Nitrate assimilation is essential for synthesizing organic nitrogen compounds like amino acids from inorganic nitrogen, primarily involving plants, fungi, and certain bacteria. This process occurs notably in roots and leaves where nitrate is absorbed and converted into ammonia for amino acid synthesis. Approximately 99% of organic nitrogen in the biosphere originates from nitrate assimilation, illustrating a continuous cycle of nitrogen between soil and plants.

1. NITRATE ASSIMILATION
(Essential for the Synthesis of
Organic Matter)
(CS 701) Advanced Biochemistry
ARNOLD V. DAMASO
MS in Crop Science- Agronomy
Central Luzon State University

2. Nitrate Assimilation
(Essential for the Synthesis of
Organic Matter)
• Introduction
• Living matter contains a large amount of nitrogen
incorporated in proteins, nucleic acids, and many
other biomolecules.
• This organic nitrogen is present in oxidation state
–III (as in NH3).
• During autotrophic growth the nitrogen demand
for the formation of cellular matter is met by
inorganic nitrogen in two alternative ways:

3. 1. Fixation of molecular nitrogen from
air; or
2. Assimilation of the nitrate or ammonia
contained in water or soil.

4. • Nitrogen assimilation is the formation
of organic nitrogen compounds like
amino acids from inorganic nitrogen
compounds present in the
environment. Organisms like plants,
fungi and certain bacteria that cannot
fix nitrogen gas (N2) depend on the
ability to assimilate nitrate or ammonia
for their needs.

5. • Other organisms, like animals, depend
entirely on organic nitrogen from their
food. Plants absorb nitrogen from the
soil in the form of nitrate (NO3
−) and
ammonia (NH3). In aerobic soils where
nitrification can occur, nitrate is usually
the predominant form of available
nitrogen that is absorbed

6. Importance and Significance

7. The reduction of nitrate to NH3
(Proceeds in two partial
reactions)
• Nitrate is assimilated in the leaves and also
in the roots.
• In most fully grown herbaceous plants,
nitrate assimilation occurs primarily in the
leaves, although nitrate assimilation in the
roots often plays a major role at an early
growth state of these plants. In contrast,
many woody plants (e.g., trees, shrubs), as
well as legumes such as soybean, assimilate
nitrate mainly in the roots.

8. • The transport of nitrate into the root
cells proceeds as symport with two
protons (Fig. 1). A proton gradient across
the plasma membrane, generated by an
H+-P-ATPase drives the uptake of nitrate
against a concentration gradient.
• The ATP required for the formation of
the proton gradient is mostly provided
by mitochondrial respiration

9. • The nitrate taken up into the root
cells can be stored there temporarily
in the vacuole. As discussed, nitrate is
reduced to NH4+ in the epidermal
and cortical cells of the root. This
NH4
+ is mainly used for the synthesis
of glutamine and asparagine

10. Figure 2. A. Nitrate reductase transfers electrons from NADH to nitrate. B. The
enzyme contains three domains where FAD, heme, and the molybdenum cofactor
(MoCo) are bound.

11. Figure 1. Nitrate assimilation in
the roots and leaves of a plant.

12. Figure 3. The molybdenum cofactor (Moco)

13. Nitrate is reduced to nitrite
in the cytosol
• Nitrate reduction uses mostly NADH as
reductant, although some plants contain a
nitrate reductase reacting with NADPH as well
as with NADH. The nitrate reductase of higher
plants consists of two identical subunits. The
molecular mass of each subunit varies from 99
to 104 kDa, depending on the species

14. Figure 4. Nitrate reductase in chloroplasts transfers electrons from ferredoxin to
nitrate. Reduction of ferredoxin by photosystem I that shown in figure.

15. The reduction of nitrite to
ammonia proceeds in the
plastids
• To a much lesser extent, the ferredoxin
required for nitrite reduction in a leaf can
also be provided during darkness via
reduction by NADPH, which is generated by
the oxidative pentose phosphate pathway
present in chloroplasts and leucoplasts.

16. • Nitrite reductase contains a covalently
bound 4Fe-4S cluster, one molecule of
FAD, and one siroheme. Siroheme (Fig.
5) is a cyclic tetrapyrrole with one Fe-
atom in the center. Its structure is
different from that of heme as it
contains additional acetyl and propionyl
residues deriving from pyrrole synthesis

17. Figure 5. Structure of siroheme

18. • The 4Fe-4S cluster, FAD, and siroheme
form an electron transport chain by which
electrons are transferred from ferredoxin
to nitrite. Nitrite reductase has a very high
affinity for nitrite. The capacity for nitrite
reduction in the chloroplasts is much
greater than that for nitrate reduction in
the cytosol

19. The fixation of NH4+ proceeds
in the same way as in
photorespiration cycle
• Glutamine synthetase in the chloroplasts
transfers the newly formed NH4
+ at the expense
of ATP to glutamate, forming glutamine. The
activity of glutamine synthetase and its affinity
for NH4+ (Km ≈510-6 mol/L) are so high that
the NH4
+ produced by nitrite reductase is taken
up completely. The same reaction also fixes the
NH4+ released during photorespiration

20. Figure 5.1. Reductase of
photosystem I

21. Figure 6.
Compartmentation of
partial reactions of
nitrate assimilation
and the
photorespiratory
pathway in mesophyll
cells.

22. • Glufosinate (Fig. 7), a substrate analogue
of glutamate, inhibits glutamine synthesis.
Plants in which the addition of glufosinate
has inhibited the synthesis of glutamine
accumulate toxic levels of ammonia and
die off. NH4+-glufosinate is distributed as
an herbicide under the trade name Liberty
(Aventis).

23. Figure 7. Glufosinate

24. Nitrate assimilation
(also takes place in the roots)
• As mentioned, nitrate assimilation occurs
in part, and in some species even mainly,
in the roots. NH4+ taken up from the soil
is normally fixed in the roots.
• The reduction of nitrate and nitrite as
well as the fixation of NH4+ proceeds in
the root cells in an analogous way to the
mesophyll cells.

25. The oxidative pentose
phosphate pathway provides
reducing equivalents for nitrite
reduction in leucoplasts
• The reducing equivalents required for the
reduction of nitrite and the formation of
glutamate are provided in leucoplasts by
oxidation of glucose 6 phosphate via the
oxidative pentose phosphate pathway
discussed in (Fig. 8).

26. • As in chloroplasts, nitrite reduction in
leucoplasts also requires reduced ferredoxin
as reductant. In the leucoplasts, ferredoxin is
reduced by NADPH, which is generated by the
oxidative pentose phosphate pathway. The
ATP required for glutamine synthesis in the
leucoplasts can be generated by the
mitochondria and transported into the
leucoplasts by a plastid ATP translocator in
counter-exchange for ADP

27. Figure 8. The
oxidative pentose
phosphate
pathway

28. Nitrate assimilation
(is strictly controlled)
• During photosynthesis, CO2 assimilation and
nitrate assimilation have to be matched to
each other. Nitrate assimilation can progress
only when CO2 assimilation provides the
carbon skeletons for the amino acids.
• Moreover, nitrate assimilation must be
regulated in such a way that the production of
amino acids does not exceed demand

29. • Finally, it is important that nitrate reduction
does not proceed faster than nitrite reduction,
since otherwise toxic levels of nitrite would
accumulate in the cells.
• Under certain conditions such a dangerous
accumulation of nitrite can indeed occur in
roots when excessive moisture makes the soil
anaerobic

30. The synthesis of the nitrate
reductase protein is regulated
at the level of gene expression
• Nitrate reductase is an exceptionally short-
lived protein. Its half-life is only a few hours.
The rate of de novo synthesis of this enzyme is
therefore very high. Thus, by regulating its
synthesis, the activity of nitrate reductase can
be altered within hours.

31. Nitrate reductase is also
regulated by reversible covalent
modification
• The regulation of de novo synthesis of nitrate
reductase (NR) allows regulation of the enzyme
activity within a time span of hours.
• This would not be sufficient to prevent an
accumulation of nitrite in the plants during
darkening or sudden shading of the plant.
Rapid inactivation of nitrate reductase in the
time span of minutes occurs via
phosphorylation of the nitrate reductase
protein (Fig. 9).

32. Figure 9. Regulation of nitrate reductase (NR).

33. 14-3-3 Proteins are important
metabolic regulators
• It was discovered that the nitrate reductase
inhibitor protein belongs to a family of
regulatory proteins called 14-3-3 proteins,
which are widely spread throughout the
animal and plant worlds.
• 14-3-3 proteins bind to a specific binding site
of the target protein with six amino acids,
containing a serine phosphate in position 4

34. The regulation of nitrate
reductase and sucrose
phosphate synthase have great
similarities
• The mechanism of the regulation of nitrate
reduction by phosphorylation of serine
residues of the enzyme protein by special
protein kinases and protein phosphatases is
remarkably similar to the regulation of sucrose
phosphate synthase.

35. The end product of nitrate
assimilation
(is a whole spectrum of amino acids)
• The carbohydrates formed as the product of
CO2 assimilation are transported from the
leaves via the sieve tubes to various parts of the
plants only in defined transport forms, such as
sucrose, sugar alcohols (e.g., sorbitol), or
raffinoses, depending on the species.

36. CO2 assimilation provides the carbon
skeletons to synthesize the end
products of nitrate assimilation
• CO2 assimilation provides the carbon
skeletons required for the synthesis of the
various amino acids. Figure 10 gives an
overview of the origin of the carbon skeletons
of individual amino acids.

37. Figure 10. Origin of carbon skeletons for various amino acids.

38. Figure 11. Carbon skeletons for the synthesis of amino acids are provided by CO2
assimilation.

39. The synthesis of glutamate requires
the participation of mitochondrial
metabolism
• That glutamate is formed from -ketoglutarate,
which can be provided by a partial sequence of
the mitochondrial citrate cycle (Fig. 11). Pyruvate
and oxaloacetate are transported from the
cytosol to the mitochondria by specific
translocators. Pyruvate is oxidized by pyruvate
dehydrogenase, and the acetyl-CoA thus
generated condenses with oxaloacetate to citrate.

40. Biosynthesis of proline and arginine
• proline has a special function as a protective
substance against dehydration damage in
leaves. When exposed to aridity or to a high
salt content in the soil (both leading to water
stress), many plants accumulate very high
amounts of proline in their leaves, in some
cases several times the sum of all the other
amino acids

41. Figure 12. Pathway for the
formation of amino acids
from glutamate.

42. Aspartate is the precursor of
five amino acids
• Aspartate is formed from oxaloacetate by
transamination with glutamate by glutamate-
oxaloacetate amino transferase (Fig. 14).
• The synthesis of asparagine from aspartate
requires a transitory phosphorylation of the
terminal carboxylic group by ATP, as in the
synthesis of glutamine. In contrast to glutamine
synthesis, however, it is not NH4+ but the amide
group of glutamine that usually serves as the
amino donor in asparagine synthesis

43. Figure 13. Two compatible substances that, like
proline, are accumulated in plants as
protective agents against desiccation and high
salt content in the soil.

44. Figure 14. The pathway for the synthesis of amino acids
from aspartate.

45. Figure 15. Feedback inhibition by end products regulates the entrance enzyme for
the synthesis of amino acids from aspartate according to demand. [-] indicates
inhibition. Aspartate kinase exists in two isoforms

46. Acetolactate synthase
participates in the synthesis of
hydrophobic amino acids
• Acetolactate synthase, catalyzing this reaction,
contains thiamine pyrophosphate (TPP) as its
prosthetic group. The reaction of TPP with
pyruvate yields hydroxyethyl-TPP and CO2, in
the same way as in the pyruvate
dehydrogenase reaction.

47. Figure 16A. Pathway for the synthesis of amino acids from pyruvate.

48. Figure 17. Synthesis of valine and leucine.

49. Figure 16B. Pathway for the synthesis of isoleucine from
threonine and pyruvate.

50. Figure 18. Herbicides: chlorsulfurone, a
sulfonyl urea, (trade name Glean,
DuPont) and imazethapyr, an
imidazolinone, (trade name Pursuit,
ACC) inhibit acetolactate synthase.
Glyphosate (trade name Roundup,
Monsanto) inhibits EPSP synthase

51. Aromatic amino acids are
synthesized via the
shikimate pathway
• Precursors for the formation of aromatic
amino acids are erythrose 4phosphate and
phosphoenolpyruvate.
• These two compounds condense to form
cyclic dehydrochinate accompanied by the
liberation of both phosphate groups (Fig. 19)

52. Figure 19. Aromatic amino acids are synthesized by the shikimate pathway.
PEP = phosphoenolpyruvate.

53. Figure 20. Several steps in the
synthesis of aromatic amino acids

54. A large proportion of the total
plant matter can be formed by the
shikimate pathway
• The function of the shikimate pathway is not
restricted to the generation of amino acids for
protein biosynthesis. It also provides
precursors for a large variety of other
substances (Fig. 21) formed by plants in large
quantities, particularly phenylpropanoids such
as flavonoids and lignin

55. Figure 21. Several secondary
metabolites are synthesized
via the shikimate pathway.

56. Glutamate is the precursor
(for synthesis of chlorophylls
and cytochromes)
• Chlorophyll amounts to 1% to 2% of the dry
matter of leaves. Its synthesis proceeds in the
plastids. Chlorophyll consists of a tetrapyrrole
ring with magnesium as the central atom and
with a phytol side chain as a membrane
anchor. Heme, likewise a tetrapyrrole, but
with iron as the central atom, is a constituent
of cytochromes and catalase.

57. Figure 22. In chloroplasts, glutamate is the precursor for the synthesis of
-amino levulinate, which is condensed to porphobilinogen.

58. Protophorphyrin is also a
precursor for heme synthesis
• By assembling the heme with apoproteins,
chloroplasts are able to synthesize their
own cytochromes. Also, mitochondria
possess the enzymes for the biosynthesis of
their cytochromes from protoporphyrin IX,
but the corresponding enzyme proteins are
different from those in the chloroplasts.

59. Figure 23. Protoporphyrin
synthesis.

60. Figure 24 Overview of the
synthesis of chlorophyll and heme
in chloroplasts.

61. Summary
• Nitrate assimilation is the formation of organic
nitrogen compounds like amino acids from
inorganic nitrogen compounds present in the
environment. Organisms like plants, fungi and
certain bacteria that cannot fix nitrogen gas (N2)
depend on the ability to assimilate nitrate or
ammonia for their needs.
• Plants like castor reduce a lot of nitrate in the
root itself, and excrete the resulting base. Some
of the base produced in the shoots is transported
to the roots as salts of organic acids while a small
amount of the carboxylates are just stored in the
shoot itself

62. • However, about 99% of the organic
nitrogen in the biosphere is derived
from the assimilation of nitrate. NH4
+ is
formed as an end product of the
degradation of organic matter,
primarily by the metabolism of animals
and bacteria, and is oxidized to nitrate
again by nitrifying bacteria in the soil.
Thus a continuous cycle exists between
the nitrate in the soil and the organic
nitrogen in the plants growing on it.

63. • While nearly all the ammonia in the
root is usually incorporated into amino
acids at the root itself, plants may
transport significant amounts of
ammonium ions in the xylem to be
fixed in the shoots. This may help avoid
the transport of organic compounds
down to the roots just to carry the
nitrogen back as amino acids.

64. End of Report