isoform (GS2) is less active. The opposite happens in the leaf, where the chloroplastic GS2 is the
most active isoform.

6. Nitrate is absorbed by most plants and reduced
to ammonia with the help of two different enzymes. The
first step conversion of nitrate to nitrite is catalyzed by an
enzyme called nitrate reductase. This enzyme has several
other important constituents including FAD, cytochrome,
NADPH or NADH and molybdenum.
NITRATE REDUCTASE
In higher plants, nitrate reduction is highly regulated. A
range of environmental factors influence the expression of
the corresponding genes as well as the enzyme activity
levels. NR activity expression and activity is controlled by
light, temperature, pH, CO 2, O 2, water potential and N
source.
Nitrate reduction takes place chiefly in green leaves and roots. The enzymes nitrate
reductase is found in cytosol). According to more recent findings the enzyme nitrate
reductase is in-fact a complex enzyme in higher plants as well as micro-organisms.

7. 1.Nitrate uptake
2.Nitrate reduction to ammonia -- nitrate reductase & nitrite reductase
3.Ammonia assimilation into glutamate & glutamine
 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.

8. BIOLOGICAL
NITROGEN
FIXATION
Nitrogen
Fixation in
Plants
(biologydiscuss
ion.com)

9. •Nitrate Uptake
• The nitrate uptake system in plant must be versatile and robust because
1. 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 magnitude.
2. Plants must compete for N in the soil with abiotic and biotic processes such as erosion,
leaching and microbial competition.
3. To function efficiently and the face of such environmental variation, plants have evolved 3
transport systems that are:
• active
• regulated
• multiphasic
• The energy that drives nitrate uptake comes from the proton gradient maintained across the plasma
membrane by the H+ ATPase
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.

10. •The H+-ATPase in the PM pumps protons out of the cell producing
pH and electrical ( psi ) 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.

11. •Nitrate is a signal for developmental changes in the physiology of the plant.
•The primary responses include:
1.Induction of genes for nitrate and nitrite reduction.
2.Nitrate uptake and translocation systems.
3.DNA regulatory proteins required for expression of the secondary response gene system.
•The secondary response include more complex phenomena such as
1.Proliferation of the root system.
2.Enhancement of respiration.
3.Other changes in the physiology of the plant.
•The fate of NO3
- taken up by a root epidermal cell.
•
•Once transported into an epidermal cell, NO3
- has one of four fates:
1.It may undergo efflux to the apoplast and soil environment.
2.It may enter the vacuole and by stored.
3.It may be reduced to ammonium by the combined action of NR and NiR.
4.It may be translocated via the symplast to the xylem.

12. •Nitrate Reduction
•Virtually all biologically important N-compounds contain N in a reduced form.
•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)
•Nitrate and nitrite reductase
•NO3
- + 2H+ + 2e- NO2
- + H2O
•NO2
- + 8H+ + 6e-
•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.
NH4
+ + 2H2O

13. •NR is typically a homodimer or homotetramer with 100-to 115 kD subunits.
• three functional domains
1. flavin (FAD) domain
2. heme (Fe) domain
3. molybdenum cofactor (MoCo) domain
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.
•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
The central heme containing 75-80 amino acids is similar to
heme of cytochrome b5s.

15. •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
1. a siroheme center
2. an iron-sulfur center; 4Fe-4S cluster
3. a ferredoxin binding domain
•NiR is a nuclear encoded enzyme transported to chloroplasts or proplastids with cleavage of a 30 kD transit sequence.
The c-terminal half of NiR is thought to contain the redox centers and the N-terminal half is thought to bind the reducing
agent ferredoxin.
•Ferredoxin reduced by the chloroplast non-cyclic electron transport system provides the e- for reducing nitrite. It is
thought that a ferredoxin-like protein in proplastids reduced by NADPH from the oxidative pentose phosphate pathway
provides the source of reductant in roots.
•A model for coupling photosynthetic electron flow, via ferredoxin to the reduction of nitrate by NiR to ammonia:
•Sirohemes
• Uroporphyrin derivatives that are quite polar.
• Novel in having eight carbohydrate containing side chains.

16. Ammonia Assimilation
NH4+ enters an organic linkage via one of 3 major reactions that are found in all cells:
1) Carbamoyl phosphate synthetase I
NH4+ + HCO3- + 2ATP -> H2N-CO-O-PO3-2 + 2ADP + Pi
2) 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 acids.
3) Glutamine synthetase (GS)
ATP-dependent amination of the g-carboxyl group of glutamate to form glutamine
Mg2+-dependent
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

17. The reaction:
Glutamate + ammonia + ATP -> glutamine + ADP + Pi
involves activation of the g-carboxyl group of Glu by ATP
followed by amination by NH4+
The glutamate consumed by the GS reaction is replenished by
an alternative mode of glutamate synthesis
Glutamate synthases
(=GoGAT glutamate: oxoglutarate aminotransferase)
reductant + a-KG + Gln -> 2Glu + oxidized reductant
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

18. Different organisms use different reductants
H+ + NADH: yeast, N. crassa, plants
H+ + NADPH: E. coli
H+ + reduced ferredoxin: plants (solely in chloroplasts)
The GS/GoGAT pathway of ammonium assimilation:
Summary: 2 NH4+ +α-KG + 2H+ + 2 Fdred + 2 ATP -> Glutamine
+ 2 Fdox + 2 ADP + 2 Pi
These reactions result in conversion of α-KG to glutamine at the
expense of 2 ATP and 1 NADPH