Sakshi Education
The most prominent chemical elements in living systems are O, H, C,N, and P. The elements O, H, and P occur widely in metabolically available forms (H2O, O2, and Pi). However, the major forms of C and Nitrogen (N), CO2 and N2, are extremely stable (unreactive); for example, the triple bond of nitrogen has bond energy of 945 kJ/mol_1. CO2, with minor exceptions, is metabolized (fixed) only by photosynthetic organisms. N is a critical limiting element for plant growth and production. It is a major component of chlorophyll, the most important pigment needed for photosynthesis, as well as amino acids, the key building blocks of proteins. It is also found in other important biomolecules, such as ATP and nucleic acids. Even though it is one of the most abundant elements (predominately in the form of nitrogen gas (N2) in the Earth’s atmosphere), plants can only utilize reduced forms of this element by Nitrogen fixation.
This process can occur by naturally, that fix the nitrogen through the following processes:
ü Lightning. Lightning is responsible for about 8% of the nitrogen fixed. Lightning converts water vapor and oxygen into highly reactive hydroxyl free radicals, free hydrogen atoms, and free oxygen atoms that attack molecular nitrogen (N2) to form nitric acid (HNO3). This nitric acid subsequently falls to Earth with rain.
ü Photochemical Reactions. Approximately 2% of the nitrogen fixed derives from photochemical reactions between gaseous nitric oxide (NO) and ozone (O3) that produce nitric acid (HNO3).
ü Biological Nitrogen Fixation. The remaining 90% results from biological nitrogen fixation, in which bacteria or blue-green algae (cyanobacteria) fix N2 into ammonium (NH4+).
Biological Nitrogen Fixation (BNF), discovered by Dutch Bologist and Botanist Beijerinck in 1901, is carried out by a specialized group of prokaryotes. These organisms utilize the enzyme nitrogenase to catalyze the conversion of atmospheric nitrogen (N2) to ammonia (NH3). Plants can readily assimilate NH3 to produce the aforementioned nitrogenous biomolecules. Microorganisms that fix nitrogen are called diazotrophs. These prokaryotes include aquatic organisms, such as cyanobacteria, free-living soil bacteria, such as Azotobacter, bacteria that form associative relationships with plants, such as Azospirillum, and most importantly, bacteria, such as Rhizobium and Bradyrhizobium, that form symbioses with legumes and other plants. These organisms are summarized in Table 1.
Overuse of these chemical fertilizers has led to an upset in the nitrogen cycle and consequently to surface water as well as groundwater pollution. Increased loads of nitrogen fertilizer to freshwater, as well as marine ecosystems, has caused Eutrophication, the process whereby these systems have a proliferation of microorganisms, especially algae. This “greening” of the water column has caused decreased levels of dissolved oxygen (DO) in bottom waters as planktonic algae die and fuel microbial respiration. These depleted DO levels result in massive mortality of aquatic organisms and create so-called dead zones, areas where little or no aquatic life can be found. Since the 1960’s, dead zones have increased exponentially worldwide.
Once fixed in ammonium or nitrate, nitrogen enters a biogeochemical cycle and passes through several organic or inorganic forms before it eventually returns to molecular nitrogen. The ammonium (NH4+) and nitrate (NO3–) ions that are generated through fixation or released through decomposition of soil organic matter becomes the object of intense competition among plants and microorganisms.
Diazotrophic (“nitrogen eaters”) organisms can fix N2 because they produce an enzyme called nitrogenase. Most N2 fixing organisms produce a Molybdenum-containing nitrogenase. In addition, some organisms have “alternative” systems that produce a vanadium-containing nitrogenase and/or an iron-only nitrogenase. Among these three classes of nitrogenase, the Mo-containing nitrogenase is the most prevalent and the composed of two oxygen-sensitive components under ideal conditions.
Nitrogenase, which catalyzes the reduction of N2 to NH3, is a complex of two proteins:
This process can occur by naturally, that fix the nitrogen through the following processes:
ü Lightning. Lightning is responsible for about 8% of the nitrogen fixed. Lightning converts water vapor and oxygen into highly reactive hydroxyl free radicals, free hydrogen atoms, and free oxygen atoms that attack molecular nitrogen (N2) to form nitric acid (HNO3). This nitric acid subsequently falls to Earth with rain.
ü Photochemical Reactions. Approximately 2% of the nitrogen fixed derives from photochemical reactions between gaseous nitric oxide (NO) and ozone (O3) that produce nitric acid (HNO3).
ü Biological Nitrogen Fixation. The remaining 90% results from biological nitrogen fixation, in which bacteria or blue-green algae (cyanobacteria) fix N2 into ammonium (NH4+).
Biological Nitrogen Fixation (BNF), discovered by Dutch Bologist and Botanist Beijerinck in 1901, is carried out by a specialized group of prokaryotes. These organisms utilize the enzyme nitrogenase to catalyze the conversion of atmospheric nitrogen (N2) to ammonia (NH3). Plants can readily assimilate NH3 to produce the aforementioned nitrogenous biomolecules. Microorganisms that fix nitrogen are called diazotrophs. These prokaryotes include aquatic organisms, such as cyanobacteria, free-living soil bacteria, such as Azotobacter, bacteria that form associative relationships with plants, such as Azospirillum, and most importantly, bacteria, such as Rhizobium and Bradyrhizobium, that form symbioses with legumes and other plants. These organisms are summarized in Table 1.
Overuse of these chemical fertilizers has led to an upset in the nitrogen cycle and consequently to surface water as well as groundwater pollution. Increased loads of nitrogen fertilizer to freshwater, as well as marine ecosystems, has caused Eutrophication, the process whereby these systems have a proliferation of microorganisms, especially algae. This “greening” of the water column has caused decreased levels of dissolved oxygen (DO) in bottom waters as planktonic algae die and fuel microbial respiration. These depleted DO levels result in massive mortality of aquatic organisms and create so-called dead zones, areas where little or no aquatic life can be found. Since the 1960’s, dead zones have increased exponentially worldwide.
Once fixed in ammonium or nitrate, nitrogen enters a biogeochemical cycle and passes through several organic or inorganic forms before it eventually returns to molecular nitrogen. The ammonium (NH4+) and nitrate (NO3–) ions that are generated through fixation or released through decomposition of soil organic matter becomes the object of intense competition among plants and microorganisms.
Diazotrophic (“nitrogen eaters”) organisms can fix N2 because they produce an enzyme called nitrogenase. Most N2 fixing organisms produce a Molybdenum-containing nitrogenase. In addition, some organisms have “alternative” systems that produce a vanadium-containing nitrogenase and/or an iron-only nitrogenase. Among these three classes of nitrogenase, the Mo-containing nitrogenase is the most prevalent and the composed of two oxygen-sensitive components under ideal conditions.
Nitrogenase, which catalyzes the reduction of N2 to NH3, is a complex of two proteins:
- The Fe-protein, a homodimer that contains one [4Fe–4S] cluster and 2 ATP-binding sites (Dinitrogenase reductase encoded by the nifH gene).
- The MoFe-protein, a a2ß2 heterotetramer that contains Fe and Mo. ( Dinitrogenase encode by the nifDK genes)
N2 + 8H+ + 8e- + 16 (Mg) ATP 2NH3 + H2 + 16 (Mg) ADP + 16Pi
The Fe protein, or component II, is a ~70 kDa homodimer that contains two ATP binding sites and a single [4Fe-4S] cluster bridged between each monomer through cysteine ligands. One of the functions of the Fe protein is to serve as an electron donor to the MoFe protein during catalysis. No other protein or artificial electron donor is able to replace this function. During catalysis, binding of ATP to the reduced Fe protein induces a conformational change that allows docking to the MoFe protein. Docking subsequently triggers the hydrolysis of ATP coupled with the transfer of one electron to the MoFe protein. One of the complexities of the nitrogenase system is that, although the MoFe protein receives one electron at a time from the Fe protein, it requires 2, 4, or 6 electrons to reduce various substrates. Catalysis therefore relies on multiple rounds of association/dissociation between the MoFe protein and the Fe protein. The Mo-Fe protein, or component I, is a ~240 kDa heterotetramer (a2ß2) and it contains two types of clusters, the P cluster and FeMo cofactor. The [8Fe-7S] P cluster is located at each a/ß interface. The P cluster is believed to serve as an intermediate electron carrier during electron transfer from the Fe protein to the Fe-Mo cofactor. In the crystal structure of the MoFe protein and Fe protein complex, the P cluster is located equidistant between the [4Fe-4S] cluster of the Fe protein and the Fe-Mo cofactor of the Mo-Fe protein The FeMo-cofactor is located within the MoFe protein a-subunit and it has a unique structure not identified in any other metalloprotein. This cofactor is a (7Fe-9S Mo-X-homocitrate) cluster, where X is likely to be a non-exchangeable nitrogen atom. A high-resolution crystal structure of the Mo-Fe protein has recently revealed the presence of this atom in the central cavity of the cofactor, previously thought to be unoccupied. The FeMo-cofactor is covalently attached to the protein by two amino acids residues, a-Cys275 and a-His442, and is tightly held within the protein through non-covalent interactions with the side chain of a variety of other residues. The nitrogenase complex is very sensitive to O2. Since nitrogenase is inactivated by O2, the fixation of N2 must occur under condition which is anaerobic at least locally. For anaerobes there is no problem. Facultative organisms such as purple photosynthetic bacteria fix N2 only when anaerobic. Other organisms have protective mechanisms. In cyanobacteria O2 is actually generated by photosynthesis. Fixation of N2 occurs in special cells known as HETEROCYSTS which do not photosynthesize but are devoted solely to N2 fixation. Leguminous plants maintain a very low level concentration of free O2 in their root nodules by binding O2 to LEGHHEMOGLOBIN. Nitrogen fixing organisms Nitrogen-fixing bacteria that form symbiotic associations with plants include Rhizobium, Bradyrhizobium, Frankia, Nostoc, and Anabaena. The associations are species specific. Some of the interactions between the host and Free-living bacteria that are capable of fixing nitrogen are aerobic, facultative, or anaerobic (see Table 2 bottom): (Taiz, L.; Zeiger, E. 2006) ü Aerobic nitrogen-
fixing bacteria such as Azotobacter are thought to maintain reduced oxygen conditions (microaerobic conditions) through their high levels of respiration. Others, such as Gloeothece, evolve O2 photosynthetically during the day and fix nitrogen during the night.
ü Facultative organisms, which are able to grow under both aerobic and anaerobic conditions, generally fix nitrogen only under anaerobic conditions.
ü For anaerobic nitrogen-
fixing bacteria, oxygen does not pose a problem, because it is absent in their habitat. These anaerobic organisms can be either photosynthetic (e.g., Rhodospirillum), or nonphotosynthetic (e.g., Clostridium).
Symbiotic Nitrogen Fixation
Symbiotic nitrogen-fixing prokaryotes dwell within nodules, the special organs of the plant host that enclose the nitrogen-fixing bacteria. In the case of Gunnera, these organs are existing stem glands that develop independently of the symbiont. In the case of legumes and actinorhizal plants, the nitrogen-fixing bacteria induce the plant to form root nodules. Grasses can also develop symbiotic relationships with nitrogen-fixing organisms, but in these associations root nodules are not produced. Instead, the nitrogen-fixing bacteria seem to colonize plant tissues or anchor to the root surfaces, mainly around the elongation zone and the root hairs.
Legumes and actinorhizal plants regulate gas permeability in their nodules, maintaining a level of oxygen within the nodule that can support respiration but is sufficiently low to avoid inactivation of the nitrogenase. Gas permeability increases in the light and decreases under drought or upon exposure to nitrate. The mechanism for regulating gas permeability is not yet known.
Nod Factor Structure and Synthesis
Plant genes specific to nodules are called nodulin (Nod) genes; rhizobial genes that participate in nodule formation are called nodulation (nod) genes. The Rhizobium signal molecules that play a key role in the induction of the initial stages of nodulation are lipochito-oligosaccharides known as Nod factors. The bacterial genes involved in Nod factor synthesis are the nod (nodulation) genes. These genes are not expressed in free-living bacteria, with the exception of nod D, which is expressed constitutively. Nod D has the ability to bind to specific flavonoids secreted by the roots of the host plant, upon flavonoid bindin Beijerinck g, it becomes a transcriptional activator of the other nod genes which encode enzymes involved in the synthesis of Nod factors. The first stage in the formation of the symbiotic relationship between the nitrogen-fixing bacteria and their host is migration of the bacteria toward the roots of the host plant. This migration is a chemotactic response mediated by chemical attractants, especially (iso) flavonoids, homoserine and betaines, secreted by the roots. A specific adhesion protein called rhicadhesin is present on the surface of Rhizobium species. Rhicadhesin is a calcium binding protein and plays role in plant-bacterium attachment. These attractants activate the rhizobial Nod D protein, which then induces transcription of the other nod genes.
Three of the nod genes (nodA, nodB, and nodC) encode enzymes (NodA, NodB, and NodC, respectively) that are required for synthesizing this basic structure.
ü NodA is an N-acyltransferase that catalyzes the addition of a fatty acyl chain.
ü NodB is a chitin-oligosaccharide deacetylase that removes the acetyl group from the terminal nonreducing sugar.
ü NodC is a chitin-oligosaccharide synthase that links N-acetyl-D-glucosamine monomers.
Other enzymes, such as -
ü NodE and NodF determine the length and degree of saturation of the fatty acyl chain;
ü NodL, influence the host specificity of Nod factors through the addition of specific substitutions at the reducing or nonreducing sugar moieties of the chitin backbone.
lnfection
Two processes—infection and nodule organogenesis—
occur simultaneously during root nodule formation. During the infection process, rhizobia that are attached to the root hairs release Nod factors that induce a pronounced curling of the root hair cells. After attachment of rhizobia to the root hair tips, the tips curl tightly and bacteria become entrapped in the curls. A local hydrolysis of the plant cell wall takes place in the curled region and the plasma membrane invaginates and new plant cell wall material is deposited. This results in the formation of a tubular structure, is called infection thread, by which the bacteria enter the plant. The ultrastructure of the wall of the infection thread is very similar to that of the normal plant cell wall, but the incorporation of certain nodulins may provide it with unique properties.
The proline-
rich early nodulins ENOD5 and ENOD12 are candidates for components of the infection thread wall, because cortical cells containing an infection thread express the corresponding genes. The bacteria in the infection thread are surrounded by a matrix that seems to consist of compounds secreted by both the plant and the bacteria. Concomitant with infection thread formation, cortical cells are mitotically reactivated, forming the nodule primordium. Infection threads grow toward this primordium and, once there, release bacteria into the cytoplasm. In those legumes that form indeterminate nodules, nodule primordia arise from inner cortical cells.
Hence, in the formation of this nodule type, the infection threads must traverse the outer cortex before they reach these cells. Before infection thread penetration, the outer cortical cells undergo morphological changes. The nuclei move to the center of the cells, and the microtubules and the cytoplasm rearrange to form a radially oriented conical structure, the cytoplasmic bridge, that resembles a preprophase band. The infection threads traverse the cortical cells through the radially aligned cytoplasmic bridges, which are therefore called preinfection threads. Although the preinfection thread-forming outer cortical cells never divide, the induced morphological changes are reminiscent of those seen in cells entering the cell cycle, express the S phase-specific Histone H4 gene.
However, a mitotic cyclin gene specifically expressed during the G2-to-M phase transition is not activated. Hence, the cells that form the preinfection thread reenter the cell cycle and most likely become arrested in the G2 phase, whereas the inner cortical cells progress all the way through the cell cycle and form the primordia. Thus, bacteria seem to be required for the formation of infection threads. It has been shown that pretreatment with lipopolysaccharides can improve the efficiency of infection thread induction, whereas pretreatment with lipopolysaccharides from a noninfectious Rhizobium strain leads to an increase in aborted infections. Furthermore, mutations in rhizobial exopolysaccharide biosynthesis can render the bacteria unable to induce infection threads. Thus, interaction with bacterial surface compounds seems to play an important role in infection thread formation.
When the infection thread reaches a cell deep in the cortex, it bursts and the rhizobia are engulfed by endocytosis into membrane-enclosed symbiosomes within the cytoplasm. At this time the cell goes through several rounds of mitosis — without cytokinesis — so the cell becomes polyploid. The cortex cells then begin to divide rapidly forming a nodule. This response is driven by the translocation of cytokinins from epidermal cells to the cells of the cortex. The rhizobia also go through a period of rapid multiplication within the nodule cells. Then they begin to change shape and lose their motility. The bacteroids, as they are now called, may almost fill the cell. Only now does nitrogen fixation begin. Reduction of nitrogen to ammonia occurs in bacteriods. Root nodules are not simply structureless masses of cells. Each becomes connected by the xylem and phloem to the vascular system of the plant. Thus the development of nodules, while dependent on rhizobia, is a well-coordinated developmental process of the plant. Nitrogenase catalyze this reaction is very sensitive to oxygen.
In root nodules the O2 level is regulated by leghemoglobin. In plants infected with Rhizobium, the presence of oxygen in the root nodules would reduce the activity of the oxygen-sensitive nitrogenase - an enzyme responsible for the fixation of atmospheric nitrogen. Leghemoglobin buffers the concentration of free oxygen in the cytoplasm of infected plant cells to ensure the proper function of root nodules. Leghemoglobin has a high affinity for oxygen (a Km of about 0.01 µM), about ten times higher than the ß chain of human hemoglobin. This allows an oxygen concentration that is low enough to allow nitrogenase to function but high enough so that it can provide the bacteria with oxygen for respiration. Leghemoglobin has close chemical and structural similarities to hemoglobin, and, like hemoglobin, is red in colour. The protein was believed to be a product of both plant and the bacterium in which the apoprotein is produced by the plant and the heme (an iron atom bound in a porphyrin ring) is produced by the bacterium. Newer findings however, indicate that the heme moiety is also produced by the plant (Virtanen, A. I. (1948). Although leghemoglobin was once thought to provide a buffer for nodule oxygen, recent studies indicate that it stores only enough oxygen to support nodule respiration for a few seconds. Its function is to help transport oxygen to the respiring symbiotic bacterial cells in a manner analogous to hemoglobin transporting oxygen to respiring tissues in animals Ludwig, R. A, 1995). Leghemoglobin is localized in the cytoplasamof infected plant cells and in inside the peribacteroid membrane.
Ammonium assimilation
Plant cells avoid ammonium toxicity by rapidly converting the ammonium generated from nitrate assimilation or photorespiration into amino acids. The primary pathway for this conversion involves the sequential actions of glutamine synthetase and glutamate synthase (Lea et al. 1992). For many years it was thought that bacteria and higher plants assimilate ammonia into glutamate via the Glutamate dehydrogenase (GDH) pathway, as in certain fungi and yeasts. However, in bacteria it became clear in 1970 that an alternative pathway of ammonia assimilation involving glutamine synthetase (GS) and an NADPH-dependent glutamine:2-oxoglutarate amidotransferase (GOGAT) or glutamate synthase, must be operating when ammonia is present in the growth medium at low levels (Tempest et al, 1970). Thus, N-starvation leads to derepression and activation of GS (with a high affinity for NH3) and derepression of GOGAT, and repression of GDH (with a relatively low affinity for NH3) (Tempest et al, 1970). High ammonia availability leads to repression and deactivation of GS and induction of GDH (Tempest et al, 1970).
Glutamate dehydrogenase
NH3 + 2-oxoglutarate + NADPH + H+ <--------------------------> glutamate + NADP+ GS- glutamine:2-oxoglutarate aminotransferase
NH3 + glutamate + ATP ---------------------------------------------------------> glutamine + ADP + Pi Glutamate synthase
Glutamine + 2-oxoglutarate + NADPH + H+ -----------------------> 2 glutamate + NADP+
Both the GDH and GS-GOGAT pathways produce 1 mole of glutamate from 1 mole each of NH3, 2-oxoglutarate and NADPH. But note that the GS-GOGAT pathway is energetically more costly than the GDH pathway, consuming 1 ATP.
ü Escherichia coli is now known to have two primary pathways for glutamate synthesis (Hellig, 1994; 1998). The GS-GOGAT pathway is essential for glutamate synthesis at low ammonium concentrations and for regulation of the glutamine pool, and is used when the cell is not under energy limitation (Hellig, 1994; 1998).
ü The GDH pathway is used in glutamate synthesis when the cell is limited for energy (and carbon; i.e. glucose-limited growth) but ammonium and phosphate are present in excess (Hellig, 1994; 1998).
ü The GS-GOGAT pathway as the primary pathway of ammonia assimilation, but the presence of GDH appears to offer a selective advantage for the cyanobacterium under nonexponential growth conditions (Chavez et al, 1999). These dual pathways may be common to bacteria, cyanobacteria, algae, yeasts and fungi (Huth and Liebs, 1988).
NITRATE ASSIMILATION
Plants assimilate most of the nitrate absorbed by their roots into organic nitrogen compounds. The first step of this process is the reduction of nitrate to nitrite in the cytosol (Oaks 1994). The enzyme Nitrate reductasecatalyzes this reaction:
Nitrate reductasecatalyzes NO3– + NAD(P)H + H++ 2 e– NO2–+ NAD(P)+ + H2O A model of the nitrate reductase dimer, illustrating the three binding domains whose polypeptide sequences are similar in eukaryotes: molybdenum complex (MoCo), heme, and FAD. The NADH binds at the FADbinding region of each subunit and initiates a two-electron transfer from the carboxyl (C) terminus, through each of the electron transfer components, to the amino (N) terminus. Nitrate is reduced at the molybdenum complex near the amino terminus. The polypeptide sequences of the hinge regions are highly variable among species.
ü Nitrate reductase is the main molybdenum-containing protein, deficiency of molybdenum is the accumulation of nitrate that results from diminished nitrate reductase activity.
ü Nitrate Reductase regulated by the Nitrate, Light, and Carbohydrates at the transcription and translation levels.
Nitrite Reductase Converts Nitrite to Ammonium
Nitrite (NO2–) is a highly reactive, potentially toxic ion.Plant cells immediately transport the nitrite generated by nitrate reduction (see Equation 12.1) from the cytosol into chloroplasts in leaves and plastids in roots. In these organelles, the enzyme nitrite reductase reduces nitrite to ammonium according to the following overall reaction:
NO2– + 6 Fd (Reduced) + 8 H+ + 6 e– NH4+ + 6 Fd oxidized + 2 H2O
Summary
To summarize, the processes involved in the assimilation of mineral nitrogen in the leaf, nitrate translocated from the roots through the xylem is absorbed by a mesophyll cell via one of the nitrate–proton symporters (NRT) into the cytoplasm. There it is reduced to nitrite via nitrate reductase (NR). Nitrite is translocated into the stroma of the chloroplast along with a proton. In the stroma, nitrite is reduced to ammonium via nitrite reductas (NiR) and this ammonium is converted into glutamate via the sequential action o glutamine synthetase (GS) and glutamate synthase (GOGAT). Once again in the cytoplasm, the glutamate is transaminated to aspartate via aspartate aminotransferase (Asp-AT). Finally, asparagine synthetase (AS) converts aspartate into asparagine. The approximate amounts of ATP equivalents are given above each reaction ((Taiz, L.; Zeiger, E. 2006).
The Fe protein, or component II, is a ~70 kDa homodimer that contains two ATP binding sites and a single [4Fe-4S] cluster bridged between each monomer through cysteine ligands. One of the functions of the Fe protein is to serve as an electron donor to the MoFe protein during catalysis. No other protein or artificial electron donor is able to replace this function. During catalysis, binding of ATP to the reduced Fe protein induces a conformational change that allows docking to the MoFe protein. Docking subsequently triggers the hydrolysis of ATP coupled with the transfer of one electron to the MoFe protein. One of the complexities of the nitrogenase system is that, although the MoFe protein receives one electron at a time from the Fe protein, it requires 2, 4, or 6 electrons to reduce various substrates. Catalysis therefore relies on multiple rounds of association/dissociation between the MoFe protein and the Fe protein. The Mo-Fe protein, or component I, is a ~240 kDa heterotetramer (a2ß2) and it contains two types of clusters, the P cluster and FeMo cofactor. The [8Fe-7S] P cluster is located at each a/ß interface. The P cluster is believed to serve as an intermediate electron carrier during electron transfer from the Fe protein to the Fe-Mo cofactor. In the crystal structure of the MoFe protein and Fe protein complex, the P cluster is located equidistant between the [4Fe-4S] cluster of the Fe protein and the Fe-Mo cofactor of the Mo-Fe protein The FeMo-cofactor is located within the MoFe protein a-subunit and it has a unique structure not identified in any other metalloprotein. This cofactor is a (7Fe-9S Mo-X-homocitrate) cluster, where X is likely to be a non-exchangeable nitrogen atom. A high-resolution crystal structure of the Mo-Fe protein has recently revealed the presence of this atom in the central cavity of the cofactor, previously thought to be unoccupied. The FeMo-cofactor is covalently attached to the protein by two amino acids residues, a-Cys275 and a-His442, and is tightly held within the protein through non-covalent interactions with the side chain of a variety of other residues. The nitrogenase complex is very sensitive to O2. Since nitrogenase is inactivated by O2, the fixation of N2 must occur under condition which is anaerobic at least locally. For anaerobes there is no problem. Facultative organisms such as purple photosynthetic bacteria fix N2 only when anaerobic. Other organisms have protective mechanisms. In cyanobacteria O2 is actually generated by photosynthesis. Fixation of N2 occurs in special cells known as HETEROCYSTS which do not photosynthesize but are devoted solely to N2 fixation. Leguminous plants maintain a very low level concentration of free O2 in their root nodules by binding O2 to LEGHHEMOGLOBIN. Nitrogen fixing organisms Nitrogen-fixing bacteria that form symbiotic associations with plants include Rhizobium, Bradyrhizobium, Frankia, Nostoc, and Anabaena. The associations are species specific. Some of the interactions between the host and Free-living bacteria that are capable of fixing nitrogen are aerobic, facultative, or anaerobic (see Table 2 bottom): (Taiz, L.; Zeiger, E. 2006) ü Aerobic nitrogen-
fixing bacteria such as Azotobacter are thought to maintain reduced oxygen conditions (microaerobic conditions) through their high levels of respiration. Others, such as Gloeothece, evolve O2 photosynthetically during the day and fix nitrogen during the night.
ü Facultative organisms, which are able to grow under both aerobic and anaerobic conditions, generally fix nitrogen only under anaerobic conditions.
ü For anaerobic nitrogen-
fixing bacteria, oxygen does not pose a problem, because it is absent in their habitat. These anaerobic organisms can be either photosynthetic (e.g., Rhodospirillum), or nonphotosynthetic (e.g., Clostridium).
Symbiotic Nitrogen Fixation
Symbiotic nitrogen-fixing prokaryotes dwell within nodules, the special organs of the plant host that enclose the nitrogen-fixing bacteria. In the case of Gunnera, these organs are existing stem glands that develop independently of the symbiont. In the case of legumes and actinorhizal plants, the nitrogen-fixing bacteria induce the plant to form root nodules. Grasses can also develop symbiotic relationships with nitrogen-fixing organisms, but in these associations root nodules are not produced. Instead, the nitrogen-fixing bacteria seem to colonize plant tissues or anchor to the root surfaces, mainly around the elongation zone and the root hairs.
Legumes and actinorhizal plants regulate gas permeability in their nodules, maintaining a level of oxygen within the nodule that can support respiration but is sufficiently low to avoid inactivation of the nitrogenase. Gas permeability increases in the light and decreases under drought or upon exposure to nitrate. The mechanism for regulating gas permeability is not yet known.
Nod Factor Structure and Synthesis
Plant genes specific to nodules are called nodulin (Nod) genes; rhizobial genes that participate in nodule formation are called nodulation (nod) genes. The Rhizobium signal molecules that play a key role in the induction of the initial stages of nodulation are lipochito-oligosaccharides known as Nod factors. The bacterial genes involved in Nod factor synthesis are the nod (nodulation) genes. These genes are not expressed in free-living bacteria, with the exception of nod D, which is expressed constitutively. Nod D has the ability to bind to specific flavonoids secreted by the roots of the host plant, upon flavonoid bindin Beijerinck g, it becomes a transcriptional activator of the other nod genes which encode enzymes involved in the synthesis of Nod factors. The first stage in the formation of the symbiotic relationship between the nitrogen-fixing bacteria and their host is migration of the bacteria toward the roots of the host plant. This migration is a chemotactic response mediated by chemical attractants, especially (iso) flavonoids, homoserine and betaines, secreted by the roots. A specific adhesion protein called rhicadhesin is present on the surface of Rhizobium species. Rhicadhesin is a calcium binding protein and plays role in plant-bacterium attachment. These attractants activate the rhizobial Nod D protein, which then induces transcription of the other nod genes.
Three of the nod genes (nodA, nodB, and nodC) encode enzymes (NodA, NodB, and NodC, respectively) that are required for synthesizing this basic structure.
ü NodA is an N-acyltransferase that catalyzes the addition of a fatty acyl chain.
ü NodB is a chitin-oligosaccharide deacetylase that removes the acetyl group from the terminal nonreducing sugar.
ü NodC is a chitin-oligosaccharide synthase that links N-acetyl-D-glucosamine monomers.
Other enzymes, such as -
ü NodE and NodF determine the length and degree of saturation of the fatty acyl chain;
ü NodL, influence the host specificity of Nod factors through the addition of specific substitutions at the reducing or nonreducing sugar moieties of the chitin backbone.
lnfection
Two processes—infection and nodule organogenesis—
occur simultaneously during root nodule formation. During the infection process, rhizobia that are attached to the root hairs release Nod factors that induce a pronounced curling of the root hair cells. After attachment of rhizobia to the root hair tips, the tips curl tightly and bacteria become entrapped in the curls. A local hydrolysis of the plant cell wall takes place in the curled region and the plasma membrane invaginates and new plant cell wall material is deposited. This results in the formation of a tubular structure, is called infection thread, by which the bacteria enter the plant. The ultrastructure of the wall of the infection thread is very similar to that of the normal plant cell wall, but the incorporation of certain nodulins may provide it with unique properties.
The proline-
rich early nodulins ENOD5 and ENOD12 are candidates for components of the infection thread wall, because cortical cells containing an infection thread express the corresponding genes. The bacteria in the infection thread are surrounded by a matrix that seems to consist of compounds secreted by both the plant and the bacteria. Concomitant with infection thread formation, cortical cells are mitotically reactivated, forming the nodule primordium. Infection threads grow toward this primordium and, once there, release bacteria into the cytoplasm. In those legumes that form indeterminate nodules, nodule primordia arise from inner cortical cells.
Hence, in the formation of this nodule type, the infection threads must traverse the outer cortex before they reach these cells. Before infection thread penetration, the outer cortical cells undergo morphological changes. The nuclei move to the center of the cells, and the microtubules and the cytoplasm rearrange to form a radially oriented conical structure, the cytoplasmic bridge, that resembles a preprophase band. The infection threads traverse the cortical cells through the radially aligned cytoplasmic bridges, which are therefore called preinfection threads. Although the preinfection thread-forming outer cortical cells never divide, the induced morphological changes are reminiscent of those seen in cells entering the cell cycle, express the S phase-specific Histone H4 gene.
However, a mitotic cyclin gene specifically expressed during the G2-to-M phase transition is not activated. Hence, the cells that form the preinfection thread reenter the cell cycle and most likely become arrested in the G2 phase, whereas the inner cortical cells progress all the way through the cell cycle and form the primordia. Thus, bacteria seem to be required for the formation of infection threads. It has been shown that pretreatment with lipopolysaccharides can improve the efficiency of infection thread induction, whereas pretreatment with lipopolysaccharides from a noninfectious Rhizobium strain leads to an increase in aborted infections. Furthermore, mutations in rhizobial exopolysaccharide biosynthesis can render the bacteria unable to induce infection threads. Thus, interaction with bacterial surface compounds seems to play an important role in infection thread formation.
When the infection thread reaches a cell deep in the cortex, it bursts and the rhizobia are engulfed by endocytosis into membrane-enclosed symbiosomes within the cytoplasm. At this time the cell goes through several rounds of mitosis — without cytokinesis — so the cell becomes polyploid. The cortex cells then begin to divide rapidly forming a nodule. This response is driven by the translocation of cytokinins from epidermal cells to the cells of the cortex. The rhizobia also go through a period of rapid multiplication within the nodule cells. Then they begin to change shape and lose their motility. The bacteroids, as they are now called, may almost fill the cell. Only now does nitrogen fixation begin. Reduction of nitrogen to ammonia occurs in bacteriods. Root nodules are not simply structureless masses of cells. Each becomes connected by the xylem and phloem to the vascular system of the plant. Thus the development of nodules, while dependent on rhizobia, is a well-coordinated developmental process of the plant. Nitrogenase catalyze this reaction is very sensitive to oxygen.
In root nodules the O2 level is regulated by leghemoglobin. In plants infected with Rhizobium, the presence of oxygen in the root nodules would reduce the activity of the oxygen-sensitive nitrogenase - an enzyme responsible for the fixation of atmospheric nitrogen. Leghemoglobin buffers the concentration of free oxygen in the cytoplasm of infected plant cells to ensure the proper function of root nodules. Leghemoglobin has a high affinity for oxygen (a Km of about 0.01 µM), about ten times higher than the ß chain of human hemoglobin. This allows an oxygen concentration that is low enough to allow nitrogenase to function but high enough so that it can provide the bacteria with oxygen for respiration. Leghemoglobin has close chemical and structural similarities to hemoglobin, and, like hemoglobin, is red in colour. The protein was believed to be a product of both plant and the bacterium in which the apoprotein is produced by the plant and the heme (an iron atom bound in a porphyrin ring) is produced by the bacterium. Newer findings however, indicate that the heme moiety is also produced by the plant (Virtanen, A. I. (1948). Although leghemoglobin was once thought to provide a buffer for nodule oxygen, recent studies indicate that it stores only enough oxygen to support nodule respiration for a few seconds. Its function is to help transport oxygen to the respiring symbiotic bacterial cells in a manner analogous to hemoglobin transporting oxygen to respiring tissues in animals Ludwig, R. A, 1995). Leghemoglobin is localized in the cytoplasamof infected plant cells and in inside the peribacteroid membrane.
Ammonium assimilation
Plant cells avoid ammonium toxicity by rapidly converting the ammonium generated from nitrate assimilation or photorespiration into amino acids. The primary pathway for this conversion involves the sequential actions of glutamine synthetase and glutamate synthase (Lea et al. 1992). For many years it was thought that bacteria and higher plants assimilate ammonia into glutamate via the Glutamate dehydrogenase (GDH) pathway, as in certain fungi and yeasts. However, in bacteria it became clear in 1970 that an alternative pathway of ammonia assimilation involving glutamine synthetase (GS) and an NADPH-dependent glutamine:2-oxoglutarate amidotransferase (GOGAT) or glutamate synthase, must be operating when ammonia is present in the growth medium at low levels (Tempest et al, 1970). Thus, N-starvation leads to derepression and activation of GS (with a high affinity for NH3) and derepression of GOGAT, and repression of GDH (with a relatively low affinity for NH3) (Tempest et al, 1970). High ammonia availability leads to repression and deactivation of GS and induction of GDH (Tempest et al, 1970).
Glutamate dehydrogenase
NH3 + 2-oxoglutarate + NADPH + H+ <--------------------------> glutamate + NADP+ GS- glutamine:2-oxoglutarate aminotransferase
NH3 + glutamate + ATP ---------------------------------------------------------> glutamine + ADP + Pi Glutamate synthase
Glutamine + 2-oxoglutarate + NADPH + H+ -----------------------> 2 glutamate + NADP+
Both the GDH and GS-GOGAT pathways produce 1 mole of glutamate from 1 mole each of NH3, 2-oxoglutarate and NADPH. But note that the GS-GOGAT pathway is energetically more costly than the GDH pathway, consuming 1 ATP.
ü Escherichia coli is now known to have two primary pathways for glutamate synthesis (Hellig, 1994; 1998). The GS-GOGAT pathway is essential for glutamate synthesis at low ammonium concentrations and for regulation of the glutamine pool, and is used when the cell is not under energy limitation (Hellig, 1994; 1998).
ü The GDH pathway is used in glutamate synthesis when the cell is limited for energy (and carbon; i.e. glucose-limited growth) but ammonium and phosphate are present in excess (Hellig, 1994; 1998).
ü The GS-GOGAT pathway as the primary pathway of ammonia assimilation, but the presence of GDH appears to offer a selective advantage for the cyanobacterium under nonexponential growth conditions (Chavez et al, 1999). These dual pathways may be common to bacteria, cyanobacteria, algae, yeasts and fungi (Huth and Liebs, 1988).
NITRATE ASSIMILATION
Plants assimilate most of the nitrate absorbed by their roots into organic nitrogen compounds. The first step of this process is the reduction of nitrate to nitrite in the cytosol (Oaks 1994). The enzyme Nitrate reductasecatalyzes this reaction:
Nitrate reductasecatalyzes NO3– + NAD(P)H + H++ 2 e– NO2–+ NAD(P)+ + H2O A model of the nitrate reductase dimer, illustrating the three binding domains whose polypeptide sequences are similar in eukaryotes: molybdenum complex (MoCo), heme, and FAD. The NADH binds at the FADbinding region of each subunit and initiates a two-electron transfer from the carboxyl (C) terminus, through each of the electron transfer components, to the amino (N) terminus. Nitrate is reduced at the molybdenum complex near the amino terminus. The polypeptide sequences of the hinge regions are highly variable among species.
ü Nitrate reductase is the main molybdenum-containing protein, deficiency of molybdenum is the accumulation of nitrate that results from diminished nitrate reductase activity.
ü Nitrate Reductase regulated by the Nitrate, Light, and Carbohydrates at the transcription and translation levels.
Nitrite Reductase Converts Nitrite to Ammonium
Nitrite (NO2–) is a highly reactive, potentially toxic ion.Plant cells immediately transport the nitrite generated by nitrate reduction (see Equation 12.1) from the cytosol into chloroplasts in leaves and plastids in roots. In these organelles, the enzyme nitrite reductase reduces nitrite to ammonium according to the following overall reaction:
NO2– + 6 Fd (Reduced) + 8 H+ + 6 e– NH4+ + 6 Fd oxidized + 2 H2O
Summary
To summarize, the processes involved in the assimilation of mineral nitrogen in the leaf, nitrate translocated from the roots through the xylem is absorbed by a mesophyll cell via one of the nitrate–proton symporters (NRT) into the cytoplasm. There it is reduced to nitrite via nitrate reductase (NR). Nitrite is translocated into the stroma of the chloroplast along with a proton. In the stroma, nitrite is reduced to ammonium via nitrite reductas (NiR) and this ammonium is converted into glutamate via the sequential action o glutamine synthetase (GS) and glutamate synthase (GOGAT). Once again in the cytoplasm, the glutamate is transaminated to aspartate via aspartate aminotransferase (Asp-AT). Finally, asparagine synthetase (AS) converts aspartate into asparagine. The approximate amounts of ATP equivalents are given above each reaction ((Taiz, L.; Zeiger, E. 2006).
Published date : 02 Jun 2014 12:22PM
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