Abstract

Among heavy metals, lead is a potential pollutant that readily accumulates in soils and sediments. Although lead is not an essential element for plants, it gets easily absorbed and accumulated in different plant parts. Uptake of in plants is regulated by pH, particle size and cation exchange capacity of the soils as well as by root exudation and other physico-chemical parameters. Excess Pb causes a number of toxicity symptoms in plants stunted growth, chlorosis and blackening of root system. Pb inhibits photosynthesis, upsets mineral nutrition and water balance, changes hormonal status and affects membrane structure and permeability. This book shows various morphological, physiological and biochemical effects of Pb toxicity and also strategies adopted by plants for Pb-detoxification and developing tolerance to Pb. Pb tolerance is associated with the capacity of plants to restrict Pb to the cell walls, synthesis of osmolytes and activation of antioxidant defense system. Remediation of soils contaminated with Pb using phytoremediation and rhizofiltration technologies appear to have great potential for cleaning of Pb-contaminated soils.

Key words: lead poisoning, photosynthesis, oxidative stress, growth, respiration and ATP content

Introduction

Pollution is the introduction of contaminants into the natural environment that cause adverse change. Pollution can take the form of chemical substances or energy, such as noise, heat or light. Pollutants, the components of pollution, can be either foreign substances/energies or naturally occurring contaminants. There are many types of pollutions, one major type is soil pollution. Soil pollution is the contamination of soil with harmful substances that can adversely affect the quality of the soil and the health of those living on it. Pollution can be the result of an accident or carelessness, or done on purpose through illegal dumping. Pollution is also a by-product of activities as normal as driving or maintaining a farm. Soil pollution can result from contaminated water draining into the soil. Agricultural chemicals can coat the soil, and litter can work its way into the dirt. Polluted acid rain can end up in soil, and metal-contaminated dust on roads can wash into the soil as part of rain-induced run-off. The Food and Fertilizer Technology Center warns that heavy-metal contamination can not only reduce crop yields due to poor soil quality, but result in the crops absorbing the metals. The effects of soil pollution reach across the spectrum from water and air to vegetation, and to human health and society as well. While the specific effects depend on the pollutant, in general they include further environmental contamination as the polluted soil washes into water or is kicked up into the air, and poisoning, such as from lead-tainted soil. Lead is a strong environmental pollutant and is toxic even if it is in a very low concentration that cause soil pollution and affects plants in term of photosynthesis, respiration, ATP content, oxidative stress, growth and seed germination

1. Definition of lead

Lead is a chemical element with symbol Pb (from the Latin plumbum) and atomic number 82. It is a heavy metal that is denser than most common materials. Lead is a naturally occurring metal found in the earth's crust. Lead can be found many places, much because of the human activity through burning fossil fuels, mining, and manufacturing, also it can be contaminated with water from mines, waste dumps, and industrial plants. There is many potential heavy metal where lead is one of them that is neither essential element nor has any role in the process of cell metabolism but it is easily absorbed and accumulated in different parts of a plant. Lead is a strong environmental pollutant and is toxic even if it is in a very low concentration. Pb is a major pollutant in both terrestrial and aquatic ecosystems (1).

1.1. Sources of lead

Besides natural weathering processes, the main sources of Pb are exhaust fumes of automobiles, chimneys of factories using Pb, effluent from the storage battery, mining and smelting, industry, Pb ores, metal plating and finishing operations, fertilizers, pesticides, additives in pigments and gasoline In developing countries like Pakistan Pb paints, Pb water pipes, Pb acid batteries, Pb containing eye cosmetics, Pb food cans, Pb in petroleum as anti-knocking agent and Pb oaring and mining are constant sources of Pb intoxication.

1.2. Availability and uptake of lead in plants

Pb is available to plants from soil and aerosol sources. Pb uptake studies in plants have demonstrated that roots have the ability to take up huge quantities of Pb simultaneously greatly restricting its translocation to above ground parts where plants they could accumulate and translocate great quantities of Pb in the leaves in a concentration dependent manner. The extent of Pb to which it enters plants via the leaves depends on the ability of their leaves to absorb Pb from aerial sources, which depends on the specific leaf morphology. Where some plants absorb heavy metals from the atmosphere such as Downy plant leaves, but it is agreed that the bulk of the Pb taken up by plants remains in the roots, where Pb accumulates in the surface layers of soils and therefore it is difficult to reliably measure the portion of soil Pb directly available to plants (1).

Its availability depends highly on soil con ditions. Pb binds to organic material in the soil. There are many factors that affect the rate of the availability and uptake of Pb such as root surface area, root exudates, mycorrhization and rate of transpiration.

1.3. Absorption, localization and mobility of lead on plants

The absorption of Pb in soil follows the Langmuir relation and increases with increasing pH between 3.0 to 8.5. The pH of soil is between 5.5 and 7.5. The Pb present in the soil is always tightly bound to organic or colloidal material or in a precipitated form, where at the root surface Pb binds to carboxyl groups of mucilage uranic acids. Soil microorganisms may affect heavy metal availability by the process of biosorption, bioaccumulation and solubilization (24).

Pb retention in the roots is based on the binding of Pb to ion exchangeable sites on the cell wall and extracellular precipitation, mainly in the form of Pb carbonate deposited in the cell wall sometimes there is addition of synthetic chelates such as H-EDTA or EDTA, the addidtion of them in combination with low pH, effectively prevents cell wall retention of lead, making it available for translocation to shoots and transportation via apoplas (17). While Pb transported from the soil to the root cells has to cross the root-cell plasma membrane through PM cation channels, such as Ca-channels. The inhibition of the Ca-channel by Pb could arise from Pb blockage of the channel or due to competitive transport of Pb through the Ca-channel (24).

Pb moves predominantly into the root apoplast and thereby in a radial manner across the cortex and accumulates near the endodermis. The endodermis acts as a partial barrier to the movement of Pb between the root and shoot. This may in part account for the reports of higher accumulation of Pb in roots compared to shoots (18).

In an experiment when rice seedlings were raised in sand cultures for 10 and 20 days in nutrient medium containing 500 µM and 1000 µM Pb(NO₃)₂, root growth was reduced by 22 to 42 % and shoot growth by 25 %, whereas localization of absorbed Pb was 1.7 to 3.3 times higher in roots compared to shoots as shown in table 1 (1).

Sometimes there is a limited transport of Pb from roots to other organs is due to the barrier of the root endodermis which acts as a partial barrier since some of the Pb moves up through the vascular tissues and diffuses out into the surrounding tissues. This indicates that Pb moves into the apoplast. That movement of Pb in the root is primarily via the apoplast is also supported by the report that a large proportion of Pb is readily extractable in water. Higher concentrations of Pb cause cell injury and disturb the barrier function of the plasmalemma as well as the selective permeability of the plasma lemma and tonoplast. As a significant the amount of Pb was retained at the surface of plasma lemma rather than in the cell walls. Thus Pb enters the injured cells and do not enter the undamaged cells in the same capacity (19)

Also Pb compounds are major pollutants emitted by automobiles, as it is evident that those plants which are growing near highways are usually exposed to more Pb than other locations and are thus more affected. where the range of Pb in soil is between 400-800 mg/kg. The Pb which is accumulated in the streets and highways is transported to surface streams by rain water.

Consequently, Pb, pollutes other surface waterways and soil. Mine water also transports a huge amount of fine grained sediments contaminated with Pb.

1. Lead poisoning definition

Environmental heavy metal contamination as lead is becoming a worldwide problem that has attracted a great deal of attention. The release of heavy metals into the environment is mainly caused by various anthropogenic activities associated with agricultural practices, mineral exploration, industrial processes and solid waste management (2).

1.1. Effect of lead poisoning on humans

Lead poisoning is an acute or chronic poisoning caused by the absorption of lead or any of its salts into the body. Lead poisoning is an environmental hazard that is capable of causing mental retardation, behavioral disturbance, and brain damage. Lead poisoning is more common in children than in adults because young children often put their hands and other objects in their mouths, and these objects can have lead dust on them. Furthermore, lead poisoning is more dangerous in children than in adults because children absorb more lead and the developing brain and nervous system are more sensitive to the damaging effects of lead. Lead was used in household paint until 1978, and it was also found in leaded gasoline, some types of batteries, water pipes, and pottery glazes. Lead paint and pipes are still found in many older homes, and lead is sometimes also found in water, food, household dust, and soil (3).

There are many effects of lead poisoning on humans, where the ancients were very much aware about the effect of lead on the human reproductive system in which it causes phthisis and an excessive loss of semen (3). Also it impairs the spermatogenesis, hypothalamic pituitary testicular axis activity and sperm function which lead to the infertility in males (7). Some heavy metals have bio-importance as trace elements but, the bio toxic effects of many of them in human biochemistry are of great concern. The bio toxic effects of heavy metals refer to the harmful effects of heavy metals to the body when consumed above the bio-recommended limits. Although individual metals exhibit specific signs of their toxicity, the following have been reported as general signs associated with cadmium, lead, arsenic, mercury, zinc, copper and aluminum poisoning: gastrointestinal (GI) disorders, diarrhea, stomatitis, tremor, hemoglobinuria causing a rust–red color to stool, ataxia, paralysis, vomiting and convulsion, depression, and pneumonia when volatile vapors and fumes are inhaled. The nature of effects could be toxic (acute, chronic or sub-chronic), neurotoxic, carcinogenic, mutagenic or teratogenic. Lead is the most significant toxin of the heavy metals, and the inorganic forms are absorbed through ingestion by food and water, and inhalation. The serious effect of lead toxicity is its teratogenic effect. Lead poisoning also causes inhibition of the synthesis of hemoglobin; dysfunctions in the kidneys, joints and reproductive systems, cardiovascular system and acute and chronic damage to the central nervous system (CNS) and peripheral nervous system (PNS). Other effects include damage to the gastrointestinal tract (GIT) and urinary tract resulting in bloody urine, neurological disorder and can cause severe and permanent brain damage. While inorganic forms of lead, typically affect the CNS, PNS, GIT and other biosystems, organic forms predominantly affect the CNS. Lead affects children by leading to the poor development of the grey matter of the brain, thereby resulting in poor intelligence quotient (IQ). Its absorption in the body is enhanced by Ca and Zn deficiencies, acute and chronic effects of lead result in psychosis (4). Also the accumulation of significant amounts of Pb in liver tissue was implicated in the induction of an oxidative stress response in the liver. Although Pb is considered to be a poor inducer of oxidative stress, lipid peroxidation with concomitant inhibition of several antioxidant enzymes such as superoxide dismutase (SOD), catalase, GSH peroxidase, GSH reductase was reported. This was accompanied by a simultaneous increase in glutathione disulphide (GSSG) and a reduction in the GSH/GSSG ratio 58, 59. The mechanisms of Pb-induced oxidative stress are depicted in Fig. 2. Along with its role in Pb-induced hepatotoxicity, oxidative stress was also noted to play a significant role in the regression phase of hepatic hyperplasia with the generation of lipoperoxide (LPO) and other oxidants and the induced expression of cytokine ediators, including TNF-α. in developing countries such as India that have increased ambient levels of Pb by the addition of occupational exposures, nutritional (e.g., iron, protein) deficiencies, and infections that confound the overall impact of Pb on human health. Further, the mild to moderate dysfunction in hepatic drug metabolism associated with Pb toxicity (and its interactions with drug efficacy) may also have greater impact on general public health (5).

In addition, heavy metal exposure or dietary deficiency is associated with increased genetic damage, cancer and age-related diseases. Folate (vitamin B9) required for DNA repair and synthesis may increase cellular susceptibility to metal induced genotoxicity. the interactive effects of folic acid deficiency and sufficiency on genome instability and cytotoxicity induced by chromium, copper, manganese, lead, and their mixture (CCMP) in WIL2-NS human B lymphoblastoid cells. Thus, exposure to the tested metals increased chromosomal DNA damage in WIL2-NS cells and this was exacerbated by folate deficiency (6). Exposure of various sensitive populations to lead induces a wide variety of adverse effects in the central nervous system of children and fetuses, in various growth indexes of children, in the cardiovascular system of older people, in heme synthesis, and in calcium homeostasis and function.

1.2. Some treatments of lead poisoning on humans

A diet that is high in iron and calcium can help protect people against absorbing lead. Treatment involves chelation therapy, whereby blood is removed and metals are filtered out through a machine, then reinfused into the patient. Treatment cannot repair damage to the brain done by lead poisoning, but it may prevent further damage.

2. Effect of lead poisoning on plants

Plants are the target of a wide range of pollutants that vary in concentration, speciation, and toxicity. Such pollutants mainly enter the plant system through the soil or via the atmosphere (20). Among common pollutants that affect plants, lead is among the most toxic and frequently encountered (21). Lead continues to be used widely in many industrial processes and occurs as a contaminant in all environmental

compartments (soils, water, the atmosphere, and living organisms). The prominence of environmental lead contamination results both from its persistence and from its present and past numerous sources (22). These sources have included smelting, combustion of leaded gasoline, or applications of lead-contaminated media such as sewage sludge and fertilizers (23). Among heavy metals lead (Pb) is one of the hazardous pollutants of the environment and Pb pollution in air, water, soil is an ecological effect due to its impact on human health, plants and environment. In plants, lead affect several metabolic activities in different cell components where lead toxicity leads to the decrease in the percentage of seed germination, as well as growth dry biomass of roosts and shoots, disruption of mineral nutrition, reduction in cell division, inhibition of photosynthesis, respiration, ATP content, protein content, and phytochemicals, also Pb is reported to produce oxidative stress (ROS) and enhances antioxidant enzymes in plants.

3.Effect of lead in term of photosynthesis

Its main effect on photosynthesis and respiration where the effect of different lead (Pb) concentrations in the nutrient solution on the growth, Pb and chlorophyll content, chlorophyll fluorescence and quenching parameters in the leaves of young sunflower plants was studied. The content of Pb in the analyzed plant parts increased following the increase in Pb content in the nutrient medium. This increase was expressed to a higher extent in the roots than in the stems and leaves. In the presence of high concentration of Pb in the leaf area, the dry mass and the height of plants were reduced. Lead treatment of sunflower plants led to a pronounced reduction of chlorophyll content, accompanied by much smaller decrease of photosynthetic O evaluation rate and PSll efficiency at low light intensity. Hence, Pb effect²did not result in the destruction of the photosynthetic apparatus, but in its reduction. The highest Pb concentration in the nutrient solution induced, however, at saturating photon flux density (PFD) a decrease in photochemical quenching and in the efficiency of PSll electron transport and significantly affected nonphotochemical fluorescence quenching, indicating an increase in proton gradient across the thylakoid membrane and a decrease of photophosphorylation (12). In an experiment, Photosynthesis and transpiration rate of detached leaves of pea exposed to solution of Pb(NO₃)₂ at 1 or 5 mmol·dm−3 concentrations were inhibited. The higher concentration of this toxicant decreased photosynthesis and transpiration rates 2 and 3 times respectively, and increased respiration by about 20 %, as measured after 24 hours of treatment. Similarly, to Pb(NO₃)₂, glyceraldehyde solution, an inhibitor of phosphoribulokinase, at 50 mmol·dm−3 concentration decreased the rates of photosynthesis and transpiration during introduction into pea leaves. The rate of dark respiration, however, remained unchanged during 2 hours of experiment. The potential photochemical efficiency of PS II (Fv/Fm) and the activity of Rubisco at 5 mmol·dm−3 of Pb(NO₃)₂ were lowered by 10 % and 20 % respectively, after 24 hours. Neither changes in the activity of PEPC (EC 4.1.1.31) or protein and pigment contents were noted in Pb-treated leaves. The photosynthetic activity of protoplasts isolated from leaves treated for 24 or 48 hours with Pb(NO₃)₂ at 5 mmol·dm−3 concentration was decreased 10 % or 25 %, whereas, the rate of dark respiration was stimulated by about 40 % and 75 %, respectively. The content of abscisic acid, a hormone responsible for stomatal closure, in detached pea leaves treated for 24 h with 5 mmol·dm−3 of Pb(NO3)2 solution was increased by about 3 times; a longer (48h) treatment led to further increase (by about 7 times) in the amount of this hormone. The results of our experiments provide evidences that CO2 fixation in detached pea leaves, at least up to 24 hours of Pb(NO₃)₂treatment, was restricted mainly by stomatal closure (13). Also chlorophyll concentration and photosynthetic energy storage efficiency decreased with time and with lead concentration. Reduction of photosynthesis progressed with time and increased with lead concentration, reaching up to 80% at the highest lead concentration after seven days (14). In addition, the process of photosynthesis is adversely affected by Pb toxicity. Plants exposed to Pb ions show a decline in photosynthetic rate which results from distorted chloroplast ultrastructure, restrained synthesis of chlorophyll, plastoquinone and carotenoids, obstructed electron transport, inhibited activities of Calvin cycle enzymes, as well as deficiency of CO₂ as a result of stomatal closure. Ceratophyllum demersum plants when grown in aquatic medium containing Pb(NO₃)₂ showed distinct changes in chloroplast fine structure. Leaf cells of such plants exhibited a reduction in grana stacks together with a reduction in the amount of stroma in relation to the lamellar system as well as absence of starch grains. Pb treatment also changes the lipid composition of thylakoid membranes. Effects of Pb on various components of photosynthesis, mitotic irregularities, respiration, water regime and nutrient uptake are shown in figure 5.

4.Lead tolerance in plants

The two basic strategies of metal uptake related to tolerance in plants, as suggested by involve (i) the 'excluder' strategy in which the concentration of heavy metals is maintained at a constant low level until critical soil concentration is reached when toxicity ensues and unrestricted metal transport results and (ii) the 'accumulator' strategy in which metals are actively concentrated within the plant tissues over the full range of soil concentration implying a highly specialized physiology. where suggested three basic strategies of response: avoidance, detoxification and biochemical tolerance each of which affects tissue metal concentrations in different ways. Figure 7 describes various responses of cells, when plants are exposed to Pb. These responses include exclusion, detoxification mechanisms and non-specific defense systems (53)

Conclusion

Pb has gained considerable attention as a potent heavy metal pollutant due to the growing anthropogenic pressure on the environment. Pb contaminated soils show a sharp decline in crop productivity. Pb is taken up by plants mainly through the root system and partly, in minor amounts through the leaves. Inside the plants Pb accumulates primarily in the root but a part of it is translocated to the aerial portions. Soil pH, soil particle size, cation exchange capacity as well as plant factors such as root surface area, root exudation and mycorrhizal transpiration rate affect the availability and uptake of lead. Limited translocation of Pb occurs from root to other organs due to the barrier function of the root endodermis. At lethal concentrations this barrier is broken and the flux of Pb enters the vascular tissues. Pb deposits of various size are present mainly in the intercellular spaces, cell walls and vacuoles. Small deposits of this metal are also seen in the endoplasmic reticulum, dictyosome and dictyosome derived vesicles.

After entering the cell, Pb inhibits activities of many enzymes, upsets mineral nutrition and water balance, changes the hormonal status and affects membrane structure and its permeability. Visual non-specific symptoms of Pb toxicity are stunted growth, chlorosis and blackening of the root system. In most cases inhibition of enzyme activities due to Pb results from the interaction of the metal with enzyme –SH groups. The activities of metalloenzymes may decline due to displacement of an essential metal by Pb from enzyme active sites.

Pb decreases photosynthetic rate by distorting chloroplast ultrastructure, diminishing chlorophyll synthesis, obstructing electron transport and inhibiting activities of Calvin cycle enzymes. At low concentrations Pb stimulates respiration and increases ATP content whereas higher concentrations are inhibitory to respiration and decrease ATP. Pb causes the imbalance of the minerals K, Ca, Mg, Mn, Zn, Cu, Fe within the tissues by physically blocking the access of these ions to the absorption sites of the roots.

Pb exclusion capacity of plants is related to oxygen transport ability, radial oxygen loss from the root, efficiency to immobilize Pb in the rhizosphere. Biochemical tolerance to Pb is related to the capacity of the plants to restrict Pb to the cell walls, synthesis of osmolytes and activation of the antioxidant defense system. Such studies will enlighten the mechanism of the genetic and biochemical basis of Pb tolerance in crops and based on biotechnological tools it should be possible to produce plants with enhanced Pb tolerance. Emerging clean-up technologies for remediation of Pb such as phytoextraction and rhizofiltration have the potential to provide environmentally sound and economically viable remedies for the cleaning of Pb-contaminated soils.

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