Role of nitrate- and sulphate-reduction in synthesis of effective substances
9.1 Nitrogen metabolism in plants
Natural circulation of nitrogen by living organisms
The most important elements of nitrogen circulation are summarized in Figure 9.1.
Pharmacognosy 1 Nitrogen fixation
Fixing of nitrogen is the following process: N2 → NH4+
, which occurs with the participation of nitrogen-fixing bacteria (Figure 9.1).
Important: If plants are able to fix nitrogen, there is no need for N-containing artificial fertilizers!
Bacteria capable of nitrogen-fixing Diazotroph bacteria
Non-symbiotic
CYANOBACTERIA (e.g. Nostoc, Anabaena, Oscillatoria)
PHOTOSYNTHETIC BACTERIA (e.g. Rhodospirillium rubrum)
CHEMOTROPHIC BACTERIA (e.g. Thiobacillus ferrooxidans)
EUBACTERIA (e.g. Azotobacter, Klebsiella pneumoniae, Azospirillium – the latter is in loose association with roots of grasses)
Symbiotic
CYANOBACTERIA (in some angiosperms, ferns, mosses)
ACTINOMYCETALES (ray fungi), e.g. Frankia – symbiosis with Alnus, Elaeagnus, Hippophae
EUBACTERIUM (Rhizobiaceae: Rhizobium, Bradyrhizobium, Azorhizobium – endosymbiosis with legumes)
Legumes – endosymbiosis
On the root of host plant: root nodules (tubers, nodules)
Bacteria can penetrate through the root-hairs: → infection thread will be formed
Bacteria penetrate through the cortical cells of the root into the internal part of the bark: → they induce mitosis
In the polyploid cells: bacteria proliferate intensively
The proliferated cells are demarcated by the membrane of the host cells (peribacteroid membrane)
This enveloped formation is: nodule (Figure 9.2-3) Root nodules
Formation of nodules – bacteria swell: bacteroid phase
In vitro: Rhizobium – they are not able to fix N2 themselves (without the legume partner), but the bacteroids isolated from root nodules are!
The genetic ability of N2-fixing is coded in Rhizobium. The host plant supplies bacteria with the suitable carbon skeleton.
Leghaemoglobin: located in the peribacteroid interstice, in plant cytosol (synthesis – hem: bacteroid; globin: host plant). It binds O2 very strongly → under micro-aerophyl conditions (low, but necessary concentration of O2).
The pink colour of a nodule shows that it contains leghaemoglobin, i.e. the nodule is viable.
Role of nitrate- and sulphate-reduction in synthesis of effective substances
Figure 9.2-3
Root nodules with nitrogen-fixing bacteria in longitudinal section Fixing of nitrogen with chemical equations
The energy of the diazo bond (:N ≡ N:): is very high.
Root nodule bacteria use about 20% of the ATP produced by the plant to break the diazo bonds.
Reduction of 1 mol N2 requires 16 mol ATP, 8 mol electron:
N2 + 10 H+ + 8 e- → 2 NH4+
+ H2
nitrogenase
Nitrogenases: proteins containing Fe and Mo (Figure 9.4)
They are formed by two subunits, which dissociate readily, but neither of them is active in itself.
Nitrogenase-reductase: smaller unit, Fe-protein complex
Nitrogenase: bigger unit, Mo-Fe-protein complex, activity: N2 → NH4+
Outside the peribacteroid membrane (in cytosol) the following processes take place:
NH4+ → glutaminic acid transaminase
Pharmacognosy 1
Figure 9.4
Structure of the nitrogenase subunit Equation of N2-fixing (reduction) (Figure 9.5):
N2 + 8 e- + 16 MgATP + 16 H2O → 2 NH4+ + H2 + 16 MgADP + 16 Pi + 6H+
Figure 9.5
Nitrogenase electrontransport Origin of ATP:
Photosynthetizing bacteria: → photosynthetic origin
Under aerob conditions (e.g. Rhizobium sp.): mitochondrial phosphorylation will take place.
Role of nitrate- and sulphate-reduction in synthesis of effective substances Nitrification
Nitrification is an important step in the nitrogen cycle in the soil (Figure 9.1). It is an aerobic process performed by autotrophic bacteria, which are quite sensitive (e.g. to acidic pH, presence of tannins).
Nitrifying bacteria:
Nitrosomonas sp.
Nitrobacter sp.
Nitrification is the biological oxidation of ammonia with oxygen, into nitrite, followed by the oxidation into nitrate:
NH4+→ NO2-→ NO3
-The function of nitrifying bacteria is hindered by this process:
NH3 → atmosphere → NH4+− absorbed in the soil Denitrification
Denitrification is a process of nitrate reduction performed by heterotrophic facultative anaerobic bacteria that will eventually produce molecular nitrogen (N2) through a series of intermediate nitrogen oxide products (Figure 9.1, 9.6).
Figure 9.6
The process of denitrification Anaerobic denitrifying bacteria:
Pseudomonas denitrificans
Micrococcus dentirificans
Thiobacillus denitrificans Nitrogen assimilation
Nitrogen assimilation is the formation of organic nitrogen compounds such as amino acids from inorganic nitrogen compounds present in the environment (Figure 9.1).
Plants that cannot fix nitrogen gas (N2) depend on the ability to assimilate nitrate or ammonia for their needs.
Pharmacognosy 1
(Figure 9.7). In some species NO3- can be reduced in the root cortex parenchyma cells;
while in the majority of plants most of the nitrate reduction is carried out in the shoots, and the roots typically reduce only a small fraction of the absorbed nitrate to ammonia.
Nitrate ions can be stored in the vacuoles until they are needed for the synthesis of amino acids (Figure 9.8). Ammonia is incorporated into amino acids via the glutamin synthetase-glutamate synthase pathway.
Figure 9.7
Transport ways of nitrate in plants Reduction of nitrate
Nitrate reduction is carried out in two steps.
1) Nitrate is first reduced to nitrite (NO2
-) in the cytosol by nitrate reductase using NADH or NADPH.
In the cytoplasm, during nitrate reduction, the nitrate-reductase enzyme is in close contact with the external membrane of the chloroplast, because in the next step further reduction will take place in the chloroplast by nitrite reductase. The presence of NO3-
increases the activity of nitrate reductase.
Role of nitrate- and sulphate-reduction in synthesis of effective substances The way of the electron:
NADH → NAD → cytochrome b557 → Mo-cofactor → NO3- → NO2-
2) Nitrite is then reduced to ammonia in the chloroplasts by a ferredoxin dependent nitrite reductase.
Nitrite-reductase:
NO2- + 8 H+ + 6 e- → NH4+ + 2 H2O
ferredoxine
The electron donor is the e- transport system in the chloroplast. Nitrate and light are the two most important factors in the regulation of nitrite reductase.
Transamination
Glutamin synthetase – glutamate synthase:
In the chloroplasts, glutamin synthetase incorporates ammonia as the amide group of glutamine, using glutamate as a substrate. Further transaminations are carried out to make other amino acids from glutamine.
The most important transport routes and pathways of nitrogen metabolism within the plant are summarized in Figure 9.8.
Pharmacognosy 1
Figure 9.8
Transport routes and pathways of nitrogen metabolism in plants