Nitrogen metabolism.pdf

Nitrogen Metabolism Role of nitrogen in plants »Major substance in plants next to water »Building blocks »Constituent element of » Chlorophyll » Cytochromes » Alkaloids » Many vitamins »Plays important role in metabolism, growth, reproduction and heredity ...

Nitrogen Metabolism Role of nitrogen in plants »Major substance in plants next to water »Building blocks »Constituent element of » Chlorophyll » Cytochromes » Alkaloids » Many vitamins »Plays important role in metabolism, growth, reproduction and heredity  Sources of nitrogen * Atmospheric Nitrogen * 78% of atmosphere * Plants cannot utilize this form * Some Bacteria, Blue Green Algae, leguminous plants * Nitrates, Nitrites and Ammonia * Nitrate is chief form * Amino acids in the soil * Many soil organisms use this form * Higher plants can also taken by higher plants * Organic Nitrogenous compounds in insects * Insectivorous plants  Nitrogen fixation *The conversion of free nitrogen into nitrogenous salts to make it available for absorption of plants Types of nitrogen fixation * Nitrogen fixation * Non * Biological biological * Non - symbiotic * Symbiotic  Non biological fixation * The micro-organisms do not take place * Found in rainy season during lightning 1. N2 + O2 lightning 2 NO (Nitric oxide) 2. 2NO + O2 oxidation 2NO2 ( Nitrogen per oxide) 3. 2NO2 + H2O HNO2 + HNO3 4. 4NO2 + 2H2O + O2 4HNO3 (Nitric acid) 5. CaO + 2HNO3 Ca (NO3)2 + H2O (Calcium nitrate) 6. HNO3 + NH3 NH4NO3 (Ammonium nitrate) 7. HNO2 + NH3 NH4NO2 (Ammonium nitrite)  Biological fixation *Fixation of atmospheric Nitrogen into nitrogenous salts with the help of micro-organisms *Two types *Symbiotic *Non-symbiotic  Non-symbiotic oFixation carried out by free living micro- organisms oAerobic, anaerobic and blue green algae oBacteria: special type (nitrogen fixing bacteria) types - oFree living aerobic : Azotobacter, Beijerenckia oFree living anaerobic : Clostridium oFree living photosynthetic : Chlorobium, Rhodopseudomonas oFree living chemosynthetic :Desulfovibro,Thiobacillus  * Contd.. oFree living fungi: yeasts and Pillularia oBlue green algae: ounicellular – Gloeothece, Synechococcus oFilamentous (non heterocystous) -Oscillatoria oFilamentous (heterocystous) – Tolypothrix, Nostoc, Anabaena * Symbiotic *Fixation of free nitrogen by micro- organisms in soil living symbiotically inside the plants *‘Symbiosis’ – coined by DeBary *Three categories *Nodule formation in leguminous plants *Nodule formation in non-leguminous plants *Non nodulation Nodule formation in leguminous plants *2500 spp. Of family leguminosae ( Cicer arientium, Pisum, Cajanus, Arachis) produce root nodules with Rhizobium spp. *They fix Nitrogen only inside the root nodules *Association provides-food and shelter to bacteria -bacteria supply fixed nitrogen to plant *Nodules may buried in soil even after harvesting – continue nitrogen fixation  Nodule formation in non-leguminous plants *Some other plants also produces root nodules * Causuarina equisetifolia – Frankia * Alnus – Frankia * Myrica gale – Frankia * Parasponia – Rhizobium *Leaf nodules are also noted * Dioscorea, Psychotria *Gymnosperms – root – Podocarpus, - leaves – Pavetta zinumermanniana, Chomelia  Non-nodulation * Lichens - cyanobacteria * Anthoceros - Nostoc * Azolla – Anabaena azollae * Cycas – Nostoc and anabaene * Gunnera macrophylla - Nostoc * Digitaria, Maize and Sorghum – Spirillum notatum * Paspalum notatum – Azotobacter paspali  Symbiotic nitrogen fixation * Small, knob-like protuberances-root nodules * Size and shape varies * Spherical, flat, finger-like or elongated * From Pin head to one centimeter in size * Various spp. Of Rhizobium noted * Named after the host plant * Pea – Rhizobium leguminosarum * Beans – R. phaseoli * Soyabeans – R. japonicum * Lupins – R. lupini * Two types of Rhizobium- * Bradyrhizobium – slow growing spp. * Rhizobium - fast growing spp.  Rhizobium *Gram negative *Non spore forming *Micro-aerobic *Show a degree of specificity *The two partners (Bacteria and Host) recognized by chemical substance LECTINS - phytoagglutinins (carbohydrate containing plant protein) Formation of root nodules in legumes *Root nodules formed due to infection of Rhizobium *Free living bacteria growing near root of legumes unable to fix nitrogen in free condition *Roots of the legumes secrete some growth factors helps in fast multiplication of bacteria *(E.g.) Pisum sativum secretes homo serine also carbohydrate containing protein Lectins over their surface * Contd.. *This helps in recognition and attachment of rhizobial cells *Rhizobial cells have carbohydrate receptor on their surface *Lectins interact with the carbohydrate receptor of rhizobial cells *Occur between root hairs and young root hair *Bacteria enter the roots through soft infected root hairs *Tips are deformed and curved *Tubular infection thread is formed in the root hair cell and bacteria enters into it * Contd.. *After entry, new cell wall is formed *Tubular infection contains mucopolysaccharides where bacteria embedded and start multiplication *It grows much and reaches the inner layers of cortex and the bacteria is released *It induces the cortical cells to multiply which result in the formation of nodule on the surface *The bacterial cells multiplies and colonize in the multiplying host cells * Contd.. * After host cells are completely filled, bacterial cells becomes dormant-bacteroids * Float in leghaemoglobin – reddish pigment in cytoplasm of host cells - Efficient O2 scavenger - Maintains steady state of oxygen - Stimulates ATP production * Nitrogenous compounds synthesized is translocated through vascular tissues * Groups of rhizobia surrounded by double membrane originated from host cell wall * Bacteroids lack firm wall (osmotically liable)  Biochemistry of nitrogen fixation * Basic requirements for Nitrogen fixation *Nitrogenase enzyme *Protective mechanism against Oxygen *Ferrodoxin *Hydrogen releasing system or electron donor (Pyruvic acid or glucose/sucrose) *Constant supply of ATP *Coenzymes and cofactors TPP, CoA, inorganic phosphate and Mg+2 *Cobalt and Molybdenum *A carbon compound  Nitrogenase enzyme *Plays key role *Active in anaerobic condition *Made up of two protein subunits * Non heme iron protein ( Fe-protein or nitrogen reductase) – 60,000 daltons -2 identical subunits. Each subunit contains 4Fe and 4S atoms * Iron molybdenum protein (Mo Fe-protein or nitrogenase) – larger subunit – 2,00,000 daltons – 4 subunits – 1-2 Mo, 12-32 Fe, 24 S atoms in each subunit *Fe protein reacts with ATP and reduces second subunit which ultimately reduces N2 into ammonia N2 + 6H+ + 6e- 2NH3  Nitrogen Fixation Process N2 + 8H+ + 8e- + 16 ATP = 2NH3 + H2 + 16ADP + 16 Pi * Contd.. * The reduction of N2 into NH3 requires 6 protons and 6 electrons * 12 mols of ATP required * One pair of electron requires 4 ATP * The modified equation N2 + 8H+ + 8e- 2NH3 + H2 * Hydrogen produced is catalyzed into protons and electrons by hydrogenase hydrogenase H2 2H+ + 2e-  Pathway of nitrogen fixation in root nodules * Glucose-6-phosphate acts as a electron donor Sucrose Sucrose ( in Glucose and Glucose-6- (synthesized roots ) fructose phosphate in leaves) * Glucose-6-phosphate is converted to phosphogluconic acid Glucose-6-phosphate + NADP+ + H2O 6-phosphogluconic acid + NADPH + H+ * NADPH donates electrons to ferrodoxin. Protons released and ferrodoxin is reduced * Reduced ferrodoxin acts as electron carrier. Donate electron to Fe-protein to reduce it. Electrons released from ferrodoxin thus oxidized * Contd.. * Reduced Fe-protein combines with ATP in the presence of Mg +2 * Second sub unit is activated and reduced * It donates electrons to N2 to NH3 * Enzyme set free after complete reduction of N2 to NH3 Mo-NΞN Mo – N=NH NΞN Mo=N-NH2 Mo + NH3 MoΞN+NH3 Ammonification Ammonification  Nitrification Nitrosomonas Nitrobacter  Nitrogen Assimilation The nitrogen compounds nitrate and ammonium are the minerals that plants need in large quantities The nitrate assimilation pathway starts with nitrate uptake followed by nitrate reduction resulting in ammonium which is fixed into the amino acids glutamine and glutamate in most plants.  Nitrogen Assimilation Nitrate is taken up by the roots and either reduced, stored in the vacuoles or translocated to the shoot for reduction and vacuolar storage (also for osmoregulation). The first step of reduction, performed in the cytosol by nitrate reductase (NR) produces nitrite, which enters the plastid (chloroplast in the shoot) and is reduced to ammonium by nitrite reductase (NIR).  Nitrogen Assimilation Ammonium is fixed by the GS/GOGAT pathway into amino acids (glutamine/glutamate) which serve as substrates for transamination reactions to produce all of the other proteinous amino acids. Nitrogen Assimilation  Nitrogen Assimilation *Glutamate dehydrogenase * reductive amination of alpha-ketoglutarate to form glutamate *Glutamine synthetase * ATP-dependent amidation of gamma-carboxyl of glutamate to glutamine  Amino Acid Biosynthesis (Anabolism) Biosynthesis of Glutamate from α-ketoglutarate Reductive amination The production of glutamine from glutamate Amidation  Amino Acid Biosynthesis *Plants and microorganisms can make all 20 amino acids and all other needed N metabolites *In these organisms, glutamate is the source of N, via transamination (aminotransferase) reactions *Transamination- the transfer of amino groups from one molecule to another; an important process in the anabolism and catabolism of amino acid.  Example of transamination reactions Transaminations involve moving an α-amino group from a donor α- amino acid to the keto carbon of an acceptor α-keto acid. Transamination involves the transfer of amino group from one amino acid to the ketogroup of keto acid. The enzyme responsible for transamination is transaminase. Denitrification