2. 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.
3. The transport of nitrate into the root cells proceeds as symport with two protons. 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 or uncouplers of respiration abolish mitochondrial ATP synthesis in the roots,
nitrate uptake normally comes to a stop.
4. these are a transporter with a relatively low affinity (half saturation >500mM nitrate) and a
transporter with a very high affinity (half saturation 20–100mM nitrate),where the latter is
induced only when required by metabolism.
The nitrate taken up into the root cells can be stored there temporarily in the vacuole.
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
5. 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. 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.
Thus, in spinach leaves, for instance, the highest nitrate content is found in the early morning.
6. 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
7.
8. Nitrate assimilation in the roots and
leaves of a plant. Nitrate is taken up from
the soil by the root. It can be stored in the
vacuoles of the root cells or assimilated in
the cells of the root epidermis and the
cortex.
Surplus nitrate is carried via the xylem
vessels to the mesophyll cells, where
nitrate can be temporarily stored in the
vacuole.
Nitrate is reduced to nitrite in the cytosol
and then nitrite is reduced further in the
chloroplasts to NH4+, from which amino
acids are formed.
H+ transport out of the cells of the root
and the mesophyll proceeds via a H+-P-
ATPase.
9. 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.
Each subunit contains an electron transport chain consisting of
1. one flavin adenine dinucleotide molecule (FAD),
2. one heme of the
3. cytochrome-b type (cyt-b557), and
4. one cofactor containing molybdenum (Fig.)
5. This cofactor is attached to molybdenum by two sulfur bonds and is called the molybdenum
cofactor, abbreviated MoCo.
Nitrate is reduced to nitrite in the cytosol
10. Nitrate is reduced to nitrite in the cytosol
Nitrate reductase transfers electrons from NADH to nitrate and convert it into nitrite
molybdenum cofactor, abbreviated MoCo
12. 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, which is located exclusively in plastids.
This enzyme utilizes reduced ferredoxin as electron donor, which is supplied by photosystem I as
a product of photosynthetic electron transport.
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
13. Nitrate reductase
P. denitrificans,
P. aeruginosa
P. stutzeri
Neisseria spp. including N. gonorrhoeae and N. meningitidis,
Rhodobacter sphaeroides and
Moraxella catarrhalis.
15. 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
16. 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.
Therefore all nitrite formed by nitrate reductase can be completely converted to ammonia. This
is important since nitrite is toxic to the cell.
17. It forms diazo compounds with amino groups of nucleobases (R–NH2), which are converted into alcohols with
the release of nitrogen.
Thus, for instance, cytosine can be converted to uracil. This reaction can lead to mutations in nucleic acids. The
very efficient reduction of nitrite by chloroplast nitrite reductase prevents nitrite from accumulating in the cell.
18. 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.
The activity of glutamine synthetase and its affinity for NH4+ are so high that the NH4+
produced by nitrite reductase is taken up completely. 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
19.
20. Nitrate assimilation also takes place 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+ occur
in the leucoplasts, a differentiated form of plastids
