Chapter No.

3 Assimilation of Nitrogen

CONTENTS:
1. The nitrogen cycle
2. Nitrogen fixation
3. Pathways of assimilation of nitrate and ammonium ions
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1. The Nitrogen Cycle: A Complex Pattern of Exchange
The global nitrogen supply is generally distributed between three major pools:
i. The atmospheric pool,
ii. The soil (and associated groundwater) pool,
iii. Nitrogen contained within the
biomass.
Central to the idea of a nitrogen cycle is the
pool of nitrogen found in the soil. Nitrogen
from the soil pool enters the biomass
principally in the form of nitrate(NO−3)
taken up by plants and microorganisms.
Once assimilated, nitrate nitrogen is
converted to organic nitrogen in the form
of amino acids and other nitrogenous
building blocks of proteins and other
macromolecules. Nitrogen moves further up
the food chain when animals consume
plants. Nitrogen is returned to the soil
through animal wastes or the death and
successive decomposition of all organisms.
 Some Essential Processes in The Nitrogen Cycle
a. Ammonification:
In the process of decomposition, organic nitrogen is converted to ammonia by a variety of
microorganisms including the fungi. This process is known as ammonification.
Some of the ammonia may volatilize and re-enter the atmosphere, but most of it is recycled
to nitrate by soil bacteria. The first step in the formation of nitrate is the oxidization of
ammonia to nitrite(NO-2) by bacteria of the genera Nitrosomonas or Nitrococcus.
b. Nitrification:
Nitrite is further oxidized to nitrate by members of the genus Nitrobacter. These two groups
are known as nitrifying bacteria and the result of their activities is called nitrification. Nitrifying
bacteria are chemoautotrophs, that is, the energy obtained by oxidizing in organic substances
such as ammonium or nitrite is used to convert CO2 to organic carbon. In taking up nitrate from
the soil, plants must compete with bacteria known as denitrifiers.
c. Denitrification:
By the process of denitrification, these bacteria reduce nitrate to di-nitrogen, which is then
returned to the atmosphere. Estimates for the amount of nitrogen lost to the atmosphere by
denitrification range from 93 million to 190 million metric tons(mmt) annually.

2. Biological Nitrogen Fixation

The loss of nitrogen from the soil pool through denitrification is largely balanced by additions
to the pool through conversion of atmospheric di-nitrogen to a combined or fixed form. The
process of reducing di-nitrogen to ammonia is known as nitrogen fixation or dinitrogen
fixation.
 Approximately 10% of the dinitrogen fixed annually is accounted for by nitrogen oxides in
the atmosphere. Lightning strikes and UV-radiation each provide sufficient energy to
convert dinitrogen to atmospheric nitrogen oxides (NO, N2O).
 Another 30% of the total dinitrogen fixed, 80mmt, is accounted for by industrial nitrogen
fixation for the production of agricultural fertilizers through the Haber-Bosch process.
 The bulk of the nitrogen fixed on a global scale, about 60% or 150 to 190mmt annually, is
accounted for by the reduction of dinitrogen to ammonia by living organisms.
 Plants are eukaryotic organisms, distinguished by the presence of a membrane-limited
nuclear compartment. Eukaryotic organisms are unable to fix dinitrogen because they do not
have the appropriate biochemical machinery.
 Bacteria and cyanobacteria are prokaryotic organisms; the genetic material is not
contained within a membrane-limited organelle. Nitrogen fixation is a prokaryote domain,
because only prokaryote organisms have the enzyme complex, called dinitrogenase, that
catalyzes the reduction of dinitrogen to ammonia.
Biological nitrogen fixation turns out to be a complex biochemical and physiological process.
Prokaryotes that fix nitrogen, called nitrogen- fixers, include both free-living organisms and
those that form symbiotic associations with other organisms.
 Nitrogen Fixation by N2-fixing free living Bacteria
Free-living, nitrogen-fixing bacteria are widespread. Their habitats include marine and
freshwater sediments, soils, leaf, and bark surfaces, and the intestinal tracts of various animals.
Although some species are aerobic (e.g., Azotobacter), most will fix dinitrogen only under
anaerobic conditions or in the presence of very-low-oxygen partial pressures (micro-aerobic).
These include both non-photosynthetic genera (Clostridium, Bacillus) and photosynthetic genera
(Rhodospirillum) of bacteria. In addition to the bacteria, several genera of cyanobacteria
(principally Anabaena, Nostoc) are represented by nitrogen-fixing species.
 Symbiotic Nitrogen Fixation
Several types of symbiotic nitrogen-fixing associations are known, including the well-known
association between various species of bacteria and leguminous plants. In symbiotic
associations the plant is identified as the host and the microbial partner is known as the
microsymbiont.
The most common form of symbiotic association results in the formation of enlarged,
multicellular structures, called nodules, on the root of the host plant. In the case of legumes,
the microsymbiont is a bacterium of one of three genera: Rhizobium, Bradyrhizobium, or
Azorhizobium. The legumes are a heterogeneous group traditionally assigned to the family
Leguminosae.
Modern treatments split the group into three families: Mimosaceae, Caesalpiniaceae,
and Fabaceae. Most of the economically important, nitrogen-fixing legumes are assigned to the
Fabaceae. Nitrogen-fixing nodules on roots of soybean are referred to as rhizobia. Curiously,
only one non-leguminous genus, Parasponia (of the family Ulmaceae), is known to form root
 nodules with a rhizobia symbiont. The rhizobia are further divided into species and sub-groups
called biovars according to their host range.
Nodules are also found in certain non-leguminous Plants such bayberry(Myrica),
Australian pine (Casuarina), some members of the family Rosaceae, and certain tropical
grasses. However, the microsymbiont in these non-leguminous nodules is a filamentous
bacterium (Frankia) of the group actinomycetes. Both Rhizobium and Frankia live freely in the
soil but fix dinitrogen only when in symbiotic association with an appropriate host plant e.g.,
Azolla is a small aquatic fern that harbors Anabaena in pockets within its leaves.
 Nodule Formation Through Rhizobia Infection to The Host Roots,
The sequence of events beginning with bacterial infection of the root and ending with formation
of mature, nitrogen-fixing nodules has been studied extensively in the legumes, historically
from the morphological outlook and more recently from the biochemical/molecular genetic
perspective. Overall the process involves a sequence of multiple interactions between the
bacteria and the host roots. In effect, the rhizobia and the roots of the prospective host plant
establish a dialogue in the form of chemical messages passed between the two partners. Based
on studies carried out primarily with Glycine, Trifolium, and Pisum, as many as nine or ten
separate developmental stages have been recognized. In order to simplify our discussion,
however, we will consider the events in four principal stages:
i. Multiplication of the rhizobia, colonization of the rhizosphere, and attachment to
epidermal and root hair cells.
ii. Characteristic curling of the root hairs and invasion of the bacteria to form an infection
thread.
iii. Nodule initiation and development in the root cortex. This stage is concurrent with
stage 1.
iv. Release of the bacteria from the infection thread and their differentiation as specialized
nitrogen-fixing cells.

(A) Rhizobia colonize the soil in the vicinity of the root hair in response to signals sent out from the host root. The
rhizobia in turn stimulate the root hair to curl while, at the same time, sending mitogenic signals that stimulate cell
division in the root cortex. (B) Rhizobia invade the root by digesting the root hair cell wall and forming an infection
thread. The rhizobia continue to multiply as the infection thread elongates toward the root cortex. (C) The infection
thread branches to penetrate numerous cortical cells as a visibly evident nodule develops on the root .)
 Stages involved in the Nodule formation
 Colonization and Nodule Initiation:
Rhizobia are free-living, saprophytic soil bacteria. Their numbers in the soil are highly
variable, from as few as zero or 10 to as many as 10 7gram−1 of soil, depending on the structure
of the soil, water content, and a variety of other factors. In the presence of host roots, the bacteria
are encouraged to multiply and colonize the rhizosphere. The initial attraction of rhizobia to
host roots appears to involve positive chemotaxis, or movement toward a chemical
stimulant(Chemotaxis).
Chemotaxis is an important adaptive feature in microorganisms generally. It allows the
organism to detect nutrients and other chemicals that are either beneficial or required for their
growth and reproduction. A group of chemicals that have been implicated in attraction of
rhizobia are the flavonoids.
Nod factor: Once rhizobia have colonized the rhizosphere, they begin to synthesize
morphogenic signal molecules called nodulation factors, or nod factors. Nod factors are
derivatives of chitin found in the cell walls of fungi and exoskeletons of insects. Nod factors
secreted into the soil solution by the rhizobia induce several significant changes in the growth
and metabolism of the host roots as a prelude to rhizobial invasion of the root hair and
subsequent nodule development. These changes include increased root hair production and the
development of shorter, thicker roots. Stimulated by the nod factors to re-new their growth, the
root hairs develop branching and curl at the tip. Before actually invading the host, rhizobia also
release mitogenic signals that stimulate localized cell divisions in the root cortex. These cell
divisions form the primary nodule meristem, defining the region in which the nodule will
eventually develop second center of cell division arises in the pericycle. Eventually these two
masses of dividing cells will fuse to form the complete nodule.
 Penetration of bacteria into the root hair by forming an infection thread
In the second stage of nodulation, the bacterium must penetrate the host cell wall in
order to enter the space between the wall and the plasma membrane. In pea, the preferred
attachment site is the tip of the growing root hair. The root hairs of pea grow by tip growth; that
is, new wall material is laid down only at the tip of the elongating hair cell.
There is some evidence that rhizobia release enzymes such as pectinase, hemicellulase,
and cellulase, which degrade cell wall materials. Once the rhizobia reach the outer surface of the
plasma membrane, tip growth of the root hair ceases and the cell membrane begins to invaginate.
The result is a tubular interruption into the cell called an infection thread, which contains the
invading rhizobia. The infection thread elongates by adding new membrane material by fusion
with vesicles derived from the Golgi apparatus.
 Finally, bacteria are released
The final Step in the infection process occurs when the bacteria are ‘‘released’’ into the host
cells. Actually the membrane of the infection thread buds off to form small vesicles, each
containing one or more individual bacteria. Shortly after release, the bacteria cease dividing,
enlarge, and differentiate into specialized nitrogen-fixing cells called bacteroids. The bacteroids
remain surrounded by a membrane, now called the peribacteroid membrane.

3. Pathways of assimilation of nitrate and ammonium ions

The first stable product of nitrogen fixation is ammonia (NH3), although at physiological pH
ammonia is almost certainly protonated to form ammonium ion:
NH3 + H + ↔ NH4
Plants that cannot fix dinitrogen meet their nutritional needs by taking in nitrogen from
the soil. While there are exceptions, most plants are able to assimilate either NH4+ or NO3−
depending on their relative availability in the soil. In most soils, ammonia is rapidly converted to
nitrate by the nitrifying bacteria.
Nitrifying bacteria do not grow well under anaerobic conditions and consequently
ammonia will accumulate in soils that are poorly drained. Nitrification itself is also inhibited in
strongly acidic soils. Some members of the family Ericaceae, typically found on acidic soils,
have adapted by preferentially utilizing ammonium as their nitrogen source. One extreme
example is the cranberry (Vaccinium macrocarpon), native to swamps and bogs of eastern
North America, which cannot exploit NO −3 as a nitrogen source and must take up nitrogen in
the form of ammonium ion. Regardless of the route taken, assimilation of mineral (inorganic)
nitrogen into organic molecules is a complex process that can be very energy intensive. It has
been estimated, for example, that assimilation of ammonium nitrogen consumes from 2 to 5
percent of the plant’s total energy production. Nitrate, on the other hand, must first be
reduced to ammonium before it can be assimilated, at a cost of nearly 15 percent of total energy
production. In this section, we will review the assimilation first of ammonium nitrogen and then
of nitrate nitrogen.

AMMONIUM IS ASSIMILATED BY GS/GOGAT

Although NH +4 Is readily available to many plants, either as the product of nitrogen fixation
or by uptake from the soil, it is also quite toxic to plants. In nitrogen-fixing systems, NH +4
will inhibit the action of dinitrogenase. Ammonium also interferes with the energy
metabolism of cells, especially ATP production. Even at low concentrations, NH +4 has the
potential to uncouple ATP formation from electron transport in both mitochondria and
chloroplasts.
It appears that plants can ill afford to accumulate excess free NH + 4 . It is assumed
that most plants avoid any toxicity problem by rapidly incorporating the NH +4 into amino
acids. The general pathway for NH +4 assimilation in nitrogen-fixing symbionts has been
worked out largely by supplying nodules with labeled dinitrogen ( 13 N 2 or 15 N 2 ). The
initial organic product is the amino acid glutamine. Assimilation of NH +4 into glutamine by
legume nodules is accomplished by the glutamate synthase cycle, a pathway involving the
sequential action of two enzymes: glutamine synthetase (GS) and glutamate synthase
(GOGAT) 2. Both GS and GOGAT are nodulin proteins that are expressed at high levels in the
host cytoplasm of infected cells, outside the peribacteroid membrane. The NH +4 formed in
the bacteroid must therefore diffuse across the peribacteroid membrane before it can be
assimilated. In the first reaction of the glutamate synthase cycle, catalyzed by GS, the addition
of an NH +4 group to glutamate forms the corresponding amide, glutamine:

glutamate + NH + 4 + ATP → glutamine + ADP + P i (11.3) 2

The acronym GOGAT refers to glutamine-2-oxoglutarate- amino-transferase. 2-Oxoglutarate
is an alternative name for α-ketoglutarate. Energy to drive the amination of glutamate is
provided by ATP, yet an additional cost of nitrogen fixation. Glutamine is then converted back
to glutamate by the transfer of the amide group to a molecule of α-ketoglutarate
.
glutamine + α-ketoglutarate + NADH → 2glutamate + NAD + (11.4)
Reaction 11.4 is catalyzed by GOGAT and requires reducing potential in the form of NADH.
The α-ketoglutarate is probably derived from photosynthetic carbon through respiration in the
host cell. α-Ketoglutarate is an intermediate in the respiratory pathway for the oxidation of
glucose. This reaction gives rise to two molecules of glutamate.
Since only one molecule of glutamate is required to keep the cycle going, the other is available
for export to the host plant. Overall, then, carbon skeletons originating with photosynthesis and
nitrogen fixed by the micros. There is a possible alternative pathway for nitrogen
assimilation, involving the direct reductive amination of α-ketoglutarate by the enzyme
glutamate dehydrogenase (GDH). However, although GDH activity has been detected in
nodules, there is no convincing evidence that it plays a significant role in NH +4 assimilation
under normal circumstances.
Both the quantities and activities of GS and GOGAT are much higher than GDH.GS alone may
account for as much as 2 percent of the total soluble protein outside the bacteroid. In addition,
GDH has a much lower affinity for NH +4 than does GS and could hardly be expected to
compete with GS for available NH + 4 . The cost of the glutamate synthase cycle is one ATP for
each NH +4 assimilated, but the benefit is rapid assimilation. The high affinity of GS for NH +
4 , together with the high concentration of the enzyme, ensure symbiont are combined to form
organic nitrogen that the free NH +4 concentration is kept below toxic levels. Before leaving
the glutamate synthase cycle, it is important to note that GS and GOGAT are not restricted to
nodules. These enzymes are located in the roots and leaves of non-nitrogen-fixing plants
where they also catalyze the assimilation of NH +4 nitrogen.

PII PROTEINS REGULATE GS/GOGAT

GS/GOGAT represents a critical metabolic point of coordination between nitrogen
assimilation (NH +4 ) and carbon metabolism (α-ketoglutarate). Cellular carbon/nitrogen
status is sensed by the signal sensing protein, PII. PII proteins are one of the most ubiquitous
and highly conserved regulatory proteins found in nature. They are present in the ancient
archea bacteria, eubacteria as well as eukaryotes such as plants. This relatively small
Chloroplastic protein of about 14kD a is nuclearen coded and senses both the ATP status and
α-ketoglutarate levels by allosteric. Cellular nitrogen status is sensed through changes in
glutamine availability which results in post-translational modification of PII by uridylylation
Like the light-regulated enzymes of the Calvin Cycle, GS/GOGAT can exist in either an
active or an inactive state. When glutamine levels in the GS/GOGAT cycle are low, PII
proteins are uridylylated using uridine triphosphate (UTP) to form PII-UMP.This modified PII
protein interacts with inactive GS/GOGAT to convert the latter from the inactive to the active
form which stimulates glutamine synthesis and hence the stimulates assimilation of NH + 4 .
Conversely, when glutamine levels are high, PII-UMP is converted back to PII which converts
the active GS/GOGAT to the inactive form which slows the rate of NH +4 assimilation.
FIXED NITROGEN IS EXPORTED AS ASPARAGINE
AND UREIDES
The final step in nitrogen fixation is the export of the fixed nitrogen from the nodule to other
regions of the host plant. Export of the organic nitrogen products from nodules is primarily
through the xylem. Consequently, the form in which the nitrogen is exported has been identified
primarily by analysis of xylem sap. There are some pit falls to such analyses, however, as there
is no guarantee that all of the nitrogen present in sap represents current nodule production.
Some of the better analyses have been conducted directly on detached nodules or by
monitoring the flow of organic nitrogen following fixation of 15 N 2 . Glutamine is the
principal organic product of nitrogen fixation, it rarely accounts for a significant fraction of
the nitrogen exported, at least in legumes. In some groups of legumes, largely those of
temperate origins, such as pea and clover, the amino acid asparagine is the predominant form
translocated.
 Legumes of tropical origins, for example, soybean and Cow pea, appear to export
predominantly derivatives of urea, known as ureides .The biosynthetic pathway for asparagine
in nodules involves two transamination reactions. A transamination reaction is the transfer of
an amino group from an amino acid to the carboxyl group of a keto acid.
Transamination reactions, catalyzed by a class of enzymes known as aminotranferases,
enable nitrogen initially fixed in glutamate to be incorporated into other amino acids and,
ultimately, into protein. Aminotransferases are found throughout the plant in the cytosol, in
chloroplasts, and in microbodies wherever protein synthesis activity is high. The enzymes
involved in asparagine biosynthesis in nodules appear to be similar to those found elsewhere in
the plant.

First step
The first step is the transfer of an amino group from glutamate to oxaloacetate, catalyzed by the
enzyme aspartate aminotransferase.
glutamate + oxaloacetate→ α-ketoglutarate + aspartate
The glutamate used in this reaction is derived from the GS-GOGAT reactions in the nodule.
In order to continue the synthesis and export of asparagine, the nodule requires a continued
supply of the 4-carbon acid oxaloacetate. The oxidation of carbon in the nodule; oxaloacetate is
another intermediate in the respiratory oxidation of glucose. However, nodules from a number
of species exhibit high activities of the enzyme phosphoenolpyruvate carboxylase (PEP
carboxylase). PEP carboxylase catalyzes the addition of a carbon dioxide the 3-carbon
phosphoenolpyruvate (PEP) to form oxaloacetate (OAA).PEP carboxylase is involved in a
number of important metabolic pathways in plants and animals, including respiration and
photosynthesis. PEP + CO 2 → OAA (11.6)

Second step:
In the second step of asparagine biosynthesis, the amide nitrogen is transferred from glutamine
to aspartate.
glutamine + aspartate + ATP→ glutamate + asparagine + ADP + P i
The enzyme for this reaction is asparagine synthetase and the reaction is driven by the energy
of one molecule of ATP for each asparagine synthesized. The synthesis of ureides is more
complex, both biochemically and with respect to the division of labor between the micro
symbiont and tissues of the host plant. Allantoin and allantoic acid are formed by the
oxidation of purine nucleotides, which apparently requires an active symbiosis. Ureides
apparently serve specifically for the transport of nitrogen. They are translocated through the
xylem to other regions of the plant, where they are rapidly metabolized. In the process, NH +4
is released, which is then reassimilated via GS and GOGAT in the target tissue.
Although the synthesis of asparagine, and especially the ureides, both appear to be complex
processes, there are some advantages relating to the energy costs and efficiencies of nitrogen
export. It has been estimated that the carbon metabolism associated with nitrogen Export may
consume as much as 20 percent of the photosynthate diverted to nitrogen fixation.. The ureides,
with a carbon to nitrogen ratio of 1 (C:N 1), are the most economic in the use of carbon. Both
asparagine and citrulline (C:N 2) require more carbon in their transport and glutamine (C:N
2.5) would be the least economic. The energy costs of ureides, asparagine, and citrulline, in
terms of ATP consumed, are about the same, so the principal advantage to be gained by the
ureide-formers appears to be a favorable carbon economy.

PLANTS GENERALLY TAKE UP NITROGEN

IN THE FORM OF NITRATE
Nitrate (NO −3 ) is the more abundant form of nitrogen in soils and is most available to plants
that do not form nitrogen-fixing associations. However, in spite of numerous studies describing
the physiology of NO −3 uptake, there is a great amount of uncertainty surrounding the
mechanism of NO −3 transport into roots. It has been shown in various studies that uptake of NO
−3 is sensitive to:
(1) low temperature,
(2) inhibitors of both respiration and protein synthesis,
(3) anaerobic conditions.
All of these results support the hypothesis that NO −3 transport across the root cell membrane
is an energy-dependent process mediated by a carrier protein. In root cells that have never
been exposed to nitrate, there appears to be a limited capacity for NO −3 uptake. This suggests
a small amount of carrier is present in the membrane at all times (that is, a constitutive
protein). On exposure to external nitrate, the rate of uptake Increases from two to five fold, but
addition of inhibitors of protein synthesis causes the rate to fall rapidly back to the constitutive
level. This pronounced sensitivity of NO −3 uptake to inhibitors of protein synthesis suggests
that the bulk of the carrier protein is inducible, that is, the presence of NO −3 in the soil
stimulates the synthesis of new carrier protein. Once inside the root, NO −3 may be stored in
the vacuole, assimilated in the root cells, or translocated in the xylem to the leaves for
assimilation. Nitrate cannot be assimilated directly but must first be reduced to NH +4. In order
to be assimilated into organic compounds.
This is a two-step process, the first being the reduction of NO −3 to nitrite (NO −2 ) by the
enzyme nitrate reductase (NR).
2H + + NO −3 + 2e − → NO −2 + H 2 O (11.8)
NR is generally assumed to be a cytosolic enzyme. The product NO −2 then moves into plastids
(in roots) or chloroplasts (in leaves) where it is quickly reduced to NH + 4 by the enzyme
nitrite reductase (NiR). In leaves, the electrons required for the reduction of NO −2 to NH +4
are generated by photosynthetic electron transport.
8H + + NO −2 + 6e − → NH + 4 + 2H 2 O (11.9)
Thus, the assimilation of NO −3 competes with the assimilation of CO 2 for photosynthetic
electrons. As a consequence, NO −3 assimilation in leaves can also be considered a
photosynthetic process similar to CO 2 assimilation. The interactions between carbon and
nitrogen metabolism indicate the importance of photosynthetic electron transport in overall
primary reductive metabolism.
Nitrite is toxic and is rarely found at high concentrations in plants. This is no doubt because the
activity of NiR (per gram dry weight of tissue) is normally several times higher than the activity
 of NR. The resulting ammonia is then rapidly assimilated into organic compounds via the
GS/GOGAT system already described. In non-nitrogen-fixing systems, both GS and GOGAT
are commonly found in root and leaf cells. GS is found in the cytosol of root cells and in both
the cytosol and chloroplasts of leaf cells. GOGAT is a plastid enzyme, localized in the
chloroplasts of leaves and in plastids in roots. Depending on its location, GOGAT may use
ferredoxin, NADH, or NADPH as electron donors. Nitrate reductase is a ubiquitous enzyme
found in both prokaryote and eukaryote cells.
In prokaryotes, the principal electron donor is ferredoxin, while in higher plants electrons are
donated by the reduced forms of one of the pyrimidin enucleotides, nicotinamide adenine
dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP).The enzyme
isolated from a variety of higher plants is composed of two identical subunits with a
molecular mass of approximately 115kD.
NR :
A key constituent of NR is molybdenum; NR is the principal Mo-protein in non-nitrogen-
fixing plants. One of the results of Mo deficiency is markedly reduced levels of nitrate
reductase activity and consequent nitrogen starvation. NR is a highly regulated, inducible
enzyme. It has long been recognized that both substrate (NO −3 ) and light are required for
maximum activity and that induction involves an increase in the level of NR messenger RNA
followed by de novo synthesis of NR protein.
Treatment of cereal seedlings such as barley or maize with nitrate in the dark induces relatively
low levels of NR activity, but activity is strongly promoted if seedlings are also exposed to
light. Induction by light is eliminated by various treatments that interfere with chloroplast
development or photosynthetic energy transformations, implying a requirement for
photosynthetic energy. NR protein kinase. Protein kinases, first characterized by Edwin Krebs
and Edmund Fischer in the 1950s, are a ubiquitous class of enzymes that phosphorylate other
proteins by transferring a phosphate group from adenosine triphosphate (ATP). The
phosphate group can then be removed by a second enzyme called protein phosphatase. It is
increasingly evident that, by switching enzymes and other proteins on and off, reversible
protein phosphorylation plays a central role in regulating metabolism. The fundamental role of
protein kinases was recognized by the award of the Nobel Prize to Krebs and Fischer in 1992.
In the case of NR, the enzyme appears to be active in both the phosphorylated and
nonphosphorylated states. When NR is phosphorylated and the leaf is transferred from the
light to dark, however, the enzyme is rapidly inactivated by binding with a small inhibitor
protein.NR activity is slowly restored on return to light by are lease of the inhibitor protein and
subsequent phosphatase action. It ensures that nitrate reduction is engaged only after
photosynthesis is fully active and able to provide both the energy required and the carbon
skeletons necessary for incorporation of ammonia.
As indicated earlier, nitrate assimilation can be carried out in either the root or shoot tissues in
most plants. Several studies have shown that the proportion of NO −3 reduced in the root or
shoot depends to a large extent on the external NO −3 concentration.
 At low concentrations, most of the NO −3 can be reduced within the root tissues and
translocated to the shoot as amino acids or amides.
 At higher concentrations of NO −3 , assimilation in the roots becomes limiting and a higher
proportion of the NO −3 finds its way into the translocation stream.
Thus, at higher concentrations, a higher proportion of the nitrogen is assimilated in the leaves.

Not all plants have the same capacity to metabolize NO −3 in their roots. In the extreme, NO −3
is virtually the sole nitrogen source in the xylem sap of cocklebur. This is because cocklebur has
no detectable NR in its roots. On the other hand, plants such as barley and sunflower
translocate roughly equal proportions of NO −3 and amino acid/amide nitrogen, and radish
translocates only about 15 percent of its nitrogen as NO −3 .