nitrogen assimilation
- 2. The reduction of nitrate to NH3 proceeds in two partial reactions Nitrate is assimilated in the leaves and also in the roots. In most fully grown herbaceous plants, nitrate assimilation occurs primarily in the leaves, although nitrate assimilation in the roots often plays a major role at an early growth state of these plants. In contrast, many woody plants (e.g., trees, shrubs), as well as legumes such as soybean, assimilate nitrate mainly in the roots.
- 3. The transport of nitrate into the root cells proceeds as symport with two protons. A proton gradient across the plasma membrane, generated by a H+-P-ATPase drives the uptake of nitrate against a concentration gradient. The ATP required for the formation of the proton gradient is mostly provided by mitochondrial respiration. When inhibitors or uncouplers of respiration abolish mitochondrial ATP synthesis in the roots, nitrate uptake normally comes to a stop.
- 4. these are a transporter with a relatively low affinity (half saturation >500mM nitrate) and a transporter with a very high affinity (half saturation 20–100mM nitrate),where the latter is induced only when required by metabolism. The nitrate taken up into the root cells can be stored there temporarily in the vacuole. nitrate is reduced to NH4+ in the epidermal and cortical cells of the root. This NH4+ is mainly used for the synthesis of glutamine and asparagine
- 5. However, when the capacity for nitrate assimilation in the roots is exhausted, nitrate is released from the roots into the xylem vessels and is carried by the transpiration stream to the leaves. There it is taken up into the mesophyll cells, probably also by a proton symport. Large quantities of nitrate can be stored in a leaf by uptake into the vacuole. Sometimes this vacuolar store is emptied by nitrate assimilation during the day and replenished during the night. Thus, in spinach leaves, for instance, the highest nitrate content is found in the early morning.
- 6. The nitrate in the mesophyll cells is reduced to nitrite by nitrate reductase present in the cytosol and then to NH4+ by nitrite reductase in the chloroplasts
- 8. Nitrate assimilation in the roots and leaves of a plant. Nitrate is taken up from the soil by the root. It can be stored in the vacuoles of the root cells or assimilated in the cells of the root epidermis and the cortex. Surplus nitrate is carried via the xylem vessels to the mesophyll cells, where nitrate can be temporarily stored in the vacuole. Nitrate is reduced to nitrite in the cytosol and then nitrite is reduced further in the chloroplasts to NH4+, from which amino acids are formed. H+ transport out of the cells of the root and the mesophyll proceeds via a H+-P- ATPase.
- 9. Nitrate reduction uses mostly NADH as reductant, although some plants contain a nitrate reductase reacting with NADPH as well as with NADH. The nitrate reductase of higher plants consists of two identical subunits. The molecular mass of each subunit varies from 99 to 104 kDa, depending on the species. Each subunit contains an electron transport chain consisting of 1. one flavin adenine dinucleotide molecule (FAD), 2. one heme of the 3. cytochrome-b type (cyt-b557), and 4. one cofactor containing molybdenum (Fig.) 5. This cofactor is attached to molybdenum by two sulfur bonds and is called the molybdenum cofactor, abbreviated MoCo. Nitrate is reduced to nitrite in the cytosol
- 10. Nitrate is reduced to nitrite in the cytosol Nitrate reductase transfers electrons from NADH to nitrate and convert it into nitrite molybdenum cofactor, abbreviated MoCo
- 11. The reduction of nitrite to ammonia proceeds in the plastids
- 12. The reduction of nitrite to ammonia proceeds in the plastids The reduction of nitrite to ammonia requires the uptake of six electrons. This reaction is catalyzed by only one enzyme, the nitrite reductase, which is located exclusively in plastids. This enzyme utilizes reduced ferredoxin as electron donor, which is supplied by photosystem I as a product of photosynthetic electron transport. To a much lesser extent, the ferredoxin required for nitrite reduction in a leaf can also be provided during darkness via reduction by NADPH, which is generated by the oxidative pentose phosphate pathway present in chloroplasts and leucoplasts
- 13. Nitrate reductase P. denitrificans, P. aeruginosa P. stutzeri Neisseria spp. including N. gonorrhoeae and N. meningitidis, Rhodobacter sphaeroides and Moraxella catarrhalis.
- 14. Nitrite reductase Nitrite reductase contains a covalently bound 4Fe-4S cluster, one molecule of FAD, and one siroheme.
- 15. Siroheme is a cyclic tetrapyrrole with one Fe-atom in the center. Its structure is different from that of heme as it contains additional acetyl and propionyl residues deriving from pyrrole synthesis
- 16. The 4Fe-4S cluster, FAD, and siroheme form an electron transport chain by which electrons are transferred from ferredoxin to nitrite. Nitrite reductase has a very high affinity for nitrite. The capacity for nitrite reduction in the chloroplasts is much greater than that for nitrate reduction in the cytosol. Therefore all nitrite formed by nitrate reductase can be completely converted to ammonia. This is important since nitrite is toxic to the cell.
- 17. It forms diazo compounds with amino groups of nucleobases (R–NH2), which are converted into alcohols with the release of nitrogen. Thus, for instance, cytosine can be converted to uracil. This reaction can lead to mutations in nucleic acids. The very efficient reduction of nitrite by chloroplast nitrite reductase prevents nitrite from accumulating in the cell.
- 18. The fixation of NH4+ proceeds in the same way as in photorespiration Glutamine synthetase in the chloroplasts transfers the newly formed NH4+ at the expense of ATP to glutamate, forming glutamine. The activity of glutamine synthetase and its affinity for NH4+ are so high that the NH4+ produced by nitrite reductase is taken up completely. The same reaction also fixes the NH4+ released during photorespiration. Because of the high rate of photorespiration, the amount of NH4+ produced by the oxidation of glycine is about 5 to 10 times higher than the amount of NH4+ generated by nitrate assimilation. Thus only a minor proportion of glutamine synthesis in the leaves is actually involved in nitrate assimilation. Leaves also contain an isoenzyme of glutamine synthetase in their cytosol
- 20. Nitrate assimilation also takes place in the roots The reduction of nitrate and nitrite as well as the fixation of NH4+ proceeds in the root cells in an analogous way to the mesophyll cells. However, in the root cells the necessary reducing equivalents are supplied exclusively by oxidation of carbohydrates. The reduction of nitrite and the subsequent fixation of NH4+ occur in the leucoplasts, a differentiated form of plastids
- 21. 5
- 22. Glutamate thus formed is the precursor for other amino acids