BOTANY VOLUME 2 - PLANT PHYSIOLOGY - 2007

6. PHYSIOLOGY OF METABOLISM

6.6. Nitrate Assimilation

Plants absorb nitrogen predominantly in the form of nitrate (NO^-₃) through The ROOT System (see 6.2.3.2). If available ammonium (NH₄⁺) is present in the soil, it can also be absorbed, being converted into Amino Acids directly in the roots. Ammonium is released in the soil As a result of the decomposition of Organic compounds upon the death of living organisms, or it is formed from atmospheric N₂ through The activity of nitrogen-fixing prokaryotes (see 9.2.1). Due to the activity of nitrifying microorganisms, NH₄⁺ is oxidized first to nitrite (NO2-) and then to nitrate (NO₃^-) (see 6.9.1). As a result of denitrification ("nitrate Respiration"), NO₃^- is reduced to N₂ According to the scheme NO^-₃ —> NO₂^-—> NO —> N₂O —> N₂. The latter escapes the biosphere. Annually, about 80 — 120 • 10⁶ t of N₂ are converted by atmospheric nitrogen fixers into NH₄⁺, and almost the same amount is lost during denitrification. To this nitrogen cycle (Fig. 6.87), humans annually add about 30 • 10⁶ t of N₂, which is converted into ammonia via the Haber–Bosch process¹ and used in agriculture for The production of synthetic fertilizers (Table 6.21).

¹ The names of the German chemists who proposed an inexpensive method for synthesizing ammonia from nitrogen and hydrogen under pressure over an iron catalyst. This name of the process is primarily used in German literature — Editor's note.

Class="center">Fig. 6.87. The Nitrogen Cycle in nature

Table 6.21. Global nitrogen balance

Nitrogen is a constituent of many organic compounds (see Ch. 1, 6.2.2.2; 6.13 — 6.16) exclusively in its reduced form (oxidation state -III, ammonium nitrogen). It is used for the synthesis of organic substances and, if necessary, can undergo secondary oxidation (example: the nitro group of aristolochic acid arises from The oxidation of an amino group)².

² A more common example is oxidation to NO, which serves as a second messenger in signal Transduction in almost all plants — Editor's note.

The reduction of nitrate to ammonium occurs via a two-step process with The formation of nitrite (NO^-₂) as an intermediate (the oxidation state is given in parentheses):

It occurs in both green and non-green PARTS OF THE plant, predominantly in the leaves and roots. The resulting ammonium is used directly for the Biosynthesis OF AMINO Acids, primarily glutamine and glutamate. Animals cannot reduce nitrate; they must consume reduced nitrogen compounds provided by plants.

6.6.1. Photosynthetic Nitrate Assimilation

In photosynthetically active Cells (in the leaves of C₄ plants, exclusively in the mesophyll), nitrate is reduced to nitrite by the cytoplasmic enzyme nitrate reductase (Fig. 6.88). The electron donor is mostly NADH + H⁺ (in Fungi, NADPH + H⁺; in Bacteria, reduced ferredoxin). Nitrate reductase is a homodimer. The monomer (molecular mass of about 100 kDa) consists of three domains, each covalently bound to a distinct cofactor, resulting in an Electron Transport Chain within a single molecular complex (Fig. 6.89). From NADH, via FAD and cytochrome b, electrons reach molybdenum, which probably changes its oxidation state from +VI to +IV. This molybdenum of the catalytic center, which interacts with NO₃^- ions, is a constituent of the molybdenum cofactor molybdopterin, which is present in sulfite reductase (see 6.7), as well as in xanthine oxidase and aldehyde oxidase.

Fig. 6.88. Photosynthetic nitrate assimilation and Regulation of the initial reaction — nitrate reductase (Fd — ferredoxin)

Both the formation of NH₃⁺ from NO₂^- and its further utilization are directly light-dependent (see Fig. 6.88). The resulting nitrite is reduced in METABOLISM/14.html">Chloroplasts to ammonium by a sequential supply of 6 electrons, mediated by a highly active nitrite reductase present in the stroma, without the formation of free intermediates. The very high affinity of the enzyme for its substrate ensures that no accumulation of the highly reactive nitrite ion occurs. Electrons are provided by reduced ferredoxin and transferred to nitrite to form NH₄ via a Fe₄S₄-siroheme cofactor, which constitutes the catalytic center of the monomeric enzyme (see Fig. 6.89).

Fig. 6.89. Structure and reaction schemes of nitrate and nitrite reductases (nitrate reductase: only one monomer of the enzyme, which is active as a dimer, is shown)

In the cofactor, which is also present in the structurally very similar sulfite reductase (see 6.7), the iron-sulfur cluster is linked to the central atom (iron) of siroheme via Cysteine-sulfur bridges. Siroheme (see Fig. 6.112) is a precursor heme that carries the acetyl and propionyl side chains of the first closed tetrapyrrole ring in heme biosynthesis, uroporphyrinogen III (see Fig. 6.113).

NH₄⁺, which is an inhibitor of Photosynthesis (see 6.4.9), is used for glutamate synthesis in a sequence of irreversible reactions with The intermediate formation of glutamine (see Fig. 6.88) and therefore does not accumulate in harmful concentrations. The Enzymes involved in glutamate synthesis, Glutamine Synthetase and glutamate synthase (also called glutamine:2-oxoglutarate aminotransferase, GOGAT), catalyze a closed loop driven by ATP and reduced ferredoxin, in which NH₄⁺ is first transferred to an amide bond on the γ-carboxyl group of glutamate, and from there to a 2-oxoglutarate molecule to form L-glutamate. As with all transaminases, Pyridoxal phosphate acts as the coenzyme of glutamate synthase, binding the amino group (pyridoxamine phosphate). L-glutamate leaves the chloroplast in exchange for 2-oxoglutarate, or presumably in some cases, malate. In addition to NH₄⁺ synthesized in chloroplasts, NH₄⁺ derived from Photorespiration is also converted into glutamate (see Fig. 6.88).

NO₂^- is highly reactive chemically. Therefore, it must be strictly ensured that nitrite does not accumulate in the chloroplasts, for example, in the dark. This is achieved by precise regulation of nitrate reductase (see Fig. 6.88). The enzyme has a short biological half-life of a few hours, so its synthesis is matched to demand through regulated Gene Expression. Both nitrate and light activate the Transcription of the nitrate reductase gene. Additionally, light exerts rapid control over enzyme activity: a light-activated phosphatase converts the phosphorylated enzyme, which is inactive in the dark, into the active dephosphorylated form. Glutamine represses the transcription of the nitrate reductase gene. These regulatory mechanisms ensure that nitrite synthesis occurs only when needed, and that the removal of nitrite from chloroplasts is guaranteed. In the dark, accumulated nitrate is stored in the vacuoles of mesophyll cells.

6.6.2. Nitrate Assimilation in Photosynthetically Inactive Tissues

In cells lacking chloroplasts (such as in roots, fungal cells, and bacteria), nitrate is also converted to ammonium via a nitrite formation step. The nitrite reductase reaction in roots takes place in leucoplasts, where the enzyme utilizes electrons from NADH + H⁺; NADH + H⁺ is supplied by the oxidative Pentose Phosphate Pathway (see 6.10.3.5). Non-photosynthetic nitrate assimilation occurs in shoots. It is also observed in woody plants (trees, shrubs) and, to a minor extent, in most mature herbaceous plants (with the exception of many legumes). Plants that predominantly undergo photosynthetic nitrate assimilation store significant amounts of NO₃ in the stem and root system (e.g., Chenopodium, Xanthium, Beta). Ammonium nitrogen synthesized in the roots is converted there into Amino Acids and is transported to the SHOOT via the xylem, primarily in the form of glutamine and asparagine.