Mochammad Roviq, 2011
References
• Hans-Walter Heldt. 2005. Plant Biochemistry.
Third edition. , Elsevier Inc
Nitrogen contribution
• Nitrogen ranks behind only carbon, hydrogen, and oxygen
in its contribution to the mass of living systems.
• Most of this nitrogen is bound up in amino acids and
nucleotides
• Nitrogen (N) is a major element of all organisms and it
accounts for 6.25% of their dry mass on average
• Although nitrogen gas (N2) accounts for about eighty
percent of the Earth's atmosphere, plants and animals do
not have an easy time obtaining all the nitrogen they need
for growth.
• This situation arises because the N2 molecule is very stable
chemically and so is unusable by most biological organisms.
It must be "fixed" before it can be assimilated.
Concept
Nitrate assimilation is essential for the synthesis of organic
matter
Living matter contains a large amount of nitrogen incorporated
in proteins, nucleic acids, and many other biomolecules.
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:
• 1. Fixation of molecular
nitrogen from air; or
• 2. Assimilation of the
nitrate or ammonia
contained in water or soil.
Assimilation of nitrate
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.
NH4+ accumulates only in poorly aerated soils with insufficient
drainage, where, due to lack of oxygen, nitrifying bacteria cannot
grow.
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.
Transport nitrate
• The transport of nitrate into the root cells proceeds as
symport with two protons (Gambar 1)
• A proton gradient across the plasma membrane,
generated by a 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.
• When inhibitors abolish mitochondrial ATP synthesis in
the roots, nitrate uptake normally comes to a stop.
Asimilasi nitrat dalam
akar dan daun tanaman.
Nitrat diambil dari tanah oleh
akar.
Nitrat dapat disimpan dalam
vakuola sel root atau diasimilasi
dalam sel-sel epidermis akar
dan korteks.
Surplus nitrat dibawa melalui
pembuluh xilem ke sel-sel
mesofil, dimana nitrat dapat
disimpan sementara dalam
vakuola.
Nitrat direduksi menjadi nitrit
dalam sitosol dan kemudian
nitrit direduksi lebih lanjut
dalam kloroplas menjadi NH4+,
dimana asam amino terbentuk
Nitrate uptake
• Root cells contain several nitrate transporters in
their plasma membrane
• The capacity of nitrate uptake into the roots is
adjusted to the environmental conditions.
• The efficiency of the nitrate uptake systems
makes it possible that plants can grow when the
external nitrate concentration is as low as 10 µM.
• The nitrate taken up into the root cells can be
stored there temporarily in the vacuole.
The synthesis of
amide
NH4 + is mainly used for the
synthesis of glutamine and
asparagine (collectively named
amide).
They can be transported to the
leaves via the xylem vessels.
However, when the capacity for
nitrate assimilation in the roots is
exhausted, nitrate is released from
the roots into the xylem vessels and
is carried by the transpiration
stream to the leaves.
There it is taken up into the
mesophyll cells, probably also by a
proton symport.
Nitrate assimilation
Large quantities of nitrate can be
stored in a leaf by uptake into the
vacuole.
Sometimes this vacuolar store is
emptied by nitrate assimilation
during the day and replenished
during the night.
In spinach leaves, the highest nitrate
content is found in the early
morning.
The nitrate in the mesophyll cells is
reduced to nitrite by nitrate
reductase present in the cytosol
and then to NH4+ by nitrite
reductase in the chloroplasts
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.
Each subunit contains an electron
transport chain (Gb. 2) consisting of
one flavin adenine dinucleotide
molecule (FAD), one heme of the
cytochrome-b type (cyt-b557), and
one cofactor containing
molybdenum
Cofactor is a pterin with a side chain to
which the molybdenum is attached by
two sulfur bonds and is called the
molybdenum cofactor, abbreviated
MoCo
The reduction of nitrite to ammonia proceeds
in the plastids
The reduction of nitrite to ammonia requires the uptake of six electrons.
This reaction is catalyzed by only one enzyme, the nitrite reductase (Gb. 4),
which is located exclusively in plastids.
Nitrite reductase contains a covalently bound 4Fe-4S cluster (see Figure
3.26), one molecule of FAD, and one siroheme.
Siroheme 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
Nitrite is toxic
The capacity for nitrite reduction in the chloroplasts is
much greater than that for nitrate reduction in the cytosol.
Therefore all nitrite formed by nitrate reductase can be
completely converted to ammonia.
This is important since nitrite is toxic to the cell.
The very efficient reduction of nitrite by chloroplast nitrite
reductase prevents nitrite from accumulating in the cell.
The fixation of NH4+ proceeds in the same way as in
photorespiration
• Glutamine synthetase in the chloroplasts transfers the
newly formed NH4+ at the expense of ATP to glutamate,
forming glutamine (Gb. 6).
• The same reaction also fixes the NH4+ released during
photorespiration.
• Because of the high rate of photorespiration, the amount
of NH4+ produced by the oxidation of glycine is about 5 to
10 times higher than the amount of NH4+ generated by
nitrate assimilation.
• Thus only a minor proportion of glutamine synthesis in the
leaves is actually involved in nitrate assimilation.
• Leaves also contain an isoenzyme of glutamine synthetase
in their cytosol.
Glufosinate
Glufosinate (Gb.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
(section 3.6) under the trade name Liberty (Aventis).
It has the advantage that it is degraded rapidly in the soil, leaving behind no toxic
degradation products.
The glutamine formed in the chloroplasts is converted via glutamate synthase
(also called glutamine-oxoglutarate amino transferase, abbreviated GOGAT),
Azaserine
Some chloroplasts and leucoplasts also
contain an NADPH-dependent glutamate
synthase.
Glutamate synthases are inhibited by the
substrate analogue azaserine , which is toxic
to plants.
a-Ketoglutarate, which is required for the glutamate synthase
reaction, is transported into the chloroplasts by a specific
translocator in counterexchange for malate, and the glutamate
formed is transported out of the chloroplasts into the cytosol
by another translocator, also in exchange for malate (Fig. 10.6).
Nitrate assimilation also takes place in the
roots
• 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.
• However, in the root cells the necessary reducing
equivalents are supplied exclusively by oxidation of
carbohydrates.
• The reduction of nitrite and the subsequent fixation of NH4+
(Gb. 8) occur in the leucoplasts, a differentiated form of
plastids .
Reduction of nitrite
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 (Gb.8).
• The uptake of glucose 6-phosphate proceeds in
counterexchange for triose phosphate.
• The glucose 6-phosphate-phosphate translocator of
leucoplasts differs from the triose phosphatephosphate translocator of chloroplasts in transporting
glucose 6-phosphate in addition to phosphate, triose
phosphate, and 3-phosphoglycerate.
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.
Nitrate assimilation must be regulated in such a way that the
production of amino acids does not exceed demand.
It is important that nitrate reduction does not proceed faster than
nitrite reduction, since otherwise toxic levels of nitrite would
accumulate in the cells.
Nitrate assimilation is strictly controlled
Under certain conditions such a dangerous accumulation of
nitrite can indeed occur in roots when excessive moisture
makes the soil anaerobic.
Flooded roots are able to discharge nitrite into water, avoiding
the buildup of toxic levels of nitrite, but this escape route is not
open to leaves, making the strict control of nitrate reduction
there especially important.
The NADH required for nitrate reduction in the cytosol can also
be provided during darkness (e.g., by glycolytic degradation of
glucose).
Reduction of nitrite and fixation of NH4+ in the chloroplasts
depends largely on photosynthesis providing reducing
equivalents and ATP.
Tamat
The end product of nitrate assimilation
is a whole spectrum of amino acids
All amino acids
present in the
mesophyll cells
are exported via
the sieve tubes.
Synthesis of
these amino
acids takes place
mainly in the
chloroplasts.
The sum of
amino acids can
be regarded as
the final product
of nitrate
assimilation.
The pattern of the
amino acids
synthesized varies
largely, depending
on the species and
the metabolic
conditions.
In most cases
glutamate and
glutamine
represent the major
portion of the
synthesized amino
acids.
Serine and
glycine, represent
a considerable
portion of the
total amino acids
present in the
mesophyll cells.
Glutamate is
exchange for malate
and glutamine in
exchange for
glutamate
Large
amounts of
alanine are
often formed
in C4 plants.
The end products of nitrate assimilation
• CO2 assimilation provides the carbon skeletons
required for the synthesis of the various amino acids.
3-Phosphoglycerate is the
most important carbon
precursor for the synthesis of
amino acids.
It is generated in the Calvin
cycle and is exported from
the chloroplasts to the
cytosol by the triose
phosphate-phosphate
translocator in exchange for
phosphate (Gb.11).
3-Phosphoglycerate is
converted in the cytosol by
phosphoglycerate mutase and
enolase to
phosphoenolpyruvate (PEP).
From PEP two pathways
branch off, the reaction via
pyruvate kinase leading to
pyruvate, and via PEPcarboxylase to oxaloacetate.
Oxaloacetate
• Oxaloacetate formed by PEP-carboxylase has two
functions in nitrate assimilation:
1. It is converted by transamination to aspartate,
which is the precursor for the synthesis of five
other amino acids (asparagine, threonine,
isoleucine, lysine, and methionine).
2. Together with pyruvate it is the precursor for the
formation of aketoglutarate, which is converted
by transamination to glutamate, being the
precursor of three other amino acids (glutamine,
arginine, and proline).
Special amino acid; Proline
• 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.
• Proline has no inhibitory effect on enzymes
even at very high concentrations
Biosynthesis of proline
• Glutamate is the precursor for the synthesis of
proline (Gb.12).
Compatible substance
Proline is classified as a compatible substance.
Other compatible substances, formed in certain plants
in response to water stress, are sugar alcohols such as
mannitol and betains, consisting of amino acids, such
as proline, glycine, and alanine, of which the amino
groups are methylated.
The latter are termed proline-, glycine-, and
alaninebetains.
The accumulation of such compatible substances,
especially in the cytosol, chloroplasts, and
mitochondria, minimizes in these compartments the
damaging effect of water shortage or a high salt
content of the soil.
Biosynthesis of Arginin
• The conversion of
ornithine to arginine
proceeds in the same
way as in the urea
cycle of animals, by
condensation with
carbamoyl phosphate
to citrulline
Aspartate is the precursor of five amino acids
• Aspartate is formed from oxaloacetate by
transamination with glutamate by glutamateoxaloacetate amino transferase (Fig. 10.14).
• The synthesis of asparagine from aspartate requires a
transitory phosphorylation of the terminal carboxylic
group by ATP, as in the synthesis of glutamine.
• Asparagine is formed to a large extent in the roots
(section 10.2), especially when NH4+ is the nitrogen
source in the soil.
• Synthesis of asparagine in the leaves often plays only a
minor role.
Aspartate is
the precursor
of five amino
acids
For the synthesis of lysine,
isoleucine, threonine, and
methionine, the first two
steps are basically the same as
for proline synthesis
For the synthesis of lysine the
semi-aldehyde condenses
with pyruvate and, in a
sequence of six reactions
involving reduction by NADPH
and transamination by
glutamate,
For the synthesis
of threonine,
the semialdehyde is
further reduced
to homoserine
Biosynthesis of leucine, valine, and
isoleucine
• The synthesis of
leucine, valine, and
isoleucine is also
subject to feedback
control by the end
products.
• Isopropylmalate
synthase is inhibited
by leucine and
threonine deaminase
is inhibited by
isoleucine
Aromatic
amino acids
Several steps in the synthesis of
aromatic amino acids are
regulated by product feedback
inhibition, thus adjusting the
rate of synthesis to demand.
Tryptophan stimulates the
synthesis of tyrosine and
phenylalanine [+].
The herbicide glyphosate (Gb
berikutnya) inhibits EPSP
Glyphosate acts as an herbicide
Glyphosate a structural analogue of
phosphoenolpyruvate, is a very strong inhibitor of
EPSP synthase.
Interruption of the shikimate pathway by
glyphosate has a lethal effect on plants.
Since the shikimate pathway is not present in
animals, glyphosate (under the trade name
Roundup, Monsanto) is used as an herbicide
Due to its simple structure, glyphosate is relatively
rapidly degraded by bacteria present in the soil.
Glyphosate is the herbicide with the highest sales
worldwide.
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.
Heme, likewise a tetrapyrrole, but with iron as the central atom, is a
constituent of cytochromes and catalase.
Porphobilinogen, a precursor for the synthesis of tetrapyrroles, is
formed by the condensation of two molecules of d-amino levulinate.
The synthesis of d-amino levulinate in plastids, cyanobacteria, and
many eubacteria proceeds by reduction of glutamate.
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