Remember from the previous class discussion, nitrogen (N₂) must first be fixed usually into a reduced form such as ammonia. Ammonia is usually rapidly oxidized into nitrate by nitrifying bacteria in soils so nitrate is the usual form of nitrogen available to most plants.

The N cycle:

• Nitrate Uptake
• The nitrate uptake system in plant must be versatile and robust because
1. Plants have to transport sufficient nitrate to satisfy the total demand for nitrogen in the face of external nitrate concentrations that can vary by five orders of magnitude.
2. Plants must compete for N in the soil with abiotic and biotic processes such as erosion, leaching and microbial competition.
3. To function efficiently and the face of such environmental variation, plants have evolved 3 transport systems that are:
• active
• regulated
• multiphasic
• The energy that drives nitrate uptake comes from the proton gradient maintained across the plasma membrane by the H⁺ ATPase
• See Fig. 1 of Crawford (1995) The Plant Cell 7: 859-868 and Fig. 16.34 of the class text.



 

• The H⁺-ATPase in the PM pumps protons out of the cell producing pH and electrical () gradients.
• The nitrate transporters (Ntr) cotransport two or more protons per nitrate into the cell.
• Nitrate can be transported across the tonoplast membrane and stored in the vacuole.
• Nitrate in the cytosol is reduced to nitrite that enters the plastid and is reduced to ammonia.
• Ammonia is fixed into glutamate (Glu) to produce glutamine (Gln) by the action of glutamine synthetase (GS).
• Nitrate also acts as a signal to increase the expression of nitrate reductase (NR), nitrite reductase (NiR) and Ntr genes.
• The initial uptake of nitrate occurs across the plasma membrane of epidermal and cortical cells of the root.  Subsequent transport across the tonoplast membrane and the PM of cells in the vascular system and leaf distributes NO₃^- throughout leaf and shoot tissue.  Ultimately N can be stored in the seed or other storage organ.  Figure 1 of Crawford `95 and Fig. 16.34 of the class text shows a brief outline of the major routes of metabolism of nitrogen within a plant:



 

1. Constitutive high-affinity transport system (CHATS)
• Responsible for nitrate uptake at low concentrations (below ~1 mM).
• Saturation kinetics, with Km values below 300 µM.
2. Inducible high-affinity transport system (IHATS)
• Induced by NO₃^- taken up by CHATS.
• Increases overall NO₃^- uptake transiently.
3. Low affinity transport system (LATS)
• Uptake of nitrate at high concentration (> 0.2 mM).
• Like the high affinity systems, the low affinity systems are electrogenic and involve proton cotransport.
• Show linear rather than saturation kinetics.
• Not saturated even at 50 mM NO₃^-.

1. ¹³N has a half-life of only 10 min.
2. ¹⁵N is not radioactive.

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3. When NO₃^- is supplied to cells it is metabolized and effluxed as equilibrium is established with preexisting pools.

• An alternative substrate of the NO₃^- uptake mechanism.
• ³⁶Cl can be used as the radioactive isotope to study nitrate uptake properties.
• ClO₃^- inhibits NO₃^- uptake and vice versa.
• ClO₃^- is not assimilated.
• ClO₃^- uptake into roots is extremely sensitive to the inhibitor FCCP, an uncoupler of energy-dependent ion transport.

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• ¹³NO₃^- influx displays typical Michaelis-Menten kinetics
• The rate of uptake is depending upon the length of the previous treatment with nitrate, thus suggesting that a nitrate transport system is induced by nitrate.

Not only is the nitrate uptake system induced, but also nitrate induces nitrate and nitrite reductase activities by altering gene expression mainly by enhancing transcription of the respective genes.  Nitrate, light and sugars are inducers; glutamate and glutamine are repressors.

1. Induction of genes for nitrate and nitrite reduction.
2. Nitrate uptake and translocation systems.
3. DNA regulatory proteins required for expression of the secondary response gene system.

1. Proliferation of the root system.
2. Enhancement of respiration.
3. Other changes in the physiology of the plant.

1. It may undergo efflux to the apoplast and soil environment.
2. It may enter the vacuole and by stored.
3. It may be reduced to ammonium by the combined action of NR and NiR.
4. It may be translocated via the symplast to the xylem.

• Virtually all biologically important N-compounds contain N in a reduced form.
• The principal inorganic forms of N in the environment are in an oxidized state.  Thus, the entry of N into organisms depends on the reduction of oxidized organic forms (N₂ and NO₃^-) to NH₄⁺.
• The reactions involving inorganic N-compounds occur only in microorganisms and green plants.  Animals acquire their N from the catabolism of organic N-compounds mainly proteins, obtained in the diet.
• Nitrate is reduced to ammonia by a two-step process catalyzed by the enzymes nitrate reductase (NR) and nitrite reductase (NiR) mentioned previously.

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• Nitrate and nitrite reductase
• NO₃^- + 2H⁺ + 2e- NO₂^- + H₂O
• NO₂^- + 8H⁺ + 6e- NH₄⁺ + 2H₂O
• 4-1. Nitrate reductase (NR)
• Located primarily in the cytosol of root epidermal and cortical cells and shoot mesophyll cells.
• Transfers 2 e- from NAD(P)H to nitrate via three redox centers composed of two prosthetic groups (FAD and heme). It also has a molybdenum cofactor (MoCo), a complex of molybdate and pterin, which catalyzes the actual nitrate reduction.



• See Fig. 2 of Crawford (1995) The Plant Cell 7: 859-868 and Figs. 16.38 - 40 of the class text.



• NR is typically a homodimer or homotetramer with 100-to 115 kD subunits.
• three functional domains
1. flavin (FAD) domain
2. heme (Fe) domain
3. molybdenum cofactor (MoCo) domain

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The NR catalyzed reduction of NO₃^- starts with e^- transport from NAD(P)H to the flavin domain, through heme and finally onto NO₃^- via the molybdenum cofactor.  Cytochrome c can be an alternative e^- acceptor (Fig. 2, 16.38 above).

• The FAD domain has been crystallized and found to contain 2 lobes.  One lobe contains 6 parallel ß-strands.  The other lobe, which binds to the FAD molecule tethered by several hydrogen bonds, also contains ß-strands, but is antiparallel (Fig. 2B Crawford, 1995, Fig. 16.40 class text).



The central heme containing 75-80 amino acids is similar to heme of cytochrome b₅s.  See illustration at the Directory of P450-containing Systems web page.

 

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• Green tissues have >>> NR than NiR activity so NR is rate limiting.  What might be the advantages of this?

 

 

 

• It is very important that plants regulate the process of nitrate reduction because it is a very energy intensive process consuming ~20% of the e^- produced by photosynthetic electron transport.  Also, the intermediate product of nitrate reduction, nitrite, is cytotoxic and mutagenic.  As mentioned above NR genes can be regulated by nitrate, light, sugars, glutamate and glutamine.  Also plant hormones and other factors such as Ca⁺⁺ can affect NR transcription.  NR can be regulated post translationally by reversible protein phosphorylation (see Fig. 16.42 text).



• Nitrite Reduction via Nitrite Reductase (NiR)
• Reduction of nitrite to ammonia:
• NiR enzyme
• the holoenzyme is a monomer, 60-70 kD with two redox centers
1. a siroheme center
2. an iron-sulfur center; 4Fe-4S cluster
3. a ferredoxin binding domain

• NiR is a nuclear encoded enzyme transported to chloroplasts or proplastids with cleavage of a 30 kD transit sequence.



The c-terminal half of NiR is thought to contain the redox centers and the N-terminal half is thought to bind the reducing agent ferredoxin.

• Ferredoxin reduced by the chloroplast non-cyclic electron transport system provides the e^- for reducing nitrite.  It is thought that a ferredoxin-like protein in proplastids reduced by NADPH from the oxidative pentose phosphate pathway provides the source of reductant in roots.
• A model for coupling photosynthetic electron flow, via ferredoxin to the reduction of nitrate by NiR to ammonia:

 

 

 

 

• Sirohemes
  • Uroporphyrin derivatives that are quite polar.
  • Novel in having eight carbohydrate containing side chains.

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  • NiR regulation:

• Ammonia Assimilation
• 5-1 NH₄⁺ enters an organic linkage via one of 3 major reactions that are found in all cells:
1. Carbamoyl phosphate synthetase I
   

   NH₄⁺ + HCO₃^- + 2ATP H₂N-CO-O-PO₃^-2 + 2ADP + Pi

2. Glutamate dehydrogenase (GDH) reaction:

 

 

GDH has a significantly higher km for NH₄⁺ than does glutamine synthetase (GS).  Consequently in organisms confronting N-limitation GDH is not effective and GS is the only NH₄⁺ assimilation reaction.  It also appears that GS is the sole port of entry of N into amino acids.
3. Glutamine synthetase (GS)
• ATP-dependent amination of the g-carboxyl group of glutamate to form glutamine
• Mg²⁺-dependent
• Very high affinity for ammonia (K_m = 3-5 µM)
• Glutamine is the major N-donor in the biosynthesis of many organic N compounds such as
• purines, pyrimidines
• other amino acids

The reaction:  Glutamate + ammonia + ATP glutamine + ADP + Pi
involves activation of the g-carboxyl group of Glu by ATP followed by amination by NH₄⁺: