Plant Physiology - Musiyenko, M. M. 2001

Plant Root Nutrition
Modern concepts of the molecular nitrogen reduction mechanism. Organisms involved in nitrogen fixation

Atmospheric molecular nitrogen is assimilated exclusively by microorganisms. There are known two genera of archaebacteria, 38 genera of Bacteria, and 20 genera of cyanobacteria capable of Nitrogen Fixation. All of them are collectively referred to as diazotrophs. The most important among them are bacteria of the genera Azotobacter and Clostridium, certain actinomycetes (Frankia), cyanobacteria (Anabaena, Nostoc), and symbiotic bacteria of the genus Rhizobium. Recently, it has been discovered that methane-producing bacteria (methanogens such as Methanobacterium) are also capable of nitrogen fixation. It should be noted that radioactive isotope screening failed to confirm the nitrogen-fixing ability of Yeast and mold species isolated from soil, contrary to previous beliefs. Nitrogen fixers are typically categorized into:

·free-living organisms (Azotobacter, Clostridium);

·symbiotic associations of these organisms with higher plants.

Among free-living organisms in the soil, bacteria predominate, whereas in aquatic environments, cyanobacteria act as the primary nitrogen fixers. Examples of symbiotic systems include bacteria of the genus Rhizobium and legumes, as well as associative symbioses between bacteria and higher plants.

Nonsymbiotic bacteria are capable of adding about 7 kg of nitrogen per hectare of arable land annually.

The process by which microorganisms assimilate molecular nitrogen from the air is called Biological Nitrogen Fixation, and the respective microorganisms are termed nitrogen fixers. Among free-living nitrogen fixers, the aerobic Azotobacter and anaerobic Clostridium are well known. Among symbiotic ones are nodule bacteria, particularly those of the genus Rhizobium, which form distinctive growths on the roots of leguminous plants. In addition to legumes, this ability is also found in Lichens, mosses, alder, sea buckthorn, certain ferns, rosaceous plants, grasses, and sedges.

The ability to fix nitrogen resides not in the plant itself, but in the microorganisms that exist in Symbiosis with it (bacteria, actinomycetes, cyanelles).

Symbiotic systems. From an agricultural perspective, these systems play a crucial role because:

·nitrogen is fixed directly adjacent to the ROOT systems that heavily rely on it;

·although a portion of the ammonium produced via fixation is utilized by the microsymbiont for its own GROWTH AND DEVELOPMENT, the majority of it is exported to the host Cells.

Let us examine the symbiosis in legumes, which exhibit Specificity toward the bacteria that colonize them (genus Rhizobium). This type of bacteria is present in all soils, but in a free-living state, they are incapable of nitrogen fixation. The bacteria colonize plant roots during The Emergence of the first leaves. The initial step in colonization involves the recognition of the host plant by various species of the genus Rhizobium. A vital role in this process is played by Lectins—specific plant Proteins capable of binding to sugar (carbohydrate) residues, thereby adhering adjacent Cell surfaces together by linking their polysaccharide groups. These lectins can interact with specific bacteria of a given genus, ensuring that the plant recognizes and contacts the appropriate species.

Bacteria enter the plant through root hairs, which become deformed under METABOLISM/18.html">The Influence of a hormone-like substance secreted by the bacterium. From the site of bacterial entry into the root Hair, an infection thread develops, through which the bacteria migrate into the root tissue while multiplying. Once the plant is colonized by a single strain, other strains are prevented from entering (hence, from a practical standpoint, the necessity arises to utilize the most efficient nitrogen-fixing strains).

The colonization of the host plant's root cells begins after the bacteria are released from the infection thread and is mediated by two Cell wall-hydrolyzing Enzymes: bacterial pectinase and plant cellulase. These enzymes facilitate the penetration of bacteria into plant cells, where they continue to divide. The plant cell increases in volume, becomes packed with bacteria, alters its Structure and function, and transforms into a bacteroid, which is ultimately responsible for nitrogen fixation.

Simultaneously with the bacterial invasion of the host cell, active division of both infected and neighboring uninfected cells begins, which facilitates the spread of bacteria and leads to The formation of root nodules characteristic of symbiotic associations (Fig. 132).

Furthermore, the inner core of infected and uninfected cells is consistently surrounded by a cortical layer of uninfected host cells.

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Fig. 132. a — nitrogen-fixing root nodules on soybean (Glycine max) formed As a result of symbiotic interactions between root cells of this legume and Rhizobium bacteria; b, c — scanning Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF white clover (Trifolium repens) nodules

The infected Regions of the inner zone of a mature root nodule may contain one or several bacteroids. They are surrounded by a membranous envelope that likely harbors a red pigment protein—phytoglobin (analogous to Blood hemoprotein Hemoglobin, Muscle Myoglobin, and plant phytoglobin). It is synthesized exclusively in nitrogen-fixing nodules and localized within infected cells, accounting for up to 30–40% of the total soluble protein in The Cell. Its synthesis is encoded by the DNA of the host cell genome rather than the bacterial DNA.

It is hypothesized that the bacteroids possess a membrane-bound Respiratory Chain structurally similar to the classical Mitochondrial Electron Transport chain coupled with Oxidative Phosphorylation. This chain participates in the synthesis of ATP, which is essential for nitrogen fixation.

Thus, for a symbiotic system to fix nitrogen, three conditions are required: the formation of root nodules, the differentiation of bacteria into bacteroids, and the synthesis of phytoglobin. Fixation continues until the nodules senesce, with some surviving until the beginning of the next growing season.

Algal symbioses arise through the association of blue-green Algae with higher plants. However, unlike Rhizobium and actinomycetes, which form symbioses with flowering plants, cyanobacteria associate with lower plant forms (lichens, liverworts, etc.).

Current Concepts of molecular nitrogen reduction. Although we are surrounded by an ocean of molecular nitrogen, the nitrogen molecule is so tightly bound that breaking the triple covalent bond requires 940 kJ/mol. Consequently, industrial ammonia synthesis requires temperatures around 500 °C and pressures of 300–350 atm, whereas nitrogen-fixing microorganisms operate under ambient conditions. This is because they possess specific, highly efficient enzyme systems that catalyze the individual steps of nitrogen fixation.

For this reason, The problem of biological nitrogen fixation occupies a prominent place in research laboratories worldwide. It must be emphasized that across all diverse nitrogen fixers, the exact same enzyme system catalyzes The conversion of molecular nitrogen into ammonia.

This enzyme complex is Nitrogenase, which was first isolated by Carnahan et al. (1960) from dry cells of Clostridium pasteurianum in the laboratories of DuPont in the USA. It has been established that nitrogenase consists of two proteins: an azoprotein (Fe-protein) and a molybcoprotein (Mo-Fe-protein):

The first of these has a Molecular Weight of 56,000–67,000 and exists as a dimer containing non-heme iron, meaning iron bound directly to protein sulfur. The second, a molybdenum protein, is a tetramer composed of 4 subunits with a molecular weight of 200,000–270,000, and contains 2 molybdenum atoms in addition to non-heme iron. Currently, both nitrogenase proteins have been isolated and studied from a range of microorganisms.

The reduction of one mole of nitrogen requires 12–16 ATP, which is 4–5 ATP per pair of electrons transferred from the reductant to nitrogen during the reduction process. In reality, energy expenditures exceed these values, reaching up to 30–40 moles of ATP per mole of fixed nitrogen. Photosynthesis, Respiration, and Fermentation serve as the sources of electrons (ferredoxins, flavodoxins) and ATP.

Enzyme function requires the formation of a 1:1 complex between the iron protein and the molybdenum-iron protein. This two-component nitrogenase complex exists only during the Electron transfer from the iron protein to the molybdenum of the molybdenum protein, which is coupled with ATP Hydrolysis. The energy required for nitrogenase activity is supplied not by free ATP, but by the Mg-ATP complex, which binds to the iron protein. Interestingly, the Fe-protein is structured from two subunits in such a way that a cleft is formed between them, which presumably accommodates the ATP molecule.

The molybdenum protein exists in three different states, each characterized by a specific degree of reduction of the iron atoms within the molecule. Although The Mechanism of Nitrogen fixation is not yet fully elucidated, it is believed that the molybdenum protein itself reacts with N₂, the potential substrate of nitrogenase, through the binding of the latter by the protein's molybdenum atoms (Fig. 133). Schematically, the fixation process involves the reduction of iron atoms in the iron protein, resulting in the hydrolysis of ATP to ADP and P_i. An electron from an iron atom in the molybdenum protein is used to reduce the substrate bound to it. Several such transfers must occur before the enzyme releases the final reduced product, ammonium. Electron transfer is accompanied by ATP hydrolysis.

Fig. 133. Nitrogen fixation in bacteroids. Malate oxidation in the Krebs cycle yields five NADH and one FADH₂. The synthesis of two molecules of NH₃ from N₂ is accompanied by the reduction of 2H⁺ to H₂ at the expense of 16 ATP molecules. The synthesis of these ATP molecules is localized in the respiratory chain of the bacteroid membrane and is driven by The oxidation of eight NADH molecules. For each molecule of fixed N₂, four O₂ molecules are consumed to oxidize NADH in the respiratory chain of the bacteroid membrane.

It should be remembered that The Nitrogenase Enzyme is destroyed (inactivated) upon contact with O₂. For this reason, facultative nitrogen fixers fix nitrogen only under anaerobic conditions. Aerobic nitrogen fixers (such as Azotobacter) require significant O₂ consumption to maintain a high rate of respiration and ensure ATP synthesis. This may be what prevents O₂ from entering the nitrogen fixation centers without adversely affecting the enzyme's activity. Another hypothesis suggests that the protection of nitrogenase is likely carried out by associated proteins, particularly through conformational changes.

Free-living cyanobacteria fix nitrogen in heterocysts, whose thick cell walls protect against oxygen influx. Symbiotic organisms, on the other hand, possess a specific regulatory system to control O₂ concentration in the nitrogen fixation zone—phytoglobin (leghemoglobin), synthesized by the host plant. It is quite sensitive to O₂ and therefore prevents oxygen accumulation. Synthesized by the host plant cell, it is integrated into the bacteroid membrane and facilitates O₂ transport to the bacteroid, thus providing a protective effect for nitrogenase.

Thus, the bacteroid should be viewed as a highly specialized compartment that creates an environment where Nitrogen Fixation and oxidative processes are physiologically compatible. The Krebs cycle functioning within the bacteroid serves as a source of substrates for oxidation in the ETS, which drives ATP synthesis, supplies nitrogenase with electrons via ferredoxin, and provides keto acids for the synthesis of Amino Acids that are subsequently transported into the host plant cells.

Regulation of Nitrogenase. In terms of Energy Expenditure, nitrogenase must be considered an economically unfavorable enzyme, as maintaining its activity requires ATP. Therefore, nitrogen-fixing organisms have evolved mechanisms to regulate the synthesis and activation of nitrogenase.

The ability to fix nitrogen is governed by a set of genes known as the nitrogen fixation (nif) genes. Free-living nitrogen fixers synthesize ammonia concurrently with the biosynthetic processes of the bacterial cell, meaning it is practically assimilated and not released into the environment. When fixed nitrogen (NH₄⁺, NO₃^-) is present in the soil and can be utilized for cellular growth processes, the nif genes are repressed, and nitrogenase is not produced.

Conversely, in some symbiotic bacteria, such as Rhizobium, the nif genes are normally derepressed, resulting in nitrogenase being produced and remaining active even in the presence of ammonium, while the fixed nitrogen is released into the host Cell Cytoplasm. In such a symbiotic association, the derepresion of nif genes drives the fixation of significant amounts of atmospheric nitrogen.

Another mode of regulating nitrogen fixation in symbiosis involves the supply of ATP. It is required for enzyme activation, whereas "in vitro" experiments have shown that ADP inhibits its activity. There is evidence that Glutamine Synthetase—an enzyme responsible for ammonia assimilation into glutamine within the cell—participates in the Transcriptional Regulation of nitrogenase genes. This enzyme exists in an active (deadenylated) form and an inactive (adenylated) form. When ammonium levels are high, the cell adenylates glutamine synthetase and thereby inactivates it, leading to the shutdown of nitrogen fixation Gene Transcription.

The regulation of glutamine synthetase activity via adenylation (the covalent attachment of 5-AMP to the hydroxyl groups of tyrosyl residues on glutamine synthetase subunits) is carried out indirectly through changes in The activity of adenylyl transferase, which catalyzes the adenylation and deadenylation reactions. When this reaction is unregulated, a cycle arises that causes fluctuations of glutamine synthetase between its active and inactive forms, resulting in wasteful ATP hydrolysis. This process can be prevented by the action of so-called regulatory proteins, which also exist in complex with ATPase in two forms.

Other mechanisms of cellular control over nitrogen fixation Gene Expression also exist.