NITRATE UPTAKE, REDUCTION AND ASSIMILATION

NITRATE UPTAKE, REDUCTION AND ASSIMILATION -
nitrogen cycle.
essential for all life science students.

1. NITRATE UPTAKE AND ASSIMILATION

2. NITRATE UPTAKE AND ASSIMILATION Schematic and simplified representation of the nitrate uptake, transport and assimilation in plants.

3. Nitrate movements through cellular membranes are always mediated by specific transporters. The translocation of NO3 ions is active and involves both high and low affinity proton symporters . In roots, three distinct groups of nitrate transporters have been identified. Two of them have been classified as high affinity transporter system (HATS) and include the inducible ( iHATS ) and constitutive ( cHATS ) transport systems. Another group is represented by a constitutive low affinity transporter system (LATS). All these transporters are up-regulated by nitrate availability. Nitrate influx into the root cells is proton coupled, as above mentioned, therefore nitrate transport is dependent on the H+ -ATPase pumps and it requires energy. It has been proposed that in both symporter systems, for each NO3 - loaded, two H+ cross the plasma membrane and that this mechanism is tightly regulated by cellular pH . Nitrate in plants can be reduced to nitrite both in roots and in shoots or loaded and stored inside the vacuole. NO3 - reduction is catalysed by the enzyme Nitrate Reductase .

4. NR in the leaf is a cytoplasmic enzyme and works in the presence of NAD(P)H, whereas in the root two distinct types of nitrate reductase are present: a cytoplasmic isoform ( cNR ) and a plasmamembrane -bound isoform (PMNR), the latter also able to oxidize succinate. In normal conditions, NR reduces nitrates to nitrites , which are then transferred to the chloroplast/plastid where they are reduced to ammonium by nitrite reductase . At last, ammonium is organicated to glutamate by glutamine synthetase to produce glutamine. Therefore, nitrate is assimilated into amino acids via the GS-GOGAT pathway (GS1, GS2, GOGAT), resulting in glutamine and glutamate as primary N organic compounds. Glutamine synthetase (GS) and glutamate synthase (GOGAT,) are the enzymes involved in ammonium assimilation, either derived from nitrate reduction, photorespiration or, in case it is externally supplied, as NH3 and or NH4 +. In roots, NO2 - is converted into NH4 + mainly by the cytoplasmic GS isoform (named GS1), whereas the plastidial isoform (GS2) is less active. The opposite happens in the leaf, where the chloroplastic GS2 is the most active isoform.

5. Nitrate is absorbed by most plants and reduced to  ammonia  with the help of two different enzymes. The first step conversion of nitrate to nitrite is catalyzed by an enzyme called nitrate reductase . This enzyme has several other important constituents including FAD, cytochrome, NADPH or NADH and molybdenum. NITRATE REDUCTASE In higher plants, nitrate reduction is highly regulated. A range of environmental factors influence the expression of the corresponding genes as well as the enzyme activity levels. NR activity expression and activity is controlled by light, temperature, pH, CO 2, O 2, water potential and N source. Nitrate reduction takes place chiefly in green leaves and roots. The enzymes nitrate reductase is found in cytosol). According to more recent findings the enzyme nitrate reductase is in-fact a complex en­zyme in higher plants as well as micro-organisms.

6. Nitrate uptake Nitrate reduction to ammonia -- nitrate reductase & nitrite reductase Ammonia assimilation into glutamate & glutamine nitrogen (N 2 ) 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.

7. BIOLOGICAL NITROGEN FIXATION Nitrogen Fixation in Plants (biologydiscussion.com)

8. Nitrate Uptake The nitrate uptake system in plant must be versatile and robust because 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. Plants must compete for N in the soil with abiotic and biotic processes such as erosion, leaching and microbial competition. 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 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 NO 3 -  throughout leaf and shoot tissue.  Ultimately N can be stored in the seed or other storage organ. 

9. The H + -ATPase in the PM pumps protons out of the cell producing pH and electrical ( psi ) 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.

10. Nitrate is a signal for developmental changes in the physiology of the plant. The primary responses include: Induction of genes for nitrate and nitrite reduction. Nitrate uptake and translocation systems. DNA regulatory proteins required for expression of the secondary response gene system. The secondary response include more complex phenomena such as Proliferation of the root system. Enhancement of respiration. Other changes in the physiology of the plant. The fate of NO 3 -  taken up by a root epidermal cell.   Once transported into an epidermal cell, NO 3 -  has one of four fates: It may undergo efflux to the apoplast and soil environment. It may enter the vacuole and by stored. It may be reduced to ammonium by the combined action of NR and NiR . It may be translocated via the symplast to the xylem.

11. Nitrate Reduction 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 (N 2  and NO 3 - ) to NH 4 + . 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 )   Nitrate and nitrite reductase NO 3 -  + 2H +  + 2e-    NO 2 -  + H 2 O NO 2 -  + 8H +  + 6e-  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. NH 4 +  + 2H 2 O

12. NR is typically a homodimer or homotetramer with 100-to 115 kD subunits. three functional domains flavin (FAD) domain heme (Fe) domain molybdenum cofactor ( MoCo ) domain The NR catalyzed reduction of NO 3 -  starts with e -  transport from NAD(P)H to the flavin domain, through heme and finally onto NO 3 -  via the molybdenum cofactor.  Cytochrome c can be an alternative e -   acceptor. 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 The central heme containing 75-80 amino acids is similar to heme of cytochrome b 5 s. 

13. 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 a siroheme center an iron-sulfur center; 4Fe-4S cluster 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.

14. Ammonia Assimilation 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 (Km = 3-5 µM) Glutamine is the major N-donor in the biosynthesis of many organic N compounds such as purines, pyrimidines other amino acids

15. The reaction: Glutamate + ammonia + ATP -> glutamine + ADP + Pi involves activation of the g-carboxyl group of Glu by ATP followed by amination by NH4+ The glutamate consumed by the GS reaction is replenished by an alternative mode of glutamate synthesis Glutamate synthases (= GoGAT glutamate: oxoglutarate aminotransferase) reductant + a-KG + Gln -> 2Glu + oxidized reductant Two equivalents of glutamate are formed from amination of α-KG from deamination of Gln These Glu can now serve as ammonia accepts for glutamine synthesis by GS

16. Different organisms use different reductants H+ + NADH: yeast, N. crassa , plants H+ + NADPH: E. coli H+ + reduced ferredoxin : plants (solely in chloroplasts) The GS/ GoGAT pathway of ammonium assimilation: Summary: 2 NH4+ + α- KG + 2H+ + 2 Fdred + 2 ATP -> Glutamine + 2 Fdox + 2 ADP + 2 Pi These reactions result in conversion of α- KG to glutamine at the expense of 2 ATP and 1 NADPH