How do plants synthesise nitrogen?

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Introduction:

Plants are autotrophic organisms. They can synthesize their organic molecular components out of inorganic nutrients. In order to do so first they need to obtain inorganic nutrients from their surroundings. Such nutrients are obtained from the soil. These include N, S, P, Si, B, K, Ca, Mg, Cl, Mn, Na, Fe, Zn, Cu, Ni and Mo. Roots absorb these minerals from the soil and incorporate them into organic compounds that are essential for growth and development of the plant. This incorporation of mineral nutrients into organic substances such as pigments, enzyme cofactors, lipids, nucleic acids, and amino acids is termed nutrient assimilation.

Nitrogen is the mineral element that plants require in greatest amounts. It is as a constituent of many plant cell components, including amino acids and nucleic acids. Therefore, nitrogen deficiency rapidly inhibits plant growth. If such a deficiency persists, most species show chlorosis (yellowing of the leaves), especially in the older leaves near the base of the plant. Under severe nitrogen deficiency, these leaves become completely yellow and fall off the plant. Younger leaves do not show these symptoms initially because nitrogen can be mobilized from older leaves. Thus a nitrogen-deficient plant may have light green upper leaves and yellow lower leaves. When nitrogen deficiency develops slowly, plants have markedly slender and often woody stems. This woodiness is due to a build-up of excess carbohydrates that cannot be used in the synthesis of amino acids or other nitrogen compounds. Carbohydrates not used in nitrogen metabolism are also used in anthocyanin synthesis, leading to pigment accumulation leading to purple coloration in leaves, petioles, and stems of some nitrogen-deficient plants, such as tomato and certain varieties of corn.

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Assimilation of nitrogen requires a complex series of biochemical reactions that are among the most energy-requiring reactions in living organisms.

Nitrogen fixation

Nitrogen fixation is the process of converting atmospheric nitrogen into ammonium and nitrate. Acquisition of nitrogen from the atmosphere requires the breaking of an exceptionally stable triple covalent bond between two nitrogen atoms (N≡N) to produce ammonium or nitrate. In nitrate (NO3–) assimilation, the nitrogen in NO3– is converted to a higher-energy form in nitrite (NO2–), then to a yet higher-energy form in ammonium (NH4+), and finally into the amide nitrogen of glutamine. This process consumes the equivalent of 12 ATPs per nitrogen. Plants such as legumes form symbiotic relationships with nitrogen-fixing bacteria to convert molecular nitrogen (N2) into ammonia (NH3). Ammonia (NH3) is the first stable product of natural fixation; at physiological pH, however, ammonia is protonated to form the ammonium ion (NH4+). The process of biological nitrogen fixation, together with the subsequent assimilation of NH3 into an amino acid, consumes about 16 ATPs per nitrogen.

Industrially nitrogen fixation is carried out by the Haber-Bosch process. In this process N2 combines with H2. The reaction takes place at a temperature of 200°C and pressure of 200 atmospheres. These elevated conditions indicate how energy intensive the process is. Naturally nitrogen fixation occurs in the following ways

  • Lightning - Lightning converts water vapour 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 combines with rain and is brought down to the ground.
  • Photochemical reactions - Photochemical reactions between gaseous nitric oxide (NO) and ozone (O3) produce nitric acid (HNO3).
  • Biological nitrogen fixation - Strains of bacteria called diazotrophs fix N2 into ammonium (NH4+). They achieve this by the enzyme nitrogenase. Cyanobacteria (blue-green algae) are free-living photosynthetic bacteria. Bacteria belonging to the genus Azotobacter, Clostridium, Pseudomonas to name a few, also fix N2. Legumes also possess the enzyme nitrogenase. The amount if metabolically useful nitrogen they produce far exceeds the amount they require and excess leaches into the soil enriching the content of fixed nitrogen in the soil. The reaction catalysed by the enzyme nitrogenase is as follows:
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N2+ + 8H+ + 8e-1 + 16ATP + 16H2O → 2NH3 + H2 + 16ADP + 16Pi

Of the above mentioned three naturally occurring processes, a major percentage of the fixed nitrogen that is, about 90% is obtained from biological nitrogen fixation. Only a small percentage of fixed nitrogen (about 2-4%) is obtained from the other two naturally occurring processes. The nitrogen in fixed ammonium or nitrate then enters various biochemical cycles and constantly shuffles between organic and inorganic forms. There is intense competition among plants and microorganisms for the ammonium or nitrate ions that are generated through fixation or released through decomposition of soil organic matter. Plants have developed mechanisms for scavenging these ions from the soil solution as quickly as possible. Under the elevated soil concentrations that occur after fertilization, the absorption of ammonium and nitrate by the roots may exceed the capacity of a plant to assimilate these ions, leading to their accumulation within the plant’s tissues.

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Figure 1: Various steps involved in the nitrogen cycle

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Figure 2: Enzymes involved in the nitrogen cycle

Nitrate assimilation:

In the most general form nitrate assimilation can be represented by the following reaction:

HNO3 + 8H+ → NH3 + 3H2O

The scheme of events that take place and the enzymes involved in the assimilation of nitrates can be depicted as follows:

HNO3 → HNO2 → (HNO)2 → NH2OH → NH3

  1. Role of enzyme nitrate reductase:

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. The enzyme nitrate reductase catalyses this reaction:

NO3– + NAD(P)H + H+ + 2e → NO2 + NAD(P)+ + H2O

NAD(P)H indicates NADH or NADPH. The most common form of nitrate reductase uses only NADH as an electron donor; another form of the enzyme that is found predominantly in non-green tissues such as roots can use either NADH or NADPH. Nitrate reductase is a metallo-flavoprotein. It composed of two identical subunits, each containing three prosthetic groups namely, FAD (flavin adenine dinucleotide), heme, and molybdenum complexed to an organic molecule called a pterin.

Nitrate reductase is the main molybdenum-containing protein in vegetative tissues, and one symptom of molybdenum deficiency is the accumulation of nitrate that results from diminished nitrate reductase activity. The FAD-binding domain accepts two electrons from NADH or NADPH. The electrons then pass through the heme domain to the molybdenum complex, where they are transferred to nitrate. This reaction can be schematically represented as follows:

""""Figure 3: Schematic representation of nitrate reduction to nitrite by the enzyme nitrate reductase.

  1. Regulation of nitrate reductase:

Nitrate, light and carbohydrates regulate the enzyme synthesis at the transcription and translational level. Light, carbohydrate levels, and other environmental factors stimulate a protein phosphatase that dephosphorylates several serine residues on the nitrate reductase protein and thereby activates the enzyme. On the other hand darkness and Mg2+ stimulate a protein kinase that phosphorylates the same serine residues. This enables the enzyme to interact with a protein inhibitor result in the enzyme’s inactivation.

  1. Role of the enzyme nitrite reductase
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Nitrite is toxic to plant cells as it is highly reactive. It is transported to the chloroplasts in leaves and plastids in roots as soon as it produced as the enzyme nitrite reductase is present within these organelles. Nitrite reductase catalyses the conversion of nitrite to ammonium.

NO2 + 6Fdred + 8H+ + 6e → NH4+ + 6Fdox + 2H2O

Nitrite reductase is also a metallo-flavoprotein containing FAD and requires NADH2 or NADPH2 as hydrogen donor. The reduced form of ferrodoxin in the reactants in the above equation is obtained from the photosynthetic electron transport which takes place in the chloroplast.

Some experiments led to the hypothesis that the intermediate formed in the reduction of nitrite to ammonium is hyponitrite while others led to the hypothesis that nitric oxide is the intermediate produced. Some theories suggest that the enzyme nitrite reductase catalyses the reduction of nitrite to ammonium without the production of any intermediates.

  1. Regulation of nitrite reductase

Nitrite reductase is encoded in the nucleus and synthesized in the cytoplasm with an N-terminal transit peptide that targets it to the plastids. NO3– and light induce the transcription of nitrite reductase mRNA. The end products of the process asparagine and glutamine repress this induction.

  1. Enzyme location

The enzymes that carry out nitrate reduction are present mostly in the shoot and roots of the plant. In many plants, when the roots receive small amounts of nitrate, nitrate is reduced primarily in the roots. As the supply of nitrate increases, a greater proportion of the absorbed nitrate is translocated to the shoot and assimilated there. Even under similar conditions of nitrate supply, the balance between root and shoot nitrate metabolism varies from species to species. In plants such as the cocklebur (Xanthium strumarium), nitrate metabolism is restricted to the shoot; in other plants, such as white lupine (Lupinus albus), most nitrate is metabolized in the roots. Generally, species native to temperate regions rely more heavily on nitrate assimilation by the roots than do species of tropical or subtropical origins.

Fate of ammonium

Accumulation of ammonium is toxic to the plant. Plants rapidly convert the ammonium generated by nitrate reductase to amino acids. This is known as ammonium assimilation. The enzyme glutamine synthase catalyses the synthesis of glutamine. The reaction is represented as follows:

Glutamate + NH4+ + ATP → Glutamine + ADP + Pi

An elevated level of glutamine stimulates the enzyme glutamate synthase. This enzyme catalyses the transfer of the amide group of glutamine to 2-oxoglutarate, yielding two molecules of glutamate.

Glutamine + 2-oxoglutarate + NADH + H+ → 2 Glutamate + NAD+

The enzyme glutamate dehydrogenase does the opposite. It deaminates glutamate. The reaction is reversible and is represented as follows:

2-Oxoglutarate + NH4+ + NAD(P)H ↔ Glutamate + H2O + NAD(P)+

Once assimilated into glutamine and glutamate, nitrogen is incorporated into other amino acids via transamination reactions. The enzymes that catalyze these reactions are known as aminotransferases. All transamination reactions require pyridoxal phosphate (vitamin B6) as a cofactor. Aminotransferases are found in the cytoplasm, chloroplasts, mitochondria, glyoxysomes, and peroxisomes.

Nitrate reduction and cell metabolism

During respiration carbohydrates are oxidized to produce CO2. Nitrate reduction is strongly dependent on respiration as the oxidation of carbohydrates serves as a source of hydrogen for the reduction of nitrates. Thus the two processes are tightly coupled. This has been proven by experiments carried out under anaerobic conditions. In these experiments the reduction of nitrates and nitrites was greatly depressed. The reduction reactions also require NADH2 and high energy phosphate both of which are supplied by respiration.

Light favours nitrate reduction indirectly. This is because NADH2 and ferredoxin required by some of the enzymes in nitrate reduction are produced during photosynthesis. Thus, nitrate reduction is coupled with both respiration and photosynthesis.

Ecological importance of the nitrogen cycle

The nitrogen cycle consists of several major steps. In nitrogen fixation specialized bacteria in soil as well as blue-green algae (cyanobacteria) in aquatic environments combine gaseous N2 with H2 to make ammonia (NH3). The bacteria use some of the ammonia they produce as a nutrient and excrete the rest into the soil or water. Some of the ammonia is converted to ammonium ions (NH4+) that plants can use as a nutrient. Ammonia not taken up by plants may undergo nitrification. In this process, specialized soil bacteria convert most of the NH3 and NH4+ in soil to nitrate ions (NO3), which are easily taken up by the roots of plants. The plants then use these forms of nitrogen to produce various amino acids, proteins, nucleic acids, and vitamins. Animals that eat plants eventually consume these nitrogen-containing compounds, as do detritus feeders and decomposers.

Plants and animals return nitrogen-rich organic compounds to the environment as both wastes and cast-off particles of tissues such as leaves, skin, or hair, and through their bodies when they die and are decomposed or eaten by detritus feeders. In ammonification, vast armies of specialized decomposer bacteria convert this detritus into simpler nitrogen-containing inorganic compounds such as ammonia (NH3) and water-soluble salts containing ammonium ions (NH4+).

In denitrification, specialized bacteria in waterlogged soil and in the bottom sediments of lakes, oceans, swamps, and bogs convert NH3 and NH4+ back into nitrate ions, and then into nitrogen gas (N2) and nitrous oxide gas (N2O). These gases are released to the atmosphere to begin the nitrogen cycle again.

Human intervention adds large amounts of nitric oxide (NO) into the atmosphere when N2 and O2 combine when any fuel is burnt at high temperatures, such as in car, truck, and jet engines. In the atmosphere, this gas can be converted to nitrogen dioxide gas (NO2) and nitric acid vapour (HNO3), which can return to the earth’s surface called acid rain.

Nitrous oxide (N2O) is added to the atmosphere through the action of anaerobic bacteria on commercial inorganic fertilizer or organic animal manure applied to the soil. This greenhouse gas can warm the atmosphere and deplete stratospheric ozone, which keeps most of the sun’s harmful ultraviolet radiation from reaching the earth’s surface.

Large quantities of nitrogen stored in soils and plants as gaseous compounds are released into the atmosphere through destruction of forests, grasslands, and wetlands. The nitrogen cycle in aquatic ecosystems is disturbed by adding excess nitrates (NO3) to bodies of water through agricultural runoff of fertilizers and animal manure and through discharges from municipal sewage systems. This can cause excess growth of algae.

Nitrogen is also removed from topsoil when nitrogen-rich crops are harvested, crops are irrigated (washing nitrates out of the soil), and grasslands and forests are burned and cleared before planting crops

Thus, it is important that there be a balance in the intermediates of the nitrogen cycle. Plants play an important role in maintaining such a balance.

References:

  • Masclaux-Daubresse C, Daniel-Vedele F, Dechorgnat J, Chardon F, Gaufichon L, Suzuki A. Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Annals of Botany. 2010;105(7):1141-1157.
  • Miflin BJ. The Location of Nitrite Reductase and Other Enzymes Related to Amino Acid Biosynthesis in the Plastids of Root and Leaves . Plant Physiology. 1974;54(4):550-555.
  • Kessler E. Nitrate Assimilation by Plants. Annual Review of Plant Physiology. 1964;15(1):57-72. pp.15.060164.000421.
  • Taiz L, Zeiger E. Plant Physiology. Sunderland, Mass.: Sinauer Associates; 2002:259-264.

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