B.Sc. III Year Mineral Nutrition

Unit I (Botany) Study material B.Sc. III Year Mineral nutrition The process including the absorption and utilization of various mineral ions by plants for their growth and development is called mineral nutrition. 80 – 90 % of tissue is composed of water. The part of the tissue left behind is called the dry matter and typically it is about 10 -20 % of the original weight. The dry matter mainly consists of organic compounds. Its 80 % consists of plant cell walls, primarily cellulose and related carbohydrates. This can be eliminated in the form of gases on combustion at 6000 C. The residue now left is ash which vary in different plant tissues from about 1- 0.15 % of the dry weight. A careful analysis of the ash shows that it contains almost all of the chemical elements present in the soil surrounding plant All these elements are not essential for the plant . Only 16 elements have been so far considered to be essential for the growth. Criteria of essentiality of an element:- Arnon and Stout 1939 suggested certain criteria that an element must fulfill in order to be classified as essential. These criteria are. 1. An element is essential if in its absence, the plant can not complete its life cycle. 2. An element is essential if it forms a part of any molecule or a constituent of plant that in itself is essential for the plant e.g. Nitrogen in protein. Magnesium in chlorophyll. Iron in cytochrome. 3. The element must act directly inside the plant or not enhance or suppress the availability of some other element. From a practical point of view an element is considered essential if plants show deficiency symptoms when they are raised without that element in the medium, the symptom is recovered only by injecting the same deficient element. Sixteen elements have so far been found to fulfill the criteria of essentiality suggested by Arnon and Stout in 1939. Sachs and Knop have divided these sixteen elements into two categories based on the quantity in which they are required by the plant. 1. Macroelements or Major elements or Macronutrients:- These elements are required by the plants in their large quantities i.e. 1 – 10 mg./ gm of dry weight. The elements are. C, H, O, N, S, P, Mg, K, Ca. Total = 09 2. Micro elements or Micronutrients or Trace elements:- These elements are required by the plants in their lesser quantities i.e. 0.1 mg / gm of dry weight. These elements are Fe, Mn, Bo, Cu, Zn, Mo, Cl. Total = 07 Scientists have added a few more elements to the list of sixteen though they have found them essential for certain group of plants e.g. Vanadium(Va), Silicon(Si), and Iodine(I) are essential for certain algae. Almunium(Al) is essential for some ferns. Selenium(Si) is essential for weeds. Sources of essential elements:- Depending upon the source of an element for the plant the elements have been divided into. 1. Mineral elements:- These are the elements which the plants get from the soil e.g. S, P, Mg, K, Ca, Fe, Mn, Bo, Cu, Zn, Mo, Cl, N. 2. Non mineral elements:- These are the elements which plant gets from the water and air e.g. C, H, O. Carbon in the form of Co2 from air. Hydrogen in the form of water from the soil and Oxygen from the air. Nitrogen is included in both the categories mineral and non mineral element because its source is both atmosphere and soil. 2

Role and deficiency symptoms of Macro elements: 1.Carbon, Hydrogen, and Oxygen:- They are absorbed in the form of Co2 and Water. Co2 is mostly obtained from air while water is got from the soil. The three elements enter the composition of all types of organic compounds like carbohydrates, organic acids, fats, proteins, amino acids, enzymes, hormones, etc. In short they build up the protoplasm. Deficiency of either Co2 or water cause retardation of growth. Apart from being a structural element, hydrogen in he form of H+ ions is highly important. (i). the concentration of H+ ion determines the PH. PH value influences the majority of reactions going on in the cells. (ii). In respiration oxidation of organic compounds involves the transfer of hydrogen from them to certain acceptor substances (iii). There is an exchange of H+ ions with cations during salt absorption. - + 2. Nitrogen:- The available forms of the Nitrogen in the soil are No 3 and NH 4 ions .Although nitrate ion is preferred and is translocated to leaves as such. Where it is first converted into Ammonia and then to amino acids. Even plants can absorb Ammonium ions but are first + converted into amino acids in the roots and amino acids are translocated to leaves. NH 4 fertilizers when added to soil are converted to nitrates by the microbes then absorbed by the plants. Dry plant material contains 2 – 4 % of nitrogen . In green plants protein Nitrogen is by far the largest Nitrogen fraction and accounts for 80 – 85 % of the total Nitrogen. Role:- Nitrogen is the mineral element that plants require in greatest amount. It is a constituent of important organic compounds like. Amino acids, Proteins, Nucleic acids, Chlorophyll, Hormones, Vitamins, ATP, NADP and NADPH coenzymes Deficiency symptoms: 1. Poor growth of the plant. 2. The young leaves remain small and the older leaves fall off prematurely. 3. Chlorosis of leaves especially in the older leaves near the base of the plant. Young leaves may not show this symptom because nitrogen can be mobilized from older leaves. Under severe conditions they may turn yellow or Tan and fall off from the plant. 4. Under severe conditions necrosis of the leaves occurs. 5. In cereals there is poor tillering, reduction in number of ears and number of grains per ear. 6. Flower formation is either suppressed or a few flowers are formed fruits and seeds formed are small and less viable. Potato produces fewer tubers. 7. When nitrogen deficiency develops slowly plants may be markedly slender and often woody stems. This woodiness may be due to build up of excess carbohydrates that can not be used in the synthesis of amino acids and other nitrogen compounds and can be used in the synthesis of anthocyanins which lead to purple colouration of leaves petioles and stems e.g. in tomato and certain varieties of corn. 8. Branching restricted. - 3. Phosphorus:- The major phosphorus containing ions in soil solution are monovalent (H2PO4 ), 2- 3- bivalent (HPo4 ), Trivalent(Po4 ), Monovalent is present in acidic soils. Bivalent is present in neutral soils. Trivalent phosphate is present in alkaline soils. Monovalent is easily available to roots while bivalent and trivalent are present in bound form. Roots are capable of absorbing phosphate from solution low in phosphate content. The phosphate content of roots and xylem sap is about 100 – 1000 fold higher than that of the soil solution. Role:- Phosphorus is the constituent of nucleoprotein, ATP, NADP, phospholipids .Another important phosphorus containing compound is phytin found in seeds. It is regarded as 3 the phosphate reserve. During seed germination it is mobilized and converted into other phosphate forms that are needed in the metabolism of young plants. Deficiency symptoms: 1. Stunted growth. 2. In cereals there is poor tillering, reduction in number of ears and number of grains per ear. 3. Older leaves become darkish green in colour, contain small necrotic spots (dead tissue) 4. Stems of annual plants become reddish in colour due to formation of anthocyanin pigments. 5. Vascular tissues are poorly developed. 6. Death of older leaves. 7. Delay in maturation of plant 8. Production of slender (bur not woody) stems. 9. Some species produce excess anthocyanins giving the leaves a slight purple colouration but not associated with chlorosis. 10. Lateral buds show prolonged dormancy but active buds are not affected because they can function as sinks at the expense of the remaining plant 11. Plants are unable to absorb and accumulate salts. 4. Potassium:- It is absorbed as k+ ions at high rates by the plants. Role:- 1. It is necessary for meristematic growth. 2. It is involved in maintaining the water status of the plants by maintaining cell turgor. 3. It plays important role in opening and closing of stomata. 4. It is involved in the translocation of photosynthetates. 5. It is the activator of enzymes like,Diastase, Catalase, Invertase. Deficiency symptoms: 1. Reduced growth rate. 2. First chlorosis and then necrosis on the margins and tips of older leaves. 3. Decrease in turgor. 4. Leaf wilting and abscission. 5. Roots become susceptible to rotting fungi in corn and the plant easily bends to ground (Lodging). 6. Stem becomes slender and weak with abnormally short internodes. 7. Apical buds may die with the result loss of apical dominance is found. 8. In absence of K+ other ions present exert toxic effects. 5. Sulphur:- It is present in the soil in inorganic and organic form. The inorganic forms of 2- sulphur in soil consists mainly of So4 . The organic sulphur of the soil is made available to 2- plants by microbial activity and So4 is produced. Plants mainly absorb sulphur in the form of 2 So4 . Plants can utilize So2 also as a sulphur source. Role: 1. It is the constituent of most important compounds like amino acids (Cysteine and Methionine) Vitamins (Lipoic acid , Thiamin , Biotin , Coenzyme A) electron carrier Ferredoxin. 2. Sulphur forms disulphide bridges in proteins. 3. It forms an alkaloid sinigrin (Diallyl disulphide) which gives pungent odour to Onion and Garlic. 4

Deficiency Symptoms: 1. Rate of plant growth is reduced. Shoot growth is more effected than root growth 2. Chlorotic symptoms occur first in the younger then most recently formed leaves rather than in old leaves because sulphur is not easily mobilized to the younger leaves in most species.. 3. Premature leaf fall. 4. Reduction in nodule formation in legumes. 5. Anthocyanin accumulation. 6. Calcium:- It is absorbed from the soil in the form of Ca 2+ ions. Role: 1. It is required for cell division because it is used in mitotic spindle during cell division. 2. It is essential for the stabilization of newly synthesized membranes. 3. It is involved in the normal functioning of cell membranes. 4. It is used in the synthesis of new cell walls particularly the middle lamella that separates newly divided cells. 5. It is activator of enzymes like ATPase, Kinase, Phospholipase, α-amylase and succinate dehydrogenase. 6. It functions as secondary messenger for hormonal and environmental signals for this it combines with calmodulin protein present in cytosol and plays role in many metabolic processes. 7. Development of thick cuticle in potomogeton occurs when calcium is supplied in abundance. 8. It plays role in binding proteins and nucleic acids in chromosomes. 9. It controls metabolism of carbohydrates. 10. Calcium counteracts toxities of other elements eg. Oxalo-aceticacid is converted into calcium oxalate which is non toxic. Deficiency symptoms: 1. Reduced meristematic activity because of necrosis of young meristematic regions. 2. Young leaves become deformed (rolling and curling). 3. The surface of apples is pitted with small brown necrotic spots called bitter pit disease. In tomato this disease is called blossom end rot. 4. Premature drop of flower and fruit by breaking of their stalks 5. Chlorosis starts from margins to middle or in the leaf area between the veins. 7. Magnesium:- Magnesium is absorbed in the form of divalent Mg 2+. Role: 1. In plant cells magnesium ions have a specific role in the activation of enzymes involved in respiration, photosynthesis, (rubisco) and the synthesis of DNA and RNA. 2. It is the part of ring structure of chlorophyll molecule. 3. Association of two sub units of ribosomes occurs in presence of Mg. 4. It is essential for the formation of carotenoids. 5. It is involved in the synthesis of magnesium pectate of middle lamella. Deficiency symptoms: 1. Chlorois between the leaf veins occurring first in the older leaves because of mobility of this element. This pattern of chlorosis results because the chlorophyll in the vascular bundles remain unaffected for longer periods than the chlorophyll in the cells between the bundle does. If the deficiency is extensive the leaves may become yellow or white. 2. Premature leaf fall. 3. Chlorosis is followed by necrosis. Defoliation may also occur. 5

4. Phloem and pith becomes reduced or remain under developed. 5. There is reduced vegetative and reproductive growth. 6. In deficiency of Mg tomato fruits develop pale orange colour, reduced pulp and wooly flesh. Role and deficiency symptoms of Micronutrients. 1. Iron:- Plants obtain iron in the form of ferric ions Fe3+ Role: 1. Iron has an important role as a component of enzymes involved in the transfer of electrons (redox reactions) such as cytochrome. In this role it is reversibly oxidized from Fe 2+ to Fe3+ during electron transfer. 2. It is essential for the development of chloroplasts and maintenance of chlorophyll though no iron dependent enzyme is involved in chlorophyll synthesis. 3. It is essential for the formation of or activity of nucleic acids and synthesis of proteins. Deficiency symptoms: 1. Chlorosis between the leaf veins occurring first in the younger leaves because iron can not be readily mobilized from older leaves. 2. Under conditions of extreme deficiency the veins may also become chlorotic and the whole leaf may become white. 3. In the leaves of cereals the deficiency is shown by alternate yellow and green stripes along the length of leaf. 4. Reduced growth because iron deficiency disturbs various plant activities like photosynthesis. Respiration, utilization of nitrate and protein synthesis. 2. Manganese:- It is absorbed in the form of Mn2+ Role: 1. Manganese activates several enzymes in plants. In particular decarboxylases, dehydrogenases, involved in the tricarboxylic acid (Krebs) cycle are specially activated by manganese. 2. The best defined function of manganese is in the splitting of water to liberate oxygen during photosynthesis 3. It is concerned some what in the formation of chlorophyll and maintenance of lamellar structure of chloroplast. 4.It is essential for nitrogen metabolism by being required for nitrite and hydroxylamine reductase. 5. It affects absorption of Calcium and Potassium 6. It is involved in Auxin synthesis. Deficiency symptoms: 1.The major symptom of manganese deficiency is interveinal chlorosis associated with the development of small necrotic spots. This chlorosis may occur first in the younger or older leaves depending upon the species and growth rate. 2. In severe deficiency the leaves show premature fall or do not develop at all. 3. Both roots and stem apices may die back and will show stunted growth. 4. Flowers are often sterile. 5. Its deficiency causes disorganization of lamellar system of chloroplasts. 3- 3. Boron:- It is absorbed in the form of borate (Bo3 ). Role: 1. It plays role in cell elongation, nucleic acid synthesis, hormone responses and membrane function. 6

2. It plays role in carbohydrate and auxin translocation 3. Pollen germination. 4. It is required for uptake and mobilization of calcium. 5. Maintenance of sugar and starch balance. 6. Pectin formation. 7. Production of root nodules in legumes. Deficiency symptoms: 1. Death of root and shoot tip, with the result apical dominance is lost causing the plant to become highly branched. 2. Black necrosis of the young leaves and terminal buds. 3. Fruits, fleshy roots and tubers may exhibit necrosis or abnormalities related to the breakdown of internal tissues causing diseases like, Heart root in beats (disintegration of internal tissues), Brown rot or water core of turnip, Internal cork of apple etc. 4. Absence of root nodules in leguminous plants. 5. Disturbance in pollen germination. 6. Stunted growth. 7. Reduced level of pectin. 8. Accumulation of fat. 9. Sugar accumulate, starch is also formed because of their non utilization in the formation of amino acids. 10. There is a shift from common path way of respiration to pentose phosphate path way. 4. Copper:- It is absorbed as Cupric ions(Cu2+). Role: 1. It is the constituent of enzymes involved in redox reactions being reversibly oxidized from Cu+ to Cu2+ like Plastocyanin which is involved in electron transfer during the light reaction of photosynthesis.Ascorbicacid oxidase and cytochrome oxidase helping in transferring electrons to oxygen. 2. It enters in composition of enzyme rubisco which takes part in carbon dioxide assimilation. Tyrosinase which is required in the formation of chlorophyll. Superoxide - dimutase takes part in detoxification of superoxide (O2 ) radicals. 3. It is connected with maintenance of carbohydrate/nitrogen balance of plant. Deficiency symptoms: 1. Production of dark green leaves which may contain necrotic spots. The necrotic spots appear first at the tips of young leaves and then extend towards the leaf base along the margins. 2. The leaves may also be twisted or malformed 3. Under extreme copper deficiency leaves may abscise prematurely. 4. Its deficiency causes two diseases (a).Exanthema:- In this disease tree bark shows deep slits from which gum exudes also called die back disease. (b). Reclamation disease:- Tips of leaves undergo chlorosis, hence it is also called leaf tip disease. 5. Stunted growth. 6. Fruits formed are fewer. They may show necrosis and skin splitting 7. Blackening of potato is also caused by shortage of copper. 5. Zinc:- Zinc is absorbed as Zinc ions (Zn2+). Role: 1. It is a component of many enzymes like Carbonic anhydrase, alcohol dehydrogenase, lactic dehydrogenase, glutamic dehydrogenase, carboxypeptidase and alkaline phosphatase. 7

2. It is required for some processes in carbohydrate metabolism, RNA synthesis, Protein synthesis, and synthesis of auxin or its precursor. 3. It is required for activity of copper containing superoxide dimutase. 4. It is involved in chlorophyll synthesis in some plants. Deficiency symptoms: 1. Reduction in internodal growth as a result plants display a rosette habit of growth in which the leaves form a circular cluster radiating at or close to ground (called rosette disease).The leaves may also be small distorted with leaf margins having a puckered appearance (little leaf disease). This symptom may result from loss of the capacity to produce sufficient amount of auxin. 2. In some species older leaves may become intervenously chlorotic and then develop white necrotic spots called white bud. This may be an expression of zinc requirement for chlorophyll biosynthesis. 3. Protein synthesis and protein levels are markedly lowered and amino acids accumulation occurs because of reduction in RNA synthesis. 4. Seed and fruit formation is reduced 5. Accumulation of fatty material but reduction in carbohydrate content. 6. Molybdenum:- It is available in the soil solution as monovalent and divalent molybedates - 2- 4+ 6+ (HMoO4, MoO4 ) exists as Mo to Mo Role: 1. It is a component of several enzymes including nitrogenase and nitrate reductase. Nitrogenase converts nitrogen gas to ammonia in nitrogen fixing organisms. Nitrate reductase catalyses the reaction of nitrate to nitrite. 2. It plays role in the synthesis of ascorbic acid. 3. It plays role in the hydrolysis of organic phosphates. Deficiency symptoms: 1. Nitrogen deficiency in plants. 2. General chlorosis between veins. 3. Necrosis of older leaves. 4. Whip tail disease in crucifers, in which leaves twist and subsequently die. Leaf now consists of petiole midrib and some laminar tissues. 5. Flower formation is prevented or fall prematurely. 7. Chlorine:- It is absorbed as Chloride anion (Cl –) Role: 1. It is required for the water splitting reaction of photosynthesis through which oxygen is produced. 2. It is required for cell division both in leaves and roots. 3. With Na + and K+ it helps in determining solute concentration and anion cation balance in cells. 4. It is required for normal production of fruits. Deficiency symptoms: 1.Reduced growth and wilting of leaf tips 2.Chlorosis and necrosis of the leaves, turn bronze coloured. 3.Stunted roots thickened at the tips 4. Reduced fruiting 8

Absorption and uptake of minerals:- Mineral nutrients are found in soil in usually three forms. (i). Dissolved:- These mineral ions are dissolved in water in the soil, constituting soil solution. The soil solution serves as the medium for ion movement to the root surface. (ii). Adsorbed:- The adsorbed mineral ions are held by soil colloids both inorganic and inorganic . The soil colloids are mostly negatively charged and hold cations over their surface. These are not easily lost when the soil is leached by water and they provide a nutrient reserve available to plant roots. - - Mineral anions, nitrate (No 3) and Cl remain in dissolved form in the soil solution . 2- Phosphate is bound to soil particles as it replaces their hydroxyl groups. Sulphate (So4 ) in 2+ presence of calcium (Ca forms gypsum (CaSo4). It is slightly soluble but it releases sufficient sulphate to support plant growth. (iii). Combined form:-These minerals have complex combinations with inorganic or organic substances. . Minerals are released when complexes break down due to various forces (physical, Chemical or organic. They then become part of the soil solution or yet adsorbed over colloidal particles. The mineral substances available to plants from the soil occur in the form of either cations or anions. K, Mg, Ca, Fe, Mn, Cu, Mo, and Cl form anions. Nitrogen occur both as + - - cations(NH4 ) and anions (No3 or No2 ). The most common form is anion nitrate. Some of the adsorbed ions can easily be displaced from the surface of the colloid when the concentration of the same decreases in the soil solution. This maintains ionic equilibrium . Others are released by ionic exchange (cation and anion exchange). Areas of root, absorbing mineral nutrients:- Plants absorb their mineral salts from the soil through the roots. The most active areas of the root for mineral absorption are the zone of elongation and root hair. Mechanism of mineral absorption:- Absorption of minerals occurs by two mechanisms. Passive and Active. 1. Passive absorption:-The absorption of minerals by the cell along their concentration or chemical potential gradient i.e. from higher concentration / chemical potential to low concentration / chemical potential is called passive absorption. It does not involve the expenditure of energy. 2. Active absorption:- The absorption of minerals by the cell against concentration or chemical potential gradient i.e. from low concentration or low chemical potential to high concentration or high chemical potential is termed active absorption. It involves the expenditure of energy. Outer and apparent free space theory:- It is found frequently that when a plant cell or tissue is transferred from a medium of low salt concentration to the medium of relatively higher concentration, there is an initial rapid uptake of ions in the outer or free space which is followed by a slow and steady uptake in the inner space. During rapid initial uptake of ions the metabolic energy is not involved. If the above plant tissue is returned to the lower salt solution or to the pure water, some of the ions taken up will diffuse out in the external medium. In other wards a part of the cell or tissue is open to free diffusion of ions. Since free diffusion implies that ions can move freely in or out of the tissue, the part of the tissue opened to free diffusion will reach equilibrium with the external medium and the ion concentration of this part will be same as found in external medium. The part of the plant cell or tissue which allows diffusion is called an outer space. 9

This outer or free space is the primary cell wall of the cell consisting of mainly cellulose microfibrills embedded in an amorphous matrix of two polysaccharides hemicelluloses and pectin substances. Which are partly made from Polygalacturonic acids having weak carboxylic acid groups and give negative charge on which hydrogen ions are loosely held when positively charged ions such as K, Mg, and Ca pass through the cell wall, they displace hydrogen ions. Cellulose microfibrills which are loosely arranged have pores. The intercellular spaces, the negatively charged region (Donnan free space) in the amorphous matrix and the pores in the cellulose microfibrills is the free space or outer space. Entry of ions into outer space seems to be passive where as entry into and exit from the inner space is active process. Movement of ions is called flux, inflow is called influx outward is called efflux. Passive absorption of Minerals:- The various methods of passive absorption can be as follows. 1. Diffusion:- It is the movement of substances from their high concentration to their lower concentration. Diffusion of solutes to the cell interior can normally take place provided their concentration is higher in the external medium. Nutrient uptake by the roots lowers the concentration of nutrients at the root surface generating concentration gradient in the soil solution surrounding root. Diffusion is of two types. Simple diffusion and facilitated diffusion 1. Simple diffusion:- In this process of diffusion membrane do not provide any special pathway . It is believed to occur through aquaporins through which water soluble gas O2 and Co2 diffuse and lipid matrix through which lipid soluble O2, Co2 and ammonia can pass. 2. Facilitated diffusion:- It occurs through channels and carriers:- (i).Channels:- These are transmembrane proteins that function as selective pores through which molecules and ions can diffuse across the membranes. As long as channel pore is open solutes that can penetrate the pore diffuse through it extremely rapidly about 108 ions per second. These channels are gated that open and close the pore in response to external signals including voltage changes, hormone binding etc. (ii).Carriers:- These do not have pores. The substance to be transported initially bounds to a specific site on the carrier protein. This requirement for binding allows carriers to be highly selective for a particular substrate to be transported Binding causes a conformational change in the protein which exposes the substance to the solution on the other side of the membrane. The rate of transport by carriers is slower than the channel proteins because transport of individual molecules or ions occurs. Rate of transport is 102 to 104 molecules per second. Carrier mediated passive transport is some times called facilitated diffusion because the transport of the ion occur down its concentration gradient or electrochemical gradient

Channel Protein 10

2. Mass flow:- A number of workers have observed increased salt uptake in rapidly transpiring plants . Transpiration causes a mass flow of water which can also drive the salt along with it. 3. Ion exchange:- The anions and cations of the plant cells are exchanged for the anions and cations of the equivalent charge from the external medium in which tissue is immersed. It has been experimentally confirmed in excised barley roots that when the roots were placed in the solution of Potassium bromide having radioactive K+. It was then excised and placed in distilled water. Radio active K+ did not leak out; however, when the excised root was kept in solution of KBr with non radioactive K+, there was an exchange of K+ ions. An appreciable number of radioactive K+ ions come out of the root. Even then the concentration of total K+ remained constant in the external solution. The ion exchange mechanism has been explained by the following two theories. (a). Contact exchange theory:- It was proposed by Jenny and overstreet in 1939. According to this theory the ions are transferred from soil particles to the root without passing into free solution. It is well known fact that the ions are adsorbed electrostatically to the surface of the root cells or clay particles. Due to thermal agitation each of them oscillates within the small volume of the space. It is termed oscillation volume. When the oscillation volume of the two ions with same charge overlap, one ion is exchanged with the other. Suppose H+ is adsorbed on the root cell surface K+ is adsorbed on the clay micelle and both oscillate in such a way that oscillation volume of H+ overlap with that of the K+. It will result in the transfer of H+ to clay micelle and K+ to the root surface. (b). Carbonic acid exchange theory:-The theory explains that the Co2 released during respiration of the root cells combine with the water to form carbonic acid (H2Co3) in the soil + - + solution. Carbonic acid dissociates into H and HCo3 ions. A cation eg. K adsorbed on the clay micelle may be exchanged with H+ of the soil solution. The cation K+ may diffuse to the root surface in exchange for H+ ion. Bicarbonate ions may act simultaneously and release anions or may accompany released cation into root.

Mode of ion absorption through contact exchange Mode of absorption of ions through carbonic acid exchange 4. Donnan equilibrium:- According to this theory there are certain pre-existing fixed or non difusable ions inside the cells which can not diffuse out side through membrane. However the cell membrane is permeable to both anions and cations present in the external medium. If the cell is immersed in an external salt solution and on the inner side of the membrane there are fixed ions (-). The movement of equal number of anions and cations take place until product of anion and cation in the internal solution becomes equal to the product of anions and cations in the external solution. Suppose a cell has potassium salt of a large indifusable organic anion (A-). It is placed in a solution of KBr. K+ is present already in the cell, therefore the external K+ can not directly diffuse inwardly, however Br- will enter the cell due to its diffusion gradient. Since the internal (A-) does not diffuse out to balance the ionic equilibrium of the external solution the externally 11 situated K+ ions also passes inwardly to maintain electrical or Donnan equilibrium. The result will be increased K+ and decreased anion Br- concentration in the interior as compared to external solution

Passive absorption and accumulation of minerals due to Donnan equllibrium Active absorption of minerals:- Hober 1945 reported that fresh water alga Nitella accumulated K+ ions in the cell about 1,000 times more than the concentration of. K+ in the surrounding medium. The absorption of ions and their retention within the cells at higher concentration require an expenditure of energy by the plants. There is a direct relationship between metabolic energy and salt uptake. It has been demonstrated experimentally by the number of workers. 1. Steward (1932) and Hopkins91956) observed a close relationship between salt accumulation and respiration. 2. Robertson and Turner observed that respiratory inhibitors inhibit salt absorption. 3. Lundergardh observed that salt uptake or absorption increases with the increase in rate of respiration. Mechanism of active salt uptake can be explained by the carrier concept. Carrier concept:-. The substance to be transported initially bounds to a specific site on the carrier protein. This requirement for binding allows carriers to be highly selective for a particular substrate to be transported Binding causes a conformational change in the protein which exposes the substance to the solution on the other side of the membrane. This carrier mediated transport is active also involving the movement of molecules or ions against concentration gradient with the involvement of metabolic energy. Active Transport is of two types. Primary and secondary. 1. Primary active transport:- It is directly coupled to a source of energy such as ATP hydrolysis. The membrane proteins that carry out primary active transport are called pumps. In the plasma membrane of plants H+ is the principal ion that is electrogenically pumped across the membrane. The plasmamembrane H+ ATPase generates the gradient of electrochemical potential of H+ across the plasma membrane. In plant membranes the most prominent pump is H+ and Ca2+. Therefore another mechanism is needed to drive the active uptake of most mineral nutrients. The other important way that solutes can be actively transported across the membrane against their gradient of electrochemical potential is by coupling of the uphill transport of one solute to the down hill transport of another . This type of carrier mediated co transport is termed secondary active transport. 2. Secondary active transport:- Protons are extruded from the cytosol by electrogenic H+ ATPase operating in the plasma membrane. Consequently a membrane potential and a PH 12 gradient are created at the expense of ATP hydrolysis. This gradient of electrochemical potential for H+ or the proton motive force represents stored free energy in the form of H+ gradient. The proton motive force generated by electrogenic H+ transport to drive the transport of many other substances against their gradient of electrochemical potentials There are two types of secondary transport Symport (Protein = Symporter) and Antiport (Protein = Antiporter).. Symport:- In this transport two substances are moving in the same direction through the membrane. Antiport:- In this transport down hill movement of protons drives the active (up hill) transport of a solute in opposite direction.

Translocation of solutes: There are two paths through which water and dissolved ions might move into the xylem cells of the root. 1. Apoplast:- In the apoplast pathway mineral ions moves exclusively through the cell walls without crossing any membrane. The apoplast is the continuous system of the cell walls. 2. Symplast:- In the symplast pathway mineral ons move from one cell to next via the plasmodesmata. The symplast consists of the entire net work of the cell cytoplasm interconnected by the plasmodesmata. At the endodermis ion movement through the apoplast pathway is obstructed by the casparian strips. The casparian strip is a band of radial cell walls in the endodermis that is impregnated with the wax like hydrophobic substance suberin. Suberin acts as a barrier to water and solute movement. The casparian strips breaks the continuity of the apoplast pathway and forces water and solutes to cross the endodermis through symplast pathway. Most experts have assumed that both the symplastic and the apoplastic pathways contribute for the transport of most ions. Experiments with radioactive isotopes of Cl36 on the water plant vallisneria show that these ions take only symplastic pathway probably through plasmodesmata. Studies using radio active isotopes of Ca45 in Apoplast and symplast pathway of solute translocation Barley seedlings show that it is transported along apoplast pathway. Mg2+ is also transported through apoplast pathway. 13

The transport of the mineral elements from the root to the shoot is driven by the gradient of water potential developed due to transpiration. An increase in transpiration rate enhances both the uptake and the translocation of mineral elements in the xylem. The lateral transport of ions from stem xylem to leaves probably take place via xylem transfer cells whose walls facing xylem are corrugated for providing large surface area for absorption and the cells contain many mitochondria that are located close to the corrugated wall in order to supply ATP for the active transport that take place across these walls. Biological nitrogen fixation The process by which molecular nitrogen (N2) is reduced to Ammonium is called nitrogen fixation. It is carried out by prokaryotic organisms, so called biological nitrogen fixation. Principal nitrogen fixers include. 1. Free living soil Bacteria e.g. Azotobacter, Clostridium, Rhodospirillum, Beijerinkia, Chromatium. Methanococcus, Derxia, Klebsiella. 2. Free living Cyanobacteria e.g. Anabaena, Nostoc , Aulosira , Cylindrospermum Totypothrix , Stogonema Calothrix. 3. Symbiotic cyanobacteria e.g. Nostoc with Gunnera a herb, Anabaena with Azola a water fern, Acetobacter with Sugarcane. 4. Bacteria which live in symbiotic association with members of leguminous pants. E.g. Rhizobium, Azorhizobium, Bradyrhizobium, Photorhizobium, Sinirhizobium. 90 % ie. 20,000 of the members of family Leguminoceae have root nodules. And 15 % of these plants have been examined for nitrogen fixation. 5. An actinomycetes Frankia living in association with number of non leguminous trees and shrubs living in nitrogen deficient soils including members of genera Alnus, Myrica, Shepherdia , Coraria , Hipophae , Elaegnus , Causurina Datisca etc. Nitrogen fixation Occurs under anaerobic conditions: Oxygen irreversibly inactivates the nitrogenase enzyme involved in nitrogen fixation. Thus each nitrogen fixing organism either functions under natural anaerobic conditions or can create an internal anaerobic environment in the presence of oxygen. In Cyanobacetria anaerobic conditions are created in specialized cells called heterocysts. Heterocysts are thick walled cells that differentiate when filamentous Cyanobacteria +. are deprived off NH4 These cells lack oxygen producing system of chloroplasts which is photosystem II. Non heterocyst Cyanobacetria fix atmospheric nitrogen in the soil of flooded rice fields, and die when the fields dry, releasing nitrogen in the soil. Free living bacteria that are capable of fixing nitrogen are aerobic, facultative or anaerobic. 1. Aerobic:-These are thought to maintain reduced oxygen conditions through their high levels of respiration. E.g. Azolla. Other bacteria evolve oxygen photosynthetically during the day and fix nitrogen during night 2. Facultative generally fix nitrogen under anaerobic conditions 3. Anaerobic: For anaerobic nitrogen fixing bacteria oxygen does not pose any problem. These can be photosynthetic (Rhodospirillum) or non photosynthetic (Clostridium). Symbiotic nitrogen fixation in legumes:- It is completed in following steps. 1. Recognition :- The symbiosis between legume and Rhizobia is not obligatory. Legume seedling germinates without any association with Rhizobia and they may remain unassociated throughout their life cycle. Rhizobia also occur as free living organism in the soil as saprophyte. Under nitrogen limited condiotiond however the symbionts seek out one another 14 through an elaborate exchange of signals. This signaling the subsequent infection process and the development of nitrogen fixing nodules involve specific genes in both host and symbionts. The first stage in the formation of symbiotic relationship between the nitrogen fixing bacteria and their host is migration of the bacteria towards the roots of the host plant. This migration is chemo tactic response mediated through chemical attractants especially (iso) flavinoids and betaines secreted by the roots. These attractants activate the rhizobial nod D proteins which then induce transcription of other nod genes. The nod genes activated by nod D code for nodulation proteins, most of which are involved in the biosynthesis of nod factors. Nod factors are lipoprotein oligosaccharide signal molecules all of which have a chitin 1 4 linked N- acetyl glucose amine backbone varying in length from 3- 6 sugar units and fatty acid accyl. Host specific nod genes that vary among rhizobial species are involved in the modification of the fatty accyl chain or the addition of groups important in determining host specificity. A particular legume host responds to a specific nod factor. The legume receptors for nod factors appear to be special lectins (sugar binding proteins) produced in root hairs. Nod factors activate the lectins. This lectin facilitates attachment of rhizobium to the cell walls of root hair. 2. Infection : During the infection process rhizobia that are attached to the root hairs release nod factors that induce a pronounced curling of root hair cells. The curling of the thread facilitates infection by providing a close environment to the bacterium, where it can enter the root hair cell by causing its localized enzymatic dissolution. Following the entry of Bacterium the root hair wall degrades and plasma membrane invaginates and grows into a tube like structure called infection thread which goes deeper into the cortex near xylem. 3. Nodule formation:- Due to infection of the infection thread containing rhizobia the cortical cells start dividing forming a distinct area within cortex called as nodule primordia from which nodule develops. The nodule cells become tetraploid. This nodule develops vascular system which facilitates the exchange of fixed nitrogen and for nutrition contributed by plants. When the infection thread with rhizobia reaches to nodule its tip fuses wit the plasma membrane of host cell releasing bacterial cells that are packed in the membrane derived from host cell called peribacteroid membrane. Branching of the infection thread inside the nodule enables the bacteria to infect many cells. As first bacteria continue to divide soon it stops its division and enlarges to differentiate into nitrogen fixing endosymbiotic organelles called bacteroids. Nodules contain an oxygen binding heme protein called leghemoglobin. It is present in the cytoplasm of infected nodule cells at high concentrations and gives the nodule pink colour. The host plant produces the globin portion of the leghemoglobin in response to infection by the bacteria, The bacterial symbiont produces heme protein. Leghemoglobin has a high affinity for oxygen about ten times higher than the B chain of human hemoglobin. Leghemoglobin stores enough oxygen so keeps oxygen away from nitrogenase. Mechanism of nitrogen fixation:- ● The enzyme catalyzing the conversion of molecular N2 to Ammonia NH3 is called nitrogenase. It is produced by a group of bacterial genes called nif genes. ●The reduction of N2 to NH3requires electrons and protons. The source of protons are NADH, NADPH produced during oxidation of carbohydrates Tranlocated from leaves to host in 15 the form of sucrose. The electron donors to nitrogenase enzyme are reduced ferredoxin or reduced flavodoxin. The bacteria have provision for the same. ●Mo and Fe are integral constituents of nitrogenase and therefore they must be available to plant in order to ensure the formation of nitrogenase. ● Reduction of molecular nitrogen to ammonia is high energy requiring process consuming 16 -24 ATP molecules per nitrogen reduced ●Nitrogenase enzyme is made of two subunits. 1. One of these contains non heme iron protein called Fe- protein or component II or dinitrogenase reductase. 2. Other contains Fe and Mo and is therefore called Fe- Mo protein or component I or dinitrogenase. Both the components are required for conversion of N2 to NH3. In the overall nitrogen reduction reaction ferredoxin serves as an electron donor to the Fe protein which inturn hydrolyzes ATP and reduces the MoFe protein . The MoFe protein can reduce the N2 into NH3. The overall reaction for nitrogen fixation is. N2 + 8 electrons + 8H + 16 ATP 2NH3 + 16ADP + 16Pi + H2. It is estimated that two to three molecules of ATP are consumed for the transfer of one electron in the nitrogenase reaction. The symbiotic nitrogen fixing prokaryots release ammonia that to avoid toxity, must be rapidly converted into organic forms in the root before being transported to the shoot via xylem. It is exported in the form of amides eg.asparagine or glutamine by temperate region legumes eg. Pea (Pisum), Clover (Trifolium) Broad bean (Vicia) and Lentil (Lens). It is also exported in the form of Ureids (allantoin, allantoic acid, citrulline). by legumes of tropical origin eg. Soyabean (Glycin), Kidney bean (Phaseolus), Pea nut (Arachis).

C D Different stages of recognition, infection and development of nodule in biological nitrogen fixation.

Nodule structure The reaction catalyzed by nitrogenase