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1 The reduction of nitrate to NH[sub(3)] proceeds in two reactions

1 The reduction of nitrate to NH[sub(3)] proceeds in two reactions

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MESOPHYLL CELL



CHLOROPLAST

Amino

acid



NH4+

6 Ferredoxin ox



Nitrite

reductase



6 Ferredoxin red



VACUOLE



NO2–



Amino

acid



NO2–

NAD +



Nitrate

reductase



NADH + H +

NO3–

ATP



ADP + P



H+



NO3–



H+



H+



NO3–



XYLEM

Amide



ROOT CELL

LEUCOPLAST

VACUOLE

Amide



Amide



Nitrate

reductase

NO3–



NO3–



Nitrite

reductase

NO2–



NADH NAD +

+ H+

ATP



ADP + P



H+



NO2–



NH4+



3 NADPH 3 NADP +

+ 3 H+



2 H+



2 H+

NO3–



SOIL



Figure 10.1 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 stored temporarily in the vacuole. Nitrate is

reduced to nitrite in the cytosol and then nitrite is reduced further in the chloroplasts to NH4ϩ, from which amino acids

are formed. Hϩ transport out of the cells of the root and the mesophyll proceeds via an Hϩ-P-ATPase.



276



Figure 10.2 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.



10



Nitrate assimilation is essential for the synthesis of organic matter



A

Nitrate reductase



NADH + H +

FAD



NO3–



MoCo



Cyt-b557



NAD +



NO2– + H2O



B

Amino acid sequence:

HOOC



1 FAD



1 Heme



1 MoCo



Domain



Domain



Domain



Figure 10.3

The molybdenum cofactor

(MoCo).



Mo

O



HN

H2N



N



4+



S

H

N

N

H



NH2



S

O

C



C

CH



CH2



OH



O



P



O



O



Pterine



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. Each subunit comprises an electron transport chain (Fig. 10.2)

consisting of one flavin adenine dinucleotide molecule (FAD), one heme of

the cytochrome-b type (cyt-b557), and one cofactor containing molybdenum

(Fig. 10.3). The latter 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 bound Mo atom probably changes between oxidation states ϩIV and ϩVI. The three redox carriers of nitrate reductase are

each covalently bound to the subunit of the enzyme. The protein chain of

the subunit can be cleaved by limited proteolysis into three domains, each

of which contains only one of the redox carriers. These separated domains,

as well as the holoenzyme, are able to catalyze via their redox carriers electron transport to artificial electron acceptors (e.g., from NADPH to Feϩϩϩ

ions via the FAD domain or from reduced methylviologen (Fig. 3.39) to



10.1 The reduction of nitrate to NH3 proceeds in two reactions



277



Light



6 Ferredoxin

reduced

Photosystem I



Nitrite reductase

4 Fe–4 S



FAD



6e – Siroheme



6 Ferredoxin

oxidized



Figure 10.4 Nitrite reductase in chloroplasts transfers electrons from ferredoxin to

nitrite. Reduction of ferredoxin by photosystem I is shown in Figure 3.16.



nitrate via the Mo domain). Moreover, nitrate reductase reduces chlorate

(ClO3Ϫ) to chlorite (ClO2–). The latter is a very strong oxidant and therefore highly toxic to plant cells. In the past chlorate was used as an inexpensive nonselective herbicide for keeping railway tracks free of vegetation.



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

(Fig. 10.4), which is located exclusively in plastids. This enzyme utilizes

reduced ferredoxin as electron donor, which is supplied as a product of

photosynthetic electron transport by photosystem I (Fig. 3.31). To a

much lesser extent, the reduced ferredoxin can also be provided during

darkness via reduction by NADPH. The latter is generated by the oxidative pentose phosphate pathway present in chloroplasts and leucoplasts

(Figs. 6.21, 10.8).

Nitrite reductase contains a covalently bound 4Fe-4S cluster (see Fig.

3.26), one molecule of FAD, and one siroheme. Siroheme (Fig. 10.5) 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 (see section 10.5).

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



NO2– + 8 H +

NH4+ + 2 H2O



278



Figure 10.5

siroheme.



10



Nitrate assimilation is essential for the synthesis of organic matter



Structure of



COOH

COOH CH2

CH2



CH2



HOOC



CH2



N



CH3

N



HOOC



CH2



CH2



COOH



CH2



CH2 COOH



N



Fe



H



CH2



N



CH3



H

CH2



CH2



CH2



COOH



COOH



Siroheme



the cell. It forms diazo compounds with amino groups of nucleobases

(R–NH2), which are converted into alcohols with the release of nitrogen.

R Ϫ NH2 ϩ NO2Ϫ → [R Ϫ N ϭ N Ϫ OH ϩ OHϪ ] → R Ϫ OH ϩ N2 ϩ OHϪ



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

plastid nitrite reductase prevents nitrite from accumulating in the cell.



The fixation of NH4ϩ proceeds in the same way as in the

photorespiratory cycle

Glutamine synthetase in the chloroplasts transfers the newly formed NH4ϩ at

the expense of ATP to glutamate, forming glutamine (Fig. 10.6). The activity of glutamine synthetase and its affinity for NH4ϩ (KmϷ5 · 10–6 mol/L)

are so high that the NH4ϩ produced by nitrite reductase is completely

assimilated into glutamine. Glutamine synthetase also fixes the NH4ϩ

released during photorespiration (see Fig. 7.9). Due to the high rate of photorespiration, the amount of NH4ϩ produced by the oxidation of glycine

is about 5 to 10 times higher than generated by nitrate assimilation. Thus

only a minor proportion of glutamine synthesis in the leaves results from

nitrate assimilation. Leaves also contain an isoenzyme of glutamine synthetase in their cytosol.



10.1 The reduction of nitrate to NH3 proceeds in two reactions



CHLOROPLAST



CYTOSOL



Nitrite

reductase



Nitrate

reductase

NO2–



NH4+



Glutamine

synthetase



279



NO3–



NO2–



Glutamine



Glutamate

synthase



Main products of

nitrate assimilation



Glutamate

Glutamate



Glutamate



Glutamate

Malate



NH4+



ATP



2 Ferredoxinox



ADP + P



2 Ferredoxinred



Glyoxylate



PEROXISOME

Malate

Glutamine



NH4+



α-Ketoglutarate



α-Ketoglutarate



Glycine



MITOCHONDRIUM



NH4+

Photorespiration



Serine

Hydroxypyruvate



Figure 10.6 Compartmentation of nitrate assimilation reactions and the

photorespiratory pathway in mesophyll cells. NH4ϩ formed in the photorespiratory

pathway is colored black and NH4ϩ formed by nitrate assimilation is colored red. The

main products of nitrate assimilation are marked with a red arrow.



Glufosinate (Fig. 10.7), a substrate analogue of glutamate, is a strong

inhibitor of the glutamine synthetase. When glufosinate is applied to

plants the synthesis of glutamine is inhibited and subsequently toxic levels

of ammonia accumulate. Glufosinate is a herbicide (section 3.6) and commercially available under the trade name Basta (Bayer Crop Science). This

herbicide degrades rapidly in the soil, without the accumulation of toxic

degradation products. Recently glufosinate-resistant crop plants have been

generated by genetic engineering, enabling the use of glufosinate as a selective herbicide for weed control in growing cultures (section 22.6).

Glutamine together with α-ketoglutarate is converted by glutamate synthase (also called glutamine-oxoglutarate aminotransferase, abbreviated



Glycolate



280



Figure 10.7

Glufosinate (also called

phosphinotricin) is a

substrate analogue of

glutamate and a strong

inhibitor of glutamine

synthetase. Ammonium

glufosinate is a herbicide

(Basta, Bayer Crop

Science). Azaserine is also

a substrate analogue of

glutamate and an inhibitor

of glutamate synthase.



10



Nitrate assimilation is essential for the synthesis of organic matter



COO



COO



H C NH3



H C NH3



CH2



CH2



CH2



O



CH3 P OH

O



Glufosinate



C O

CH2 N NH2



Azaserine



GOGAT), to two molecules of glutamate (see also Fig. 7.9). Ferredoxin is

used as reductant in this reaction. Some chloroplasts and leucoplasts also

contain an NADPH-dependent glutamate synthase. Glutamate synthases

are inhibited by the substrate analogue azaserine (Fig. 10.7), which is toxic

to plants.

α-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). Yet another translocator in the chloroplast envelope transports glutamine in counter-exchange for glutamate, enabling the

export of glutamine from the chloroplasts.



10.2 Nitrate assimilation also takes place in

the roots

As mentioned, nitrate assimilation occurs in part, and in some species

even mainly, in the roots. NH4ϩ taken up from the soil is normally fixed

in the roots. The reduction of nitrate and nitrite as well as the fixation of

NH4ϩ proceeds in the root cells analogously to that of the mesophyll cells.

However, in the root cells the necessary reducing equivalents are supplied

exclusively by oxidation of carbohydrates. In roots the reduction of nitrite

and the subsequent fixation of NH4ϩ (Fig. 10.8) occur in the leucoplasts, a

differentiated form of plastids (section 1.3).



The oxidative pentose phosphate pathway in leucoplasts

provides reducing equivalents for nitrite reduction

In leucoplasts the reducing equivalents required for the reduction of nitrite

and the formation of glutamate are provided by oxidation of glucose



10.2 Nitrate assimilation also takes place in the roots



Triose

phosphate



LEUCOPLAST



281



2 Triose

phosphate



Triose

phosphate



1



Fructose1,6-bisphosphate



2



P



3 Ribulose 5phosphate



3 Glucose 6phosphate



6 NADPH

+ 3 CO2



6 NADP +



Fructose 6phosphate



Glucose 6phosphate



oxidative

pentose phosphate

pathway



3 NADP +



NADP + NADPH



3 NADPH



CYTOSOL

6 Fdred 6 Fdox

NO2–



Glutamate

NH4+



2 Glutamate



ATP



Nitrite

reductase



2 Fdred 2 Fdox



ADP

+P



Glutamine

synthetase



α-Ketoglutarate

Glutamate

synthase



Glutamate

α-Ketoglutarate



ATP



ATP



ADP



ADP



Figure 10.8 The oxidative pentose phosphate pathway provides the reducing

equivalents for nitrite reduction in plastids (leucoplasts) from non-green tissues. In

some plastids, glucose 1-phosphate is transported in counter-exchange for triose

phosphate or phosphate. Fd ϭ ferredoxin.



6-phosphate via the oxidative pentose phosphate pathway (section 6.5, Fig.

10.8). The uptake of glucose 6-phosphate proceeds in counter-exchange for

triose phosphate. The glucose 6-phosphate-phosphate translocator of leucoplasts differs from the triose phosphate-phosphate translocator of chloroplasts in transporting glucose 6-phosphate in addition to phosphate, triose

phosphate, and 3-phosphoglycerate. In the oxidative pentose phosphate

pathway, three molecules of glucose 6-phosphate are converted to three

molecules of ribulose 5-phosphate with the release of three molecules of

CO2, yielding six molecules of NADPH. The subsequent reactions yield one

molecule of triose phosphate and two molecules of fructose 6-phosphate;

the latter are reconverted to glucose 6-phosphate via hexose phosphate

isomerase. In the cytosol, glucose 6-phosphate is regenerated from two



282



10



Nitrate assimilation is essential for the synthesis of organic matter



molecules of triose phosphate via aldolase, cytosolic fructose 1,6-bisphosphatase, and hexose phosphate isomerase. In this way glucose 6-phosphate

can be completely oxidized to CO2 in order to produce NADPH.

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. Also, the glutamate synthase of the leucoplasts uses reduced ferredoxin as redox partner, although

some leucoplasts also contain a glutamate synthase that utilizes NADPH

or NADH directly as reductant. Nitrate reduction in the roots provides the

shoot with organic nitrogen compounds mostly as glutamine and asparagine via the transpiration stream in the xylem vessels. This is also the case

when NH4ϩ is the nitrogen source in the soil.



10.3 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 the demand. Finally, it is important that

nitrate reduction does not proceed faster than nitrite reduction, to prevent

the accumulation of toxic levels of nitrite (section 10.1). For example, dangerous levels of nitrite may accumulate under anaerobic soil conditions in

the case of excessive moisture. Flooded roots are able to release nitrite into

the soil water, avoiding the buildup of toxic levels of nitrite. This escape

route, however, does not function in leaves, and there the strict control of

nitrate reduction is especially important.

The NADH required for nitrate reduction in the cytosol can also be

provided during darkness (e.g., by glycolytic degradation of glucose).

The reduction of nitrite and fixation of NH4ϩ in the chloroplasts depends

largely on photosynthesis providing reducing equivalents and ATP,

whereas the oxidative pentose phosphate pathway can offer only limited

amounts of reducing equivalents in the dark. Therefore, during darkness

nitrate reduction in the leaves has to be slowed down or even switched off

to prevent an accumulation of nitrite. This illustrates how essential it is for

a plant to regulate the activity of nitrate reductase, which is the entrance

step of nitrate assimilation.



10.3 Nitrate assimilation is strictly controlled



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 time is

only a few hours. The rate of de novo synthesis of this enzyme is very high.

Thus, by regulating its synthesis, the activity of nitrate reductase in the tissue can be altered within hours.

Various factors control the synthesis of the enzyme at the level of gene

expression. Nitrate and light stimulate the enzyme synthesis. Part of the

light effect is caused by carbohydrates generated by photosynthesis. The

synthesis of the nitrate reductase protein is stimulated by glucose and other

carbohydrates generated by photosynthesis, and is inhibited by NH4ϩ,

glutamine and other amino acids (Fig. 10.9). Sensors seem to be present in

the cell that adjust via regulation of gene expression the capacity of nitrate

reductase both to the demand for amino acids and to the supply of carbon

skeletons from CO2 assimilation for its synthesis.



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 acute accumulation of nitrite in the plants during

darkening or sudden shading of the plant. Rapid inactivation within minutes of nitrate reductase occurs via phosphorylation of the nitrate reductase

protein (Fig. 10.9). Upon darkening, a serine residue, which is located in

the nitrate reductase protein between the heme and the MoCo domain, is

phosphorylated by a protein kinase termed nitrate reductase kinase. This

protein kinase is inhibited by the photosynthesis product triose phosphate

and other phosphate esters and is stimulated by Caϩϩ ions, a messenger

compound of many signal transduction chains (section 19.1). The phosphorylated nitrate reductase binds an inhibitor protein, which interrupts

the electron transport between cytochrome-b557 and the MoCo domain

(Fig. 10.2). The nitrate reductase phosphatase hydrolyzes the enzyme’s

serine phosphate and this causes the inhibitor protein to be released from

the enzyme and thus nitrate reductase is restored to an activated state.

Okadaic acid inhibits nitrate reductase phosphatase and in this way also

inhibits the reactivation of nitrate reductase. Since the phosphorylation of

the serine residue and the binding of the inhibitor protein are reversible,

there is a dynamic equilibrium between the active and inactive form of the

nitrate reductase. The inhibition of nitrate reductase kinase by triose phosphate and other phosphate esters ensures that nitrate reductase is active



283



284



10



Nitrate assimilation is essential for the synthesis of organic matter



Other stimuli



Light



Photosynthesis



Triose phosphate

and other

phosphate esters



Ca++

Glucose

(other

carbohydrates)



Light



Nitrate

Nitrate reductase

kinase

ATP



+



Nitrate reductase

gene







+ –



Nitrate reductase

active

Ser



Nitrate reductase

active



OH



Ser

P



O



Nitrate reductase

inactive

P



Ser



O



P





Nitrate reductase

phosphatase



+

NH4 ,



Glutamine

(other amino acids)



Inhibitor

protein



ADP



Okadaic acid



Figure 10.9 Regulation of nitrate reductase (NR). Synthesis of the NR protein is

stimulated by carbohydrates (perhaps glucose or its metabolic products) and light [ϩ],

and inhibited by glutamine or other amino acids [–]. The newly formed NR protein

is degraded within a few hours. Nitrate reductase is inhibited by phosphorylation

of a serine residue and the subsequent interaction with an inhibitor protein. After

hydrolytic liberation of the phosphate residue by a protein phosphatase, the inhibitor is

dissociated and nitrate reductase regains its full activity. There is a dynamic equilibrium

between the active and inactive form of the enzyme. The activity of the nitrate reductase

kinase is inhibited by products of photosynthesis in the light, such as triose phosphate

and other phosphate esters. In this way nitrate reductase is active in the light. Through

the effect of Caϩϩ on nitrate reductase kinase other still not identified factors may

modulate the activity of nitrate reductase. Okadaic acid, an inhibitor of protein

phosphatases, counteracts the activation of nitrate reductase. (After Huber et al., 1996.)



only when CO2 fixation is operating for delivery of the carbon skeletons for

amino acid synthesis, which is discussed in the next section.



14-3-3 proteins are important metabolic regulators

It was discovered that the nitrate reductase inhibitor protein belongs to a

family of highly conserved regulatory proteins called 14-3-3 proteins, which

are widely spread throughout the animal and plant worlds. 14-3-3 proteins



10.3 Nitrate assimilation is strictly controlled



bind to a specific binding site of the target protein with six amino acids

(Arg-X-X-SerP/ThrP-X-Pro), which contain a serine or threonine phosphate in position 4. The importance of these latter amino acids for nitrate

reductase was verified in an experiment, in which the serine in the 14-3-3

protein binding site of nitrate reductase was exchanged for alanine via site

directed mutagenesis; the altered nitrate reductase was no longer inactivated by phosphorylation. 14-3-3 proteins bind to a variety of proteins and

change their activity. They form a large family of multifunctional regulatory

proteins, many isoforms of which occur in a single plant. Thus 14-3-3 proteins regulate in plants the activity of the H؉-P-ATPase (section 8.2) of the

plasma membrane. 14-3-3 proteins regulate the function of transcription

factors (section 20.2) and protein transport into chloroplasts (section 21.3).

There are indications that 14-3-3 proteins are involved in the regulation of

signal transduction (section 19.1) as they bind to various protein kinases

and play a role in defense processes against biotic and abiotic stress. The

elucidation of these various functions of 14.3.3 proteins is at present a very

hot topic in research.

This important function of the 14-3-3 proteins in metabolic regulation is

exploited by the pathogenic fungus Fusicoccum amygdalis to attack plants.

This fungus forms the compound fusicoccin, which binds specifically to the

14-3-3 protein binding sites of various proteins and thus cancels the regulatory function of 14-3-3 proteins. In this way fusicoccin disrupts the metabolism to such an extent that the plant finally dies. This attack proceeds in a

subtle way. When F. amygdalis infects peach or almond trees, at first only

a few leaves are affected. In these leaves the fungus excretes fusicoccin into

the apoplasts, from which it is spread via the transpiration stream through

the other parts of the plant. Finally, fusicoccin arrives in the guard cells;

where it causes an irreversible transformation of the Hϩ-P-ATPase into the

active form resulting in continuously opened stomata (Fig. 8.4). This leads

to a very high loss of water; consequently, the leaves wilt, the tree dies,

which ultimately is the nutrient source of the fungus.



There are great similarities between the regulation of nitrate

reductase and sucrose phosphate synthase

The regulation principle of nitrate reduction by phosphorylation of serine

residues by special protein kinases and protein phosphatases is remarkably

similar to the regulation of sucrose phosphate synthase discussed in Chapter

9 (Fig. 9.18). Upon darkening, both enzymes are inactivated by phosphorylation, which in the case of nitrate reductase also requires a binding of an

inhibitor protein. Both enzymes are reactivated by protein phosphatases,

which are inhibited by okadaic acid. Also, sucrose phosphate synthase has



285



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