Plant-mineral nutrition: macro- and micro nutrients, uptake, functions, deficiency and toxicity symptoms

M.N.V.Prasad Department of Plant Sciences

School of Life Sciences University of Hyderabad

Hyderabad 500046 E-mail: mnvsl@uohyd.ernet.InTel 040-23011604; 23134509 Fax 040-23010120; 23010145

Mobile 9989144651

Contents: Major sedimentary nutrient cycles Phosphorus cycle Sulphur cycle Major gaseous cycles

Nitrogen Cycle Carbon Cycle Ammonification Nitrification

Nitrogen fixation Symbiotic nitrogen fixation Non-symbiotic nitrogen fixation Photosynthesis Light reaction Dark reaction Carbon dioxide cycle Micronutrients

Plant uptake of trace elements (essential and non-essential) Factors influencing micronutrient uptake by plants Model of nutrient cycling and nutrient budgets

Significant Keywords: Nutrient cycling , Biogeochemical cycling ,Bigbang theory, Macro-nutrients, Micro-nutrients, sedimentary nutrient cycles,Nitrogen fixation, Carbon dioxide cycle

Introduction Nutrient cycling or Biogeochemical cycling is one of the integral function of a sustainable ecosystem. (Figures 1 and 2) Nutrients that are required for normal metabolism of all living organism are evolved as envisaged by the bigbang theory (Figure 3 ). Macro- and micro nutrients are essential for life and are recycled in characteristic pathways from environment to organisms and back to the environment. These paths are known as biogeochemical cycles (Colinvaux 1973 ; Kormondy 1996 ; Mackenzie et al 1999; Odum 1971; Smith 1996).

There are two basic types of biogeochemical cycles: a) sedimentary nutrient cycles and b) gaseous nutrient cycles. For each nutrient cycle operate in ecosystem through two pools.

i. Reservoir pool - large source of the nutrient, non-biological component,

ii. Exchange or cycling pool - smaller - exchange between organisms and their immediate environment.

Figure 1: Generalized model of an ecosystem showing interaction between nutrient pool and biotic compartment

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Figure 2: Important components of a generalized biogeochemical cycle

Figure 3: Big bang theory and origin of elements of nutritional significance

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Major sedimentary nutrient cycles Phosphorus cycle

• Luxury consumption • An ability to use phosphate at low levels, and • Alkaline phosphatase production.

1. Luxury consumption consists in the uptake of more phosphate than is required for growth and its storage within the cell. This process is resorted to by numerous algae at times of optimal phosphorus supply.

The stored phosphorus is in the form of phosphate granules, or, for short periods, as RNA and ATP.

This internal storage then allows a certain population growth even when the phosphorus supplies in the medium are exhausted.

An obvious advantage of the cell-containing polyphosphate granules is that they do not exercise osmotic or toxic effects. In contrast, most nitrogenous products cannot be readily condensed to a suitable storage form and must be kept as protein or pigments.

2. In most lakes, phosphate growth constant is very low for phytoplankton. It is about 1 to 3 µg/l phosphate-P, which means that the enzyme system in algae is not saturated for much of the time under natural conditions. Phosphate levels are extremely low, especially in summer. On the contrary, lakes polluted with sewage or those with high natural phosphorus inputs usually have high phosphate levels, saturating the phosphorus uptake system of phytoplankton. Phosphate growth constant greatly varies between species, and as available phosphate decreases, this might play a role in species succession. Because phosphate is recycled rapidly, the rate of its uptake is also important. A higher uptake rate can thus compensate, to some extent, for lack of mechanism to remove phosphate at very low levels.

3. The enzyme alkaline phosphatase cleaves the bond between phosphate and the organic molecule to

which it is attached. The phosphate thus freed is available for plant growth. The enzyme production by phytoplankton is a remarkable adaptation to an environment low in phosphate, but relatively rich in larger organic or condensed inorganic polyphosphates. Algae deficient in phosphorus may contain up to 25 times more alkaline phosphatase than algae with surplus phosphorus. Even more remarkable is the release of phosphatases in free, dissolved form into the medium, a process which quickly hydrolyzes the otherwise unavailable total phosphorus. The releases are roughly proportional to algal biomass in the lake. It is of interest that insoluble inorganic phosphates such as FePO and AlPO can be solubilized by sediment bacteria by producing organic acids rather than phosphatases.

Sulphur cycle Sulphur is an integral components of some amino acids like cycteine and methionine which are essential for life. An important practical implication of sulphur cycle is the anaerobic corrosion of steel/iron structures in sulphate containing soils and sediments. This type of corrosion can severely corrode or destroy the iron pilings used in huge constructions. Thus, this could pose as a potential civil engineering problem since it was believed that corrosion would be minimal in anaerobic conditions, The process include spontaneous chemical and microbial mediated steps but the bacterial contribution is essential for driving the whole process (Figures 5 and 6)

a. Surface of metal reacts with water forming ferrous hydroxide and hydrogen

b. The process would tend to stop at this point but Desulphovibrio desulfuricans removes the protective hydrogen and forms hydrogen sulphide

c. The hydrogen sulphide attacks iron in a spontaneous chemical reaction forming ferrous sulphide and hydrogen sulphide

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Fe + 2H2O → Fe(OH)2 + H2

4H2 + CaSO4 → H2S + Ca(OH)2 +2H2O

2H2S + Fe → FeS + H2

──────────────────────────────────────────────── 4Fe+CaSO4 + 4H2O FeS+3Fe(OH)2 + Ca(OH)2────────────────────────────────────────────────

Figure 5: Schematic representation of sulphur cycle

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Figure 6: Inorganic and organic compartments of sulphur cycle involving chemolithrophic bateria

accomplishing oxidation and reduction reactions

Major gaseous cycles a) Nitrogen Cycle (Figire 7) b) Carbon Cycle Nitrogen cycle Nitrogen, an essential element of protoplasm, proteins and genetically important nucleic acids such as DNA, has a complicated cyclic pathway through the ecosystem. It is also a major constituent, (about 79%) of the atmosphere. Most green plants need nitrogen in the form of nitrate ions (NO3

- ) and ammonium ions (NH4- ).

Though present in abundance in the atmosphere nitrogen in its gaseous state is unavailable to most life. It must first be converted to some chemically usable form i.e., water soluble ionic compounds containing nitrate ions and ammonium ions, and getting into that form comprises a major part of the nitrogen cycle. The processes involved in the cycle are fixation, ammonification, nitrification and denitrification.

Blue-green algae are important group of non- symbiotic nitrogen fixers. Of the some 40 known species the most common are in the genera Nostoc and Calothrix found both in soil and aquatic habitats. Blue-green algae are well adopted to exist on barest requirements for living. They are often pioneers on bare mineral soil. Especially successful in water logged soil, they appear to be nitrogen fixers in rice fields, where studies indicate that they annually fix 30 to 50 kg N/ha. Blue-green algae are perhaps the only fixers of nitrogen over a wide range of temperatures in aquatic habitats from Arctic and Antarctic seas to freshwater ponds and hot springs (55oC). Like bacteria, blue-greens require molybdenum for nitrogen fixation. In humid tropical forests epiphytes growing on tree branches and bacteria and algae growing on leaves may fix appreciable amounts of nitrogen. Certain lichens (Collema tunaefirnae and Peltigera rufescens) have been implicated in nitrogen fixation. Lichens with nitrogen-fixing ability possess nitrogen-fixing blue-green species as their algal component. A more recently discovered system of symbiotic nitrogen fixation is that of grasses, wheat, corn and plants growing in association with the species of Azospirullum.

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Figure 7: Nitrogen cycle

Ammonification

Nitrogen is also made available through the breakdown of organic matter by ammonification, nitrification and denitrification, three other processes in the nitrogen cycle. In ammonification amino acids are broken down by decomposer organisms to produce ammonia, with a yield of energy. It is a one way reaction. Ammonium or ammonium ion, is absorbed directly by plant roots and directly incorporated into amino acids which are subsequently passed along through the food chain. Wastes and dead animal and plant tissues are broken down to amino acids by heterotrophic bacteria and fungi in soil and water. Amino acids are oxidized to carbon dioxide, water, ammonia, with a yield of energy:

Part of the ammfixed in both ac

Nitrification

Nitrification is Two groups of soil as their sole

CH2NH2COOH + 1 1/2O2 → 2CO2 + H2O + NH3 + 176 Kcal

onia is dissolved in water or taken up by plants; another part is trapped in the soil and id clay and certain base saturated minerals.

a biological process in which ammonia is oxidized to nitrite and nitrate yielding energy. microorganisms are involved in this process. Nitrosomonas utilizes the ammonia in the source of energy because they can promote its oxidation to nitrate ions and water:

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Nitrite ion can be oxidized furthnitrite ion is exploited by anothnitrate with a release of 18 Kcal

N

Thus, nitrification is a processNitrosomonas oxidizes 35 molesmoles. Nitrifying bacteria are oxidizers are Nitrosomonas, NiNitrospina etc. (Figures 8 and 9)

2NH3 + 3O2 → HNO2 + 2H2O

Nitrous acid

↓

H+ + N02-

Nitrite ion

er to nitrate ions in an energy releasing reaction. This energy left in the er group of bacteria, the Nitrobacter, which oxidizes the nitrite ion to of energy:

NO + 1/2 O2 → NO3

itrite ion Nitrate ion

in which the oxidation state (or valance) of nitrogen is increased. of nitrogen for each mole of CO2 assimilated; Nitrobacter oxidizes 100 common in soil, sewage and aquatic environments. Some ammonia trosococcus, Nitrosovibrio. Nitrite-oxidizing bacteria are Nitrobacter,

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Nitrogen in atmosphere ~78%

Figure 8: Nitrogen cycle involving nitrogen bacteria

Denitrification

Some bacteria are capable of reducing nitrates to gaseous nitrogen, a process called denitrification. Denitrification occurs only under anaerobic conditions, such as water logged soils. In denitrification, the reduction of nitrate to nitrogen gas occurs by a sequence of reactions in which various intermediate compounds are formed:

2NO3 → 2NO2 → 2NO → N2O → N2

Many bacteria such as Agrobacterium, Alcaligenes, Bacillus, Thiobacillus and Pseudomonas can carry out denitrification. The nitrification is useful for these bacteria for energy production. But from the stand point of agriculture denitrification in undesirable; it results in the loss of nitrogen from the soil and consequently a decline in nutrients for plant growth. Environmental conditions have a significant effect on the level of denitrification. For example, the process is enhanced in soil having an abundance of organic matter, elevated temperatures (25-60oC) and neutral or alkaline pH.

Ammonia

2NH3 + 3O2 → 2N02- + 2H+ +2H2O (Catalysed by Nitromonas, obligate aerobe)

Glutamine Carbamyl

Nitrogen metabolites in cell

Decay by microorganisms

Nitrifying organisms Haber Bosch process Denitrification

Nitrifying organisms

Oxidative deamination by decay organisms

Nitrification

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Figure 9: Role of nitrogen bacteria in sewage water treatment.

In the presence of oxygen and optimum temperature the following reactions would occur as shown below:

2NH3 + 3O2 → 2N02- +2H+ +2H2O (Catalysed by Nitromonas, obligate aerobe)

2NO2 + O2 → 2NO3- (Nitrobacter (obligate aerobe)

A well nourished adult excrete bout 8 gm combined nitrogen per day (6 g urea, 1 g uric acid and 1 g in the form of bacterial protein in faeces. Uric and bacterial proteins will be broken down to derivatives that may be assimilated by growing bacteria. Urea is quickly hydrolysed by the enzyme urease to carbon dioxide and ammonia. Ammonia is toxic to animals and interferes with brain functioning. Trout (fish) will die after 24h in water containing 2 ppm of ammonia.

Nitrogen fixation Nitrogen fixation is the conversion of atmospheric nitrogen to ammonia or nitrate. Ammonia is the product of biological fixation; nitrate is the product of high-energy fixation by lightning or occasionally cosmic radiation or meteorite burning. In high energy fixation, nitrogen and oxygen in the atmosphere combine into nitrates, which are carried to the earth as nitric acid dissolved in precipitation and as particles of solid nitrate compounds (salts). Estimates suggest that about 8.9 kg N/ha is brought to earth by high-energy fixation. Biological fixation, the more important method makes available 100 to 200 kg N/ha i.e., roughly 90% of the fixed nitrogen contributed to earth each year. In biological fixation molecular nitrogen, N2, is split into two atoms of free nitrogen: N2 → 2N

The above step requires an input of 160 Kcal of energy for each mole (28 g) of nitrogen. The free nitrogen atoms can combine with hydrogen to form ammonia, with the release of about 13 Kcal of energy resulting in a net energy input for nitrogen fixation of 147 Kcal/mole.

2N + 3H2 → 2 NH3

The importance of biological fixation is its low energy requirement regulated by two enzymes nitrogenase (an iron containing protein) and hydrogenase (a molybdenum containing protein). This is in contrast to the extremely high temperature (400oC) and pressures (200 atmospheres) required for

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industrial fixation of nitrogen through Haber and Bosch method. Nitrogen fixing bacteria can be classified as either symbiotic nitrogen fixers which fix nitrogen only when they live in association with plants or non-symbiotic nitrogen fixers which live freely and independently in soil.

Symbiotic nitrogen fixation

The biological fixation of nitrogen is accomplished by symbiotic bacteria living in association with leguminous and non-leguminous plants, by free living aerobic bacteria and by blue-green algae. In agricultural ecosystems about 200 species of nodulated legumes are the pre-eminent nitrogen fixers. In non-agricultural ecosystems some 12,000 species, from free-living bacteria and blue-green algae to nodule bearing plants, are responsible for nitrogen fixation. In many cases plants derive great benefit from the nitrogen-fixing bacteria that live in association with them as in Rhizobium - legume, Anabaena - Azolla, Nostoc - Cycas, Frankia - Alder tree, Frankia - Alnus tree and other systems. Legumes, the most conspicuous of the nitrogen - fixing plants, have a symbiotic relationship with members of the bacterial genus Rhizobium. Rhizobium are aerobic non-spore forming rod shaped bacteria. They exist in the immediate surroundings of the plant roots called rhizosphere. There they multiply due to stimulations caused by the enzymes secreted by the legumes in response to the exudates of the bacteria and loosen the fibrils of the root hair walls. Swarming rhizobia enter the root hair tops and penetrate inner cortical cells. There they multiply and increase in size, resulting in swollen infected cells, called root nodules, in which an oxygen carrying red pigment hemoprotein, termed as leghemoglobin, similar to hemoglobin of animal blood in many respects, develops. This facilitates transfer of O2 at a concentration appropriate for nitrogenase activity and adequate for bacteroid respiration. Those cells make up the central tissues of the nodules. Inside the nodules the bacteria change from rod-shaped to non-mobile forms that carry on nitrogen fixation. A large number of non-leguminous nodule bearing plants associated with actinomycetous fungus make significant contributions in fixing atmospheric nitrogen. Among such plants are Alnus, Ceanothus, Eleagnus, Casuarina etc.

Non-symbiotic nitrogen fixation

Also contributing to the fixation of nitrogen are free-living soil bacteria that can carry out non-symbiotic nitrogen fixation (Figures 10 and 11). The most prominent among them are the aerobic Azotobacter species and the anaerobic Clostridium species. Azotobacter prefers soils with a pH 6 to 7 that are rich in minerals and low in nitrogen. Less efficient in fixing nitrogen, Clostridium is ubiquitous, found in nearly all soils. It is more tolerant of acidic conditions than Azotobacter. Both genera produce ammonia as the first stable end products. The free-living non-symbiotic and symbiotic bacteria both require molybdenum as an activator and are inhibited by an accumulation of nitrates and ammonia in the soil. Some examples of nonsymbiotic nitrogen fixing bacteria are shown in Table 1.

Table 1: Some examples of Nonsymbiotic Nitrogen-fixing bacteria

Bacterial genera/species Physiological characteristics

Azatobacter chroococcum Heterotrophic

Cyanobacteria (blue-green algae) Photosynthetic

Clostridium spp. Heterotrophic

Rhodospirillum rubrum Photosynthetic

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H2 Light

Figure 10: Role of nitrogen bacteria in sewage water treatment.

Figure 11: Biological nitrogen fixation.

Biological nitrogen fixation can be represented by the following equation, in which two moles of ammonia are produced from one mole of nitrogen gas, at the expense of 16 moles of ATP and a supply of electrons and protons (hydrogen ions):

Absence of molecular nitrogen

Nitrogenase Hydrogenase [CH2O]

CO2 O2

Photosynthesis Vegetative cell Vegetative cell

[CH2O]

Heterocyst Vegetative cell

Oxidised ferredoxin reduced ferredoxin

reduced Fe protein oxidised Fe protein

4 ADP4 ATP

oxidised Mo protein reduced Mo protein 2 e-

2H+

2 e-HN=NH N2 H2N -NH2 HN=NH 2NH 3 H2N -NH2

N + 8H+ + 8e + 16 ATP = 2NH + H + 16ADP + 16 Pi2 -

3 2

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This reaction is performed exclusively by prokaryotes (the bacteria and related organisms), using an enzyme complex termed nitrogenase. This enzyme consists of two proteins - an iron protein and a molybdenum-iron protein, as shown below. The reactions occur while N2 is bound to the nitrogenase enzyme complex. The Fe protein is first reduced by electrons donated by ferredoxin. Then the reduced Fe protein binds ATP and reduces the molybdenum-iron protein, which donates electrons to N2, producing HN=NH. In two further cycles of this process (each requiring electrons donated by ferredoxin) HN=NH is reduced to H2N-NH2, and this in turn is reduced to 2NH3. Depending on the type of microorganism, the reduced ferredoxin which supplies electrons for this process is generated by photosynthesis, respiration or fermentation.There is a remarkable degree of functional conservation between the nitrogenase proteins of all nitrogen-fixing bacteria. The Fe protein and the Mo-Fe protein have been isolated from many of these bacteria, and nitrogen fixation can be shown to occur in cell-free systems in a laboratory when the Fe protein of one species is mixed with the Mo-Fe protein of another bacterium, even if the species are very distantly related.

Carbon dioxide cycle

Respiratory activity of the producer and cosumer community account for return of a considerable amount of biologically fixed carbon as CO2 to atmosphere. However, most substantial return is accomplished by the respiratory activity of decomposers in producing the waste materials and dead remains of other trophic levels (Figure 12).

Geologic component represents about 99 % of the total carbon which includes the deposition of the plant and animal material as peat, coal and oil, mollusc shells and protozoan cysts contribute to carbonate rocks. In addition, a number of aquatic plants growing in alkaline waters release calcium carbonate as a byproduct of photosynthesis. For e.g Elodea canadensis can deposit about 2 kg of calcium carbonate in 10 hrs of sunlight under natural conditions.

Carbon dioxide in atmosphere

The physical and chemical properties of CO2 make it easily accessible and available to aquatic and terrestrial ecosystems. Gaseous CO2 is colorless, odorless and slightly denser than air. It is about 0.03% by volume of air (i.e 300 ppm) and distributed more or less evenly by thermal air currents and pressure changes of air masses in the atmosphere. CO2 is released into atmosphere by the respiratory activity of living organic matter by microbes. Natural phenomena like bush and forest fires, and burning of vegetation along the path of lava outflows (due to volcanic activity) release large quantities of CO2 into atmosphere.

In the modern world, man's activities are adding enormous quantities of CO2 to the atmospheric pool, in the form of exhaust gases of aero-engines, rockets and missiles that are directly injected into upper atmosphere. Exhaust gases of automobiles, emissions from industries, and burning of fossil fuels (natural gas, petroleum products, coal, lignite and peat) add enormous quantities of CO2 to the atmosphere. A global annual estimate of 1970 reveals that about eight billion tons of CO2 was released into atmosphere by man's activities. A part of atmospheric CO2 dissolves in water at air-water interface and enters into oceans and inland water bodies.

Carbon dioxide in soil

Aerobic decomposition of litter and soil organic matter by microbial activity releases CO2 and it will be trapped partly in the air bound to the interstices of soil particles. Atmospheric CO2 goes into solution in rain drops of clouds and reaches the soil surface. Rain water, as it percolates through layers of soil receives CO2 from decomposing organic matter. By the time this soil water reaches the subterranean water streams, it is often supercharged with CO2. In the soil water, CO2 chemically transforms into carbonic acid (H2CO3

2-), bicarbonate (HCO3-), and carbonate (CO3

2-). These aspects will be discussed under the title CO2 in hydrosphere. Soil fauna and flora release CO2 in their respiratory activity.

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Carbon dioxide in hydrosphere

Despite the small proportion of CO2 among the gases of the air, it is relatively abundant in natural waters. A major reason for this abundance is its high coefficient of solubility. CO2 is more soluble than the most abundant atmospheric gases, nitrogen and oxygen. In natural dilute inland water bodies, CO2 in solution combines chemically with water molecules to form the weak carbonic acid.

CO2 +H2O → H2CO3

Carbonic acid dissociates into two ionic forms, bicarbonate and carbonate.

H2CO3 → HCO3- + H+

(bicarbonate)

HCO3- → CO3

2- + H+

(carbonate)

Hydrogen ion concentration, pH which is an index of alkalinity or acidity determines the relative concentration of free dissolved CO2 , unionized carbonic acid, bicarbonate and carbonate. The transformation of CO2 into any of the chemical forms cited above is ecologically important, as it facilitates absorption of vast quantities of carbon dioxide, Oceans are estimated to contain 130,000x10 tons of carbonates. Aquatic plant communities utilize bicarbonate as a source of carbon dioxide for their photosynthetic activity. The respiratory activity of all aquatic organisms releases CO2 into ambient environment. Theoretically when Hydrogen (H+) and Hydroxyl (OH-) ions in water are in equilibrium at the neutral pH7, bicarbonate predominates along with small amounts of unionized carbonic acid and traces of free soluble CO2. As the pH decreases with increasing acidity of water, carbonic acid (H2CO3) and free dissolved CO2 accumulate and bicarbonate values fall. As the pH rises beyond 8.3, with increasing alkalinity, carbonate values increase and bicarbonate decreases. These dynamic changes of HCO3

- and CO32- in relation to pH provides a regulatory and buffering mechanism controlling wide

fluctuations of pH which are detrimental to the normal metabolic activities of organisms. The living fauna and flora and their environment constitute the biosphere of earth.Aquatic plants utilize HCO3- for photosynthesis. Carbonic anhydrase of aquatic plants is involved on caryying HCO3 from water to palnt cell and split HCO3 into CO2 and H2O in the cell.

Photosynthesis consists of two reactions

a) Light reaction: In which light is absorbed by chlorophyll pigments and light energy is used to

synthesize APT and NADPH. This reaction occurs in the thylakoid membranes of the chloroplasts

andATP and NADPH2 are produced outside the thylakoid membranes i.e in the stroma

b) 2) Dark reaction: In this reaction CO2 is reduced to carbohydrate by using APT and NADPH

produced in the light reaction. Dark reaction is localizd in chloroplasts

Many photosynthetic organisms contain green chlorophyll pigments in the chloroplasts of their cells. Chlorophyll molecules are excited by light energy. Excited chlorophyll molecules remove electrons (e) from Hydrogen of water molecules.

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A chemicalend producwater moleino NADPH

The synthestroma of tenergy tranphotosynthreactions glyceraldeh

A simplisti

Light energy Chlorophyll → Excited chlorophyll

Excited chlorophyll H20 → 2H+ + 2e +1/2 O2

reaction involving light energy to split a molecule is known as photolysis. Oxygen is one of the ts of this reaction and it is released into environment. The electrons removed from Hydrogen of cules are held by nicotinamide adenine trinucleotide phosphate (NADP) and NADP ic converted

. The soluble ferredixin-NADP reductase sare involved in reducing NADP+ to NADPH

sis of ATP molecules in chlorophyll by light energy is termed "Photophosphorylation" (in the he chloroplast). ATP molecules are universally present in living organisms and function as sfer compounds in biochemical reactions. ATP and NADPH molecules are utilized in

esis to reduce CO2 For this synthesis, light energy is not required (dark reaction) and the take place in cytosol. In this process a stable initial compound of CO2 is yde-3-phosphate.

NADP+2-e + H+→ NADPH

2NADPH + ADP + Pi +2e → ATP + NADP

H ─ C = O

⏐

H ─ C ─ OH

⏐

H2C ─ O ─ P

c chemical reaction to indicate the production of oxygen in photosynthesis is:

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Light 6CO2 + 6H2O + 674 Kcal → → → C6H12O6 + 6O2

Chlorophyll Carbohydrate 1 mole

The gradual build up of oxygen biologically produced in water and its diffusion into atmosphere brought about tremendous changes in the geochemistry of earth. Precipitation of iron and formation of various minerals were important events.

Figure 12: Carbon dioxide cycle

All the biogeochemical cycles (sedimentary gaseous and) (=nutrient cycles) self regulatory feed back mechanisms resulting in a relatively homeostatic system. Addition and deletion can be readily equilibrated and compensated. To what degree the system will withstand or adapt to long term disturbances of the existing equilibrium is of course uncertain.(Figures 13 and 14) Here comes the reference of man and his role in biogeochemical cycles and release of anthropogenic pollutants (Figure 15)..

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Figure 13: Internal and external factors influencing the nutrient budgets in a generalized sedimentary

nutrient cycle

Figure 14: Nutrient removal and addition to the ecosystem by natural and articifial processes

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Atmosphere

Figure 15: Noosphere, phytosphere, atmosphere, hydrosphere and lithoshere cicrlulating nutrients

Micronutrients

Those of the elements such as As, Cd, Co, Cu, Cr, Hg, Mn, Ni, Pb, Se, Zn etc which constitute about 1% of the total elemental content of the soil are called trace elements. The term trace element includes metals, metalloids and radionuclides. 'As and Sn' are half-metals and 'Se' is a non-metal. Some elements can occur in different valence states and the toxicity varies in different states. One example is Cr(III) and the more toxic Cr(VI). Toxicity of essential or non-essential element depends on its concentration in the environment, speciation, pH, redox potential, and cation exchange capacity. A few metals are precious, e.g., gold and platinum. Cs, Hg and Ga are liquids at room temperature. Cadmium, Pb, and Hg have no metabolic significance and are important in material scence for e.g high energy and durable bateery production etc. Trace elements (micronutrients that have been studied most extensively in soils are those that are essential for the nutrition of higher plants i.e. B, Cu, Fe, Mn, Mo and Zn

1. An element could be considered essential if : its deficiency prevents the plant from completing its life cycle,

2. its deficiency is specific to the element and can be corrected or prevented only by supplying that element, and

3. the element has a nutritional role apart from correcting any unfavorable microbial or chemical condition of the soil

Table 2: Phytotoxicity effects of excess of essential trace elements (Prasad,and Strzałka 2002).

Trace element Effects Cr Decreases enzyme activity and plant growth; produces membrane

damage, chlorosis and root damage Cu Inhibits photosynthesis, plant growth and reproductive process;

decreases thylakoid surface area Ni Reduces seed germination, dry mass accumulation, protein

production, chlorophylls and enzymes Zn Reduces Ni toxicity and seed germination; increases plant growth and

ATP/chlorophyll ratio

Biosphere

Gaseous cycles

Detrtitus/sediments

Erosion

Lithosphere

Noosphere Man Plants Sink for pollutants

Interfacing Water cycle

Sedimentary cycles

Hydrosphere

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Table 3: Significance of mineral compounds for plant cells (Prasad and Strzałka 2002).

Nutrient/Mineral Significance Nitrate amino acids, proteins, nucleotides, chlorophyll, etc.

Potassium co-factor of many enzymes, necessary for regulatory processes (like guard cell movements) and for syntheses, for example protein biosynthesis

Calcium regulatory functions, has part in cell wall structure; stabilizes membranes, controls movements

Phosphate energetic bonds (ATP), component of nucleic acids, has part in phosphorylations, for example of sugars and proteins

Magnesium chlorophyll component, counter ion of ATP, important for protein biosynthesis

Sulphur amino acid and protein component, coenzyme A

Iron necessary for chlorophyll synthesis, component of cytochromes and ferredoxin

Chloride takes part in osmotic processes, cofactor of light reactios of photosynthesis

Copper Cco-factor of some enzymes Manganese like copper, component of protein biosynthesis

Zinc like copper (for example carboxypeptidase, DNA-dependent RNA polymerase)

Molybdenum controls nitrogen metabolism

Borate influences use of Ca2+ , translocation of sugars, pollen germination etc.

Plant uptake of trace elements (essential and non-essential)

Plants take up and accumulate mineral nutrients in their tissue from soil solution and also from air in some exceptional cases. Plants also release some nutrients back into the surrounding medium. The accumulation nutrients by the plant therefore, is referred to as net uptake and is based on both influx and efflux. Nutrients, thus taken up by plants are translocated to other plant parts (Figure 16 and 17).

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Plant root

Figure 16: Trace elements in soil solution + Ligand = related complex biogeochemical processes (rhizosphere biogeotechnology). Mucigel (grey) excreted by the plant root may facilitate or restrict the uptake of elements. Mycorrhiza and bacteria enhance or prevent uptake of elements.

The absorption area of the roots/leaves also affects uptake. The larger the absorption area the higher is the effective uptake (Prasad, 2004 ). Furthermore, increased biomass production increases the uptake of elements. However, the accumulation in the tissue will diminish due to dilution caused by biomass increase and that the uptake rate is lower than the rate of biomass production.

Uptake by roots

The movement of mineral elements to the root surface depends on i) Diffusion of elements along the concentration gradient formed due to uptake generating depletion of the element in the root vicinity. ii) Root interception, where soil volume is displaced by root volume due to root growth. iii) Mass flow, transport of bulk soil solution along the water potential gradient (driven by transpiration) (Marschner 1995, Adriano (2001).

Mycorrhizae are known to increase the uptake of phosphorus by plant roots as well as prevent heavy metal and nitrogen-fixing bacteria help the plant to use atmospheric nitrogen (Marschner, 1995) (Figure 16). Mineral nutrients are transported either apoplastically, i.e. in the cell wall system outside the plasma membrane, or symplastically, i.e. in the cytoplasm from cell to cell via plasmodesmata. Elements that penetrate into the cytoplast can also be shuttled into the vacuole via various mechanisms depending on element.

Organic matter

Organic acids

Transporter Iron plaque

Root exudate

Bacteria

Mycorrhizae Phytosiderophores

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Root exudatesd and rhizospheric processes

Inorganic ligands HCO3- H+ Enzymes

PhytosiderophoresOrganic acids

Alkalinization Acidification Chelation Reduction

Solubilization

Phytoavailability

Figure 17 : Plant-microbe interactions in acquisition of mineral nutrients

Citric acid is a naturally occurring chelating agent. It protects the micros from being tied up by the potting media, making the micros more available to the plant. The chelation process is activated throgh irrigation (Figure 18).

Figure 18: Citric acid, usually applied with irrigation for enhancing the bioavaioability of micromnutrients

Need for sophistricated analytical instrumentation for species-selective analysis of trace elements (micronutrients) in plants (Prasad 2004)

Interactions of plants with metals and metalloids have several economic and environmental implications and had gained global significance. The prominent areas being mineral exploration to solid waste management. Edible parts of the plants are the important (and sometimes major) sources of trace elements in human diet which makes legislation set limits regarding toxic metal concentrations e.g. beverages, herbal medicines and avariety of agricultural produce.

The use of specially engineered plants has been finding the growing interest in the remediation of polluted waters and soils (phytoremediation). For some elements, especially plants (e.g. algae, seaweeds, and phytoplankton) are an important link in food chains. In the environment, plants are sedentary and are exposed to the pollution risk in comparison with other living organisms since, they can not move around. Especially, plants in growing in urban areas and along busy motorways have suffered from the automotive pollution by alkyl lead

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compounds (antiknock additives to petrol) and by their substituents: organomanganese compounds, and platinum released from the catalysts. The mechanisms of metal uptake by roots, metal translocation from roots to shoots, and plant tolerance to toxic metals are dependent on the molecular forms (speciation) of metals which can be further modified by the organism studied.

Different types of metal-chelating compounds have been developed by living organisms to regulate the intracellular metal ion concentration. They include: amino acids, citric acid, malic acid, phytochelatins and metallothioneins. The synthesis of some of these compounds, e.g. phytochelatins is enzymatically mediated, i.e. requires a protein activated by the metal of interest. The identification of the complex of the metal with the enzyme and the characterization of the metal complexes with products of the enzyme-catalysed reaction is an emerging field of research in environmental speciation analysis.

In order to be of concern in terms of essentiality and/or toxicity, trace elements in the human diet must be bioavailable, i.e. readily absorbable by the gut and further utilisable in the body. Since the bioavailability depends critically on the actual species of an element present, it is becoming more and more evident that information on the concentration an element in a foodstuff tells us very little how well the element will be assimilated. Precise information regarding the identity, nature and concentration of individual metal compounds present in a sample is therefore required. This concerns, in particular, food and feed supplements aiming at supplying a given oligo element, e.g. selenium.

The success of using hyperaccumulating plants for phytoremediation of contaminated soils and waters requires a better understanding of the mechanisms of metal uptake, translocation and accumulation by plants. The prerequisite for the understanding of these mechanisms is the identification of molecules involved, notably bioligands and metal complexes synthesised by a plant to be used for metal transport and suitable for bioaccumulation.

Whereas the total element concentration status has been a long established parameter to assess the stress to plant, its nutritive value in terms of oligo element supply or its toxicity, species-related information has often been neglected despite its crucial value. The reason for this has been the lack of analytical techniques able to deliver the information of the concentration of several species in which each of trace elements is present in a sample. Other analytical techniques that advanced our knowledge in the area of mineral nutrition are listed below:

– Colorimetry – Fluorimetry – Flame photometry – Neutron activation analysis – Atomic absoption spectroscopy – Microwave excitation emission spectroscopy – Isolation and study of metalloenzymes – Micronutrients become a part of specialized compounds for e.g Metal-Biomolecule Complexes

such as Metalloenzymes – Examples of metalloenzymes: – Superoxide dismutase (Zn and Cu) – Nitrogenase (Mo and Fe) – Carboxypeptidase A (Zn) – Carbonic anhydrase (Zn) – Cytochrome oxidase (Fe and Cu) – Xanthine oxidase (Co and Fe) – Biological examples of metal-activated enzymes – Rubisco (Mg)

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– creatine kinase (Mg, Mn, Ca or Co) – glycogen phosphorylase kinase (Ca) – salivary and pancreatic alpha-amylases (Ca) – phenyl-alanine ammonia lyase (PAL) (Mn)

Foliar uptake

The outer cell layer of the leaf surface consists of epidermis cells and on top of their cell walls a cuticle layer is formed containing cutin, pectin and cuticular lipids (also called waxes). On top of this, a layer of cuticular lipids is situated, and this layer may appear very different from plant species to plant species and may also vary due to external conditions. Stomata guard cells are situated in the epidermis cell layer. Plants are able to take up lements via the leaf surface both via stomata (gases) and via the cuticle (ions) (Marschner, 1995). A high density of stomata can also promote uptake of ions since the uptake occurs to a high degree through ectodesmata. Ectodesmata are nonplasmic “channels” (which are less dense parts of the cuticle layer) that are situated foremost in the epidermal cell wall/cuticular “membrane” system between guard cells (stomata) and subsidiary cells. Elements that occur as cations will penetrate the leaves through the cuticle whereas elements that occur as anion like S, Se, N, I, also are common in gaseous form and thus will be taken up via stomata in gaseous form (Figure 19).

Figure 19: Foliar uptake of trace element

Copper

Copper is an essential component of the plant enzymes diamine oxidase, ascorbate oxidase, o-diphenol oxidase, cytochrome c oxidase, superoxide dismutase, plastocyanin oxidase and quinol oxidase. In the absence of Cu these enzymes are inactivated. Like iron, copper is involved in redox reactions (Cu2++e-<=> Cu+) in the mitochondria and in the light reactions of photosynthesis.

Salient functions:

• Catalyzes several plant processes

• Major function in photosynthesis

• Major function in reproductive stages

• Indirect role in chlorophyll production

• Increases sugar content

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• Intensifies color

• Improves flavor of fruits and vegetables

Manganese

Manganese ions (Mn2+) activate several enzymes, such as decarboxylases and dehydrogenases involved in Krebs cycle and this element is necessary for the photosynthetic oxygen evolution. Manganese is a cofactor for both phenylalanine Ammonoia-lyase, that mediates production of cinnamic acid and other phenolic compounds, and for peroxidase involved in polymerization of cinnamyl alcohols into lignin. Deficiency of Mn, therefore, inhibits the building of phenolic substances and lignin which is considered important for the defense of fungal infections (Rengel ????). Manganese deficiency causes intravenous chlorosis and small necrotic spots. As under Fe deficiency, root growth is more inhibited than shoot growth.

Salient functions:

• Functions as a part of certain enzyme systems

• Aids in chlorophyll synthesis

• Increases the availability of P and Ca

Molybdenum

Molybdenum ions are cofactors of several enzymes, such as nitrate reductase, nitrogenase, xanthine oxidase/dehydrogenase, aldehyde oxidase and sulfite oxidase. Since Mo is involved both in nitrate assimilation and nitrogen fixation, a molybdenum deficiency may also cause nitrogen deficiency, if the nitrogen source is mainly nitrate, or if the plants depend on symbiotic nitrogen fixation (Taiz and Zeiger 1998). The molybdenum requirement of root nodules in legumes and non-legumes, e.g. alder, is relatively high. Under Mo deficiency older leaves will get chlorosis between veins and necrosis. In broccoli and cauliflower the leaves do not show necrosis but become twisted and even die due to insufficient differentiation of vascular bundles (whiptail desease). Flower formation may be prevented or the flowers abscise before they are fully developed.

Salient functions

Widely found in commonly used foods (cereals, vegetables)

Mo is part of flavoproteins, xanthine oxidase, aldehyde oxidase

Nickel

Nickel (Ni) is an essential element for plant growth and is probably involved in different physiological processes. Deficiency of Ni is not very common. This element is a component of the enzyme urease and is essential for its function (Gerendas et al. 1999). Nickel is essential for plant species that use ureides in their metabolism (Marschner 1986). Nickel-deficient plants accumulate urea in their leaves and show leaf tip necrosis (Taiz and Zeiger 1998). Nitrogen-fixing microorganisms require nickel for hydrogen uptake hydrogenase and Ni-deficiency may, therefore, cause nitrogen-deficiency in the plant. Compared with other heavy metals the mobility of Ni within the plant is rather high (Gerendas et al 1999).

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Zinc

Zinc is a co-factor of more than 200 enzymes, such as oxidoreductases, hydrolases, transferases, lyases, isomerases, and ligases . Many of the metalloenzymes are involved in the synthesis of DNA and RNA and protein synthesis and metabolism. (Prasad 2002a,b) (Figure 20; Table 4)

Salient functions

• Aids plant growth hormones and enzyme system

• Necessary for chlorophyll production

• Necessary for carbohydrate formation

• Necessary for starch formation

• Aids in seed formation

• Necessary for nitrogen metabolism

Table 4: Salient symptoms of zinc deficiency (Marschner 1995)

In dicots

• stunted growth and short internodes

• leaved in "rosetting" with smaller size

• decreased shoot/root ratio

• root growth is less affected than shoot growth

• interveinal chlorosis in leaves

• reduction in crop production

In monocots

• chlorotic band along the midrib with and red necrotic spots

• Usually grain and seed yield are suppressed than total dry matter

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Figure 20: Zinc deficiency manifestations

Vanadium

• Plays a role in lipid metabolism (deficiency leads to high plasma cholesterol and triglyceride levels)

• may also function as an oxidation-reduction catalyst

Boron

Boron is one of the most essential elements for plant growth which can be applied through soils or plant's foliage. Boron is an essential element for higher plants, but its toxicity is observed when the element is present in higher concentration. Boron is an essential micronutrient for higher plants, and a deficiency causes inhibition of plant growth. It is generally up-taken by roots of a tree from soils containins boric acid solutions (Mukherjee 2005).

Boron is emitted into the atmosphere from natural and anthropogenic sources. Natural sources include volcanoes and sea salts, whereas anthropogenic sources are mining operations, glass and ceramic industries, chemical production and coal fired power plants. Boron enters into soil compartment not only from industrial and natural sources, but also through rocks, different amendments such as fertilizers, coal fly ash and residues. Research findings revealed that the lowest level of Boron concentration was in sandy and loamy soils, but the highest concentration was reported for lateritic and calcareous soils Kabata-Pendias and Pendias (2001) Boron content in soil is also affected due to rainfall for prolonged leaching loss of B in soils.

Factors influencing micronutrient uptake by plants

a) Soil factors: The availability of trace elements as nutrients depends on several factors, such as lithosphere (base-rock), soil age, soil type and the covering flora. In general, heavy metals can be accumulated in higher amounts in the sorption complex of soils rich in organic matter, but their release to soil solution is much slower

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than in mineral soils due to high affinity of soil organic compounds to heavy metals. Thus, the availability of nutrient metals to plants is controlled also by soil pH, type of mineral colloids and other important factors, like microbial activity, redox potential and aeration. Some of these factors act in several ways - increase of Ca2+ content in soil (liming) decreases heavy metals uptake by roots due to physiological antagonism of Ca2+/heavy metal ions, but even more by increased heavy metals retention by soil colloids. Availability of free-ions has been widely considered as a primary factor of metallic nutrients uptake by plants. There is evidence now that this relationship is not so obvious. Free ion-concentration dependent uptake of divalent cations is easier than when they are present in chelated form. Nonetheless, if the quantity of the metal-complexing ligand is limited (the case of soil solution ligands, which often are of low affinity and low selectivity in respect to individual elements) or the ligand is very diluted, chelating may have no influence or may even enhance a divalent cation uptake. However, chelating has a very stimulating effect on root uptake and translocation to shoots of trivalent cations, like Cr, Ga or In. It is also not possible to select the most important soil factors influencing metals availability to plants, because their importance can vary between elements - for Zn it seems to be Zn buffering mechanism in the soil, while for Fe and Mn it is soil pH and soil redox state.

b) Plant factors: Almost all heavy metals can be taken up by plants in two ways, which are mostly concentration-dependent (Siedlecka 1995):

i) Non-metabolic uptake - by energy independent mechanisms:

Intact membranes are effective barriers for ions and uncharged molecules, but when solutes are more concentrated at the one side of the membrane they can diffuse down the concentration gradient with the aid of membrane carriers or even aqueous pores. This transport is known as passive and takes place when heavy metal is present in the root environment at a high amount. It may be also stimulated by lowering the free ion level in cytoplasm due to its incorporation into organic structures, deposition in some cell compartments, transport to other cells or bounding to charged groups, this process often takes place in meristematic tissue of root tips. ii) Metabolic uptake - by energy dependent mechanisms:

This active uptake is involved in taking up ions against their concentration gradient. In this mechanism a proton motive force (ATP-driven H+ pumps) creates pH and electropotential gradient which stimulate ion passing to the cell through selective ion channels or carriers. Higher plants have sophisticated and specific mechanisms for Fe uptake. Iron, despite being widely spread in the lithosphere, usually predominates in soils in non-soluble and oxidized forms and plants have developed two strategies of its uptake encountering two main problems: availability and necessity of its presence in plants in reduced form. (Figure 19a,b)

Figure: 21 Schematic sketch of membrane

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Figure: 22 Carrier proteins that help in translocation of nutrients through membranes

Iron

Iron is required for functioning of a range of enzymes, especially those involved in oxidation and reduction processes, for synthesis of the porphyrine ring (chlorophyll and heme biosynthesis), reduction of nitrite and sulphate, N2-fixation (as part of the leghemoghlobin), etc. Iron is a constituent of feredoxin and takes part in assembling of the thylakoid units, thus having important functions in electron transport as well as in photosynthesis (photosystem I).

Iron is one of the essential micronutrients for plant and human nutrition. About 30%of the world’s population suffers from iron deficiency anemia. Properties that make iron beneficial in biological systems also render it potentially toxic . Uptake and transport of iron by the plant is an integrated process of membrane transport, reduction and trafficking between chelator species, whole-plant allocation and genetic regulation. Therefore, iron homeostasis is regulated through complex mechanisms involving various pathways to reduce the toxic effects of the iron

Iron toxicity

Aqueous iron and oxygen chemistry will produce a ferric ion trillions of times less soluble than cell iron concentrations, along with radical forms of oxygen that are toxic. Iron is both necessary for plant growth and is toxic in excess concentraions.

Properties that make iron beneficial in biological system as also render it potentially toxic especially in aerobic organisms due to the chemistry of iron and oxygen in physiological condition producing insoluble Fe compounds and soluble oxy radicals through Fenton reaction. Once after uptake the problem is not the solubility but the high reactivity that causes severe problems. To control their iron homeostasis, multicellular organisms have to balance iron uptake, intracellular compartmentalization, partitioning to the various organs, and storage. Fe excess is believed to generate oxidative stress. Toxic reduced O, species are ineviTable by-products of biological oxidations. The toxicity of the relatively unreactive superoxide radicals and H2O2 arises by the Fe-dependent conversion into the extremely reactive hydroxyl radicals (Haber-Weiss reaction) that cause severe damage on membranes, proteins, and DNA. Because hydroxyl radicals are by far too reactive to be controlled directly, aerobic organisms eliminate the less reactive O2 species as efficiently as possible. H,O, is removed by catalases, ASA peroxidases, and GSH peroxidases. The dismutation of superoxide radicals to O2 and H2O is catalyzed by metal containing SODs. Recently, evidence that Fe uptake leads to an oxidative stress has been

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acquired for E. coli and yeast. Indeed, genes coding for both Fe uptake and oxidative stress response are regulated by the same transcription factors in these organisms

Model of nutrient cycling and nutrient budgets

Nutrient cycle in the ecosystem (unlike energy flow, which is unidirectional and a given amount of energy is ultimately dissipated). Nutrients are recycled to varying degrees. Some nutrients are involved in short term cycling. Some are temporarily tiedup in organic compartment while some are lockedup in deep sediments and rocks. Foliar leaching, rainfall, percolation through soil, decomposition, uptake, transport of nutrients in and out of the ecosystem are linked to hydrological cycle and could not function without water. Hydrological cycle links the terrestrial and aquatic ecosystem and in doing so, relates the local ecosystem to the global ecosystem. In the ecosystem, the internal and external nutrients are cycled. Nutrient interchanges among biotic and abiotic components of an ecosystem. Exchanges occur externally among ecosystem through geological, meteorological and biological processes.

Conclusion

Ionomics- Transcriptome, proteome, metabolome, and ionome, the four basic biochemical pillars of functional genomics conneted to nutrient cycling. “Ionomics” is an important and broad biological phenomena, including electrophysiology, signaling, enzymology, osmoregulation and transport. An understanding of the “ionomics” and how it interacts with other cellular systems such as the genome, proteome, metabolome, and environment are integral to our full understanding of how plants integrate their organic and inorganic metabolisms (Figures 23 and 24). Compounds containing the non-metals phosphorus, sulfur, or nitrogen in the “ionome” and “metabolome” and metals such as zinc, copper, manganese, and iron in “metalloproteins” , “proteome” or in “metalloproteome”

Figures 23: Key families of metal transporters

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Figure 24: “Ionomics” is an important and broad biological phenomena, including electrophysiology, signaling, enzymology, osmoregulation and transport. Transcriptome, proteome, metabolome, and ionome, the four basic biochemical pillars of functional genomics. “Ionomics” is an important and broad biological phenomena, including electrophysiology, signaling, enzymology, osmoregulation and transport. An understanding of the “ionomics” and how it interacts with other cellular systems such as the genome, proteome, metabolome, and environment are integral to our full understanding of how plants integrate their organic and inorganic metabolisms. Non-metallic nutrients viz., phosphorus, sulfur, or nitrogen constitute the “ionome”, and metals such as zinc, copper, manganese, and iron form the “metabolome” in “metalloproteins”/“proteome” or in “metalloproteome” .

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References

1. Adriano DC (2001) Trace elements in terrestrial environments: Biogeochemistry, bioavailability, and risks of metals. 2nd ed. Springer-Verlag New York Inc. p. 866

2. Alina Kabata Pendias (2001) (3rd ed) Trace elements in soil and plants. CRC Press. Pp. 432.

3. Marschner H, 1995. Mineral nutrition of higher plants. Academic press.

4. Mukheriee A.B. An overview of boron: sources, uses and uptake by plants. International Workshop "Fate and Impact of Persistent Pollutants in Agroecosysterns" 10 - 12 March 2005, lUNG, Pu/awy. Poland

5. Prasad M.N.V., Sajwan K.S, and Ravi Naidu (eds) (2006) Trace elements in the environment: Biogeochemistry, Biotechnology and Bioremediation. CRC Press. Boca Raton.. 726 pp. Taylor and Francis Group

6. Prasad, M.N.V and K. Strzałka (2002). Physiology and biochemistry of metal toxicity and tolerance in plants. Kluwer Academic Publishers. Dordrecht. pp. 460.

7. Prasad, M.N.V. (2002) Zinc is the friend and foe of life. Zeszyty Naukowe PAN 33, Com. "Man and Biosphere". Eds:A. Kabata-Pendias and B. Szteke, Warsaw, p.49-54.

8. Prasad, M.N.V. (2002) Zinc transporting genes in plants. Zeszyty Naukowe PAN 33, Com. "Man and Biosphere". Eds: A. Kabata-Pendias and B. Szteke, Warsaw, p. 363-366.

9. Taiz L, Zeiger E, 1998. Plant Physiology. 2nd ed. Sinauer Assoc. Inc., Publ. Sunderland,

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