PLANT PHYSIOLOGY AND BIOCHEMISTRY

Lecture Notes

7. MINERAL NUTRITION OF PLANTS

Nitrogen Nutrition

Nitrogen uptake and transformation in plants

Molecular nitrogen (N₂) accounts for approximately 80% of the atmosphere. However, in this form, it is inaccessible to higher plants. They can only assimilate its oxides or ammonia, which are present in the atmosphere in very low concentrations. Therefore, the primary source of nitrogen for plants is its mineral compounds found in the soil. Plants possess the capacity for the autotrophic assimilation of not only carbon but also nitrogen.

Autotrophic nitrogen assimilation

Nitrogen exists in the soil in various chemical forms. A significant portion is incorporated into humus (a product of the incomplete decomposition of organic residues), which plants can only utilize after it has been mineralized by soil microflora into more accessible compounds, primarily ammonia. In its gaseous form, ammonia is readily absorbed by plants, but its concentration in soil is low. Typically, ammonia dissolves in soil Water and reacts with various acids to form ammonium salts, or it is oxidized by nitrifying Bacteria into nitrous and nitric acids, which then react with cations to form nitrites and nitrates. Ammonium salts and nitrates are water-soluble and constitute the main Forms of soil nitrogen available to plants.

Plants require significant amounts of nitrogen for their development.

Ammonium nitrogen enters plant roots in a reduced form and is therefore immediately incorporated into the synthesis of Amino Acids and Proteins.

In nitrates, nitrogen is in an oxidized state. Consequently, before it can enter metabolic pathways, it must be reduced to ammonia.

The conversion of nitrates into ammonia within ROOT Cells is known as chemical reduction. This process occurs through two successive reactions involving molybdenum- and iron-containing Enzymes: nitrate reductase and nitrite reductase. First, nitrates are reduced by nitrate reductase to nitrites, which are subsequently reduced by nitrite reductase to ammonia:

A necessary condition for these reactions is the presence of a reducing agent (an electron donor) and energy. NADH serves as the source of electrons, while ATP provides the energy, both generated during root Respiration. For active nitrate reduction, the root must maintain a reserve of CARBOHYDRATES, which serve as a respiratory substrate and a source of keto acids capable of binding the ammonia. Otherwise, ammonia accumulates and inhibits the nitrate reduction reaction.

In the absence of these conditions, or when large quantities of nitrates enter the roots, they are not metabolized locally. Instead, they are transported via the Transpiration stream through the stems to the leaves, where they are reduced to ammonia using NADPH₂ and ATP generated during Photosynthesis. For this reason, this process is referred to as photochemical nitrate reduction.

Ammonia synthesized through nitrate reduction is attached to primary keto acids (Pyruvate, oxaloacetate, α-ketoglutarate) via direct amination to form the corresponding primary amino acids (Alanine, aspartic acid, and glutamic acid). These reductive amination reactions are catalyzed by specific dehydrogenases (alanine, aspartate, and glutamate dehydrogenases) and require NAD(P)H₂. Through the rearrangement of their carbon skeletons, primary Amino acids are converted into Other Amino Acids, which are then utilized in the synthesis of primary proteins. This entire process constitutes the anabolic branch of Nitrogen METABOLISM. Most amino acids are used for Protein Synthesis, while redundant Polypeptides are broken down into amino acids. These can be reused for protein synthesis, undergo Transamination, or be deaminated to yield keto acids and ammonia. This process represents the catabolic branch of plant nitrogen metabolism.

Ammonia that enters the plant or is produced during biochemical transformations is toxic to the Organism. However, unlike in animals, it is not excreted into the environment; instead, it is bound by aspartic or glutamic acid to form the corresponding amides—asparagine and glutamine. This process is mediated by synthetases and requires energy.

Amides serve as a nitrogen reservoir, where ammonia—a scarce resource for the plant—is sequestered and detoxified. The amino groups of primary amino acids and their amides are transferred to various keto acids by aminotransferases, resulting in The formation of secondary amino acids. The enzymatic transfer of amino groups from amino acids and amides to keto acids without The intermediate formation of ammonia is called transamination. Transamination reactions link the anabolic and catabolic branches of nitrogen metabolism, which both originate and terminate with ammonia.

In addition to autotrophic N assimilation, there is heterotrophic assimilation, where certain plant species have adapted to meet their nitrogen requirements by absorbing nitrogenous compounds from other organisms. These include parasitic plants, hemiparasites, Saprophytes (which absorb decaying organic matter), and carnivorous plants.

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Fig. 19. Transformation of nitrogenous substances in plants.

MOLECULAR Nitrogen Fixation

Only certain bacteria are capable of fixing molecular nitrogen. They reduce it into an ammonia form accessible to other organisms. Therefore, they are known as nitrogen fixers. These are categorized into free-living and symbiotic nitrogen-fixing bacteria.

Free-living nitrogen fixers inhabit water bodies and soils and comprise a large group of diverse microorganisms. These include bacteria of the genera Clostridium and Azotobacter, photosynthetic bacteria, mycobacteria, actinomycetes, blue-green Algae (or cyanobacteria), and Lichens.

Under specific ecological conditions, all of these organisms enrich the soil with fixed nitrogen forms. For instance, blue-green algae are the primary nitrogen suppliers in rice paddies and the World Ocean. Primitive soil is enriched with nitrogen through The activity of lichens. In podzolic and peat soils, mycobacteria serve as the main nitrogen accumulators, as the activity of other nitrogen fixers is inhibited there by high acidity.

The mechanism of Biological Nitrogen Fixation is not yet fully understood. It has been established that this process is mediated by Nitrogenase, a specialized enzyme complex consisting of two protein fractions. One fraction contains azoferredoxin with non-heme iron (4FeS), while the other contains molybdoferredoxin, which includes two Mo atoms in addition to non-heme iron in the form of FeS clusters.

The source of the reducing agent required for molecular nitrogen reduction is reduced ferredoxin (which supplies ē), while the energy source is ATP, generated via Fermentation (in anaerobic nitrogen fixers) or respiration (in aerobic ones). Mg²⁺ ions serve as an activator for this reaction.

In general terms, The process of molecular nitrogen fixation occurs as follows: first, azoferredoxin, with the participation of ATP and electrons, undergoes a conformational change and becomes reduced. It then transfers electrons to molybdoferredoxin, which directly interacts with molecular nitrogen and reduces it to ammonia.

The nitrogen-fixing capacity of free-living nitrogen fixers amounts to several tens of kilograms of nitrogen per hectare.

Symbiotic nitrogen fixers represent a large group of so-called root-nodule bacteria (genus Rhizobium) that develop on the roots of legumes, as well as many woody and shrub species. This group also includes certain actinomycetes and cyanobacteria. In their free-living state, they do not fix nitrogen; they acquire this ability only upon entering into Symbiosis with higher plants. Currently, about 190 species of trees and shrubs from various families are known to form symbiotic relationships with these microorganisms. They penetrate root cells through root hairs via a specialized infection thread, which facilitates their movement into deeper tissue layers.

It is likely that these bacteria secrete hormone-like substances that stimulate root Cell Division and the formation of nodules. Within the nodule cells, the bacteria undergo physiological and morphological changes, acquire an additional membrane, and transform into so-called bacteroids.

In 1967, Bergersen, using the ¹⁵N isotope, established that Nitrogen fixation is carried out specifically by the bacteroid cells. Root Hair cells play a vital role in the nitrogen-fixing process. They contain a pink pigment called leghemoglobin, which, similar to Hemoglobin, binds oxygen and supplies it to the bacteroids located deep within the nodules.

The Mechanism of biological molecular Nitrogen fixation by symbiotic bacteria is fundamentally similar to that of free-living nitrogen fixers. This process is carried out by an analogous enzyme complex and requires a significant expenditure of energy.

Root-nodule bacteria comprise many strains, each adapted to the roots of only one or a few plant species. Nitrogen fixation productivity depends on The Nature of the strain. The most active nitrogen accumulators are symbionts of alfalfa (providing approximately 500-600 kg of fixed nitrogen per hectare per year), clover (300 kg/ha), and lupine (160 kg/ha). Symbionts of peas, beans, and lentils are less efficient (50-60 kg/ha). Root-nodule bacteria of woody species fix about 100 kg of nitrogen per hectare per year. Overall, the nitrogen-fixing productivity of root-nodule bacteria is significantly higher than that of free-living nitrogen fixers. This is attributed to the fact that, As a result of symbiosis with higher plants, root-nodule bacteria are better supplied with respiratory substrates and other essential metabolites.

The productivity of nitrogen fixation depends on the Nature of the relationship between the symbiotic partners and the activity of the strain. Effective strains exhibit clear parasitism toward the host plant during the Initial Stages of penetration and nodule formation, utilizing its metabolites without fixing nitrogen. The plants may show signs of suppressed vitality. However, as the nodules grow and bacteroids form, the nitrogen-fixing capacity of the bacteria increases, and the relationship between the partners becomes mutually beneficial. By the time annual legumes reach the flowering stage, the supply of nutrients to the nodules decreases, the bacteroids and nodule cells undergo lysis, and atmospheric nitrogen fixation ceases.

Nodules of perennial grasses fix nitrogen over several years, but toward the end of each growing season, a portion of their cells containing bacteroids degrades, which also leads to a decrease in nitrogen fixation intensity.

When plants are infected with an ineffective strain, very little leghemoglobin is produced in the nodules, and nitrogen fixation productivity is virtually zero. In such cases, the bacteria merely inhibit the GROWTH AND DEVELOPMENT of the higher plant. Therefore, in agricultural practice, legume seeds are specifically inoculated with a bacterial preparation called Nitragin, which contains a highly effective strain of root-nodule bacteria.

Biological fixation of molecular nitrogen plays an exceptionally important role in the life of an ecosystem. Nitrogen fixers are of great significance in natural biogeocenoses, where various nitrogen losses (leaching of salts, denitrification, etc.) are primarily replenished through their activity and only partially through the decomposition of animal and plant remains.

In agrobiocenoses, nitrogen losses are even greater, as a significant portion is removed during harvest. For example, a potato yield of 250-300 centners/ha removes approximately 200 kg of nitrogen from the soil. Therefore, legumes are an essential component of any crop rotation. However, a significant portion of nitrogen losses must still be replenished through the application of organic and mineral fertilizers.