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The present study was conducted to determine the influence of Pb on wheat (Triticum aestivum L. cv Shafaq-2006). The experiment was set up at GC University Botanic Garden, Lahore. Lead nitrate was applied (100, 200, 400, 600, 800, 1000 mg/Kg of soil in different treatments) to sealed plants while control plants were grown with normal soil (without lead nitrate). Existence of lead severely reduces germination (30%), seedling fresh weight (74%), seedling dry weight (77%), vigor index (89%), tolerance index (84%), plant height (33%), number of leaves (41%), root fresh weight (50%), shoot fresh weight (62%), root dry weight (62%), shoot dry weight (71%) and root length (45%). Physiological parameters viz., photosynthetic rate (74%), transpiration rate (72%), stomatal conductance (82%) and some of the biochemical attributes like chlorophyll a, chlorophyll b, total chlorophyll and carotenoids were also reduced in the range of 42%, 53%, 43% and 41%, respectively. In addition, protein contents, phosphorous and potassium was also significantly reduced by 81%, 60% and 55%, respectively. Lead accumulation was extremely higher in roots (4600%), shoots (9800%) and in seeds (119%) compared to control plants. Yield parameters like number of seeds/plant, seed weight/plant, 1000 seed weight and harvest index were reduced by 90%, 88%, 44% and 61%, respectively. In summary, present extensive investigation has reported that lead is injurious to every aspect of wheat germination, growth and yield. Lead accumulation above threshold concentrations in various plant parts and seeds is a matter of serious concern from environment and human health viewpoint.

Heavy metals are of considerable environmental concern, harmful for humans and also tend to bio-accumulate in living organisms (Stoltz and Greger. 2002). Mining activities, agronomic practices, use of industrial effluents for irrigation (Zeng et al., 2006) and sludge use as manure are the major sources of heavy metal contamination. In many developing countries, farmers are irrigating their crops with industrial effluents having high level of toxic metals (Cu, Pb, Cr, Cd, Fe, Mn, Ni, and Zn (Jarup, 2003), due to the unavailability of irrigation water.

Worldwide environmental and human health problems are increasing sharply in the biosphere due to heavy metal contamination since 1900 (Lavado et al., 2001). Cultivation of crops in or close to contaminated sites may result in both growth inhibition and tissue accumulation of heavy metals (Pb), with resulting possible risks to humans or livestock health if these tissues are ingested. Different studies showed that heavy metals inhibited the enzymatic activities of microbial community. The extent of inhibition increased significantly with increasing level of heavy metals (Hartley-Whitaker et al., 2001).

Among all the existing pollutants lead is on of the major contaminant of the soil. (Gratao et al., 2005), posing major environmental problems (Wang et al., 2007). Lead (Pb), is one of the most persistent metals in environment with soil retention time of 150 to 1500 years (Shaw, 1990) lead is also a threat for human and animal health (Ahmed et al., 1993). Lead accumulates in plants from air, water and soil (Smith et al., 1989). The harmful effects of Pb include intervention with nutrients uptake (Vogel-Mikus et al., 2005), toxicity on germination and seedling growth (Farooqi et al., 2009) growth delay (Tomar et al., 2000), alters metabolism (Wang et al., 2007), changes in enzymes (physate) activity (Zofia et al., 2006), disturbed respiration (Wai et al., 2008) and photosynthesis (Ahmed et al., 1993), creates physiological disorders, changes in root morphology (Fargasova, 2001). The primary effect of Pb toxicity in plant is a quick inhibition of root growth possibly due to inhibition of cell division in root tip (Ho et al., 2008). Heavy metal caused excessive generation of reactive oxygen species (ROS), mainly in chloroplast / mitochondria (Mittler, 2002) and alters antioxidant enzymes (Liu et al., 2003). These ROS can quickly attack all types of biomolecules, leading to cell death (Sinha and Saxena, 2006). Pb contamination thus stances a severe problem for agriculture.

Wheat, sugarcane, cotton, and rice, are most important crops in Pakistan which account more than 75% of the value of total crop output. Pakistan’s main food crop is wheat. Due to the shortage of irrigation water farmers are using industrial wastewater for irrigation. The presence of lead in food chain can caused chronic health problems (Jianjie et al., 2008). The aim of the research work is to examine the effect of Pb on growth, Pb buildup in different parts and purpose of Pb toxicity on physiological, biochemical and growth parameters.

Germination studies: Data for % age germination, Seedling vigor index and tolerance index is shown in Fig. 1. Data reveled that as concentration of Pb increases the germination parameters decreases. The maximum reduction was 30% (germination), 89% (Seedling vigor index) and 74% (tolerance index). Fig. 2 represents the data for Seedling dry weight, Seedling fresh weight, Plumule length, Seedling length, Radicle length. As the Pb concentration increases it creates significant effect compared to control

Growth assessment: Plants receiving Pb treatments had highly significant stunted growth compared to the control (Table 1). Control plants were lush green and health of plants decline successively with increase in lead concentration

Yield assay: Table 2 represents the data for the yield assay. Seed weight/plant and number of seeds/plant decreases sharply at low Pb concentrations. However, 1000 seed weight decreased sharply at higher lead concentrations.

Eco-physiological attributes: Fig. 3. shows that lead has the injurious effect on Photosynthetic rate, Transpiration rate and Stomatal conductance. Data for Chlorophyll a, Chlorophyll b, Carotenoids and Total chlorophyll is shown in Fig. 4. As depicted, the pigments gradually decrease from T1 to T6. The maximum reduction of Chlorophyll a, Chlorophyll b, Carotenoids and Total chlorophyll was, 44% 53%, 42% and 43%, respectively.

Table 3 depicts the data for minerals nutrients. Phosphorous content decreased from 11% – 58%, Potassium contents decreased from 14% to 34%, Nitrogen contents from 38% – 82% and Proteins contents from 18% – 81%. This decrease is due to the disturbance in chlorophyll synthesis and also due to the interference in the uptake of magnesium (key element of chlorophyll).

Lead determination: The experimentation depicted that Pb was accumulated in the root, shoots and seeds of wheat plants (Table. 3). Statistical analysis reveled that the treatment means were highly significant. The magnitude of Pb increase, relative to control was alarming. For instance, root shows 4600% (T6) increase in Pb concentration. Similarly, shoot and seeds shows 9800% and 118% increase in Pb concentration. Age of the plants also effect the Pb accumulation. The concentration of Pb in different parts of wheat plant decrease in the following manner; root ; shoot ; seeds.

Discussion

Propagation parameters are very important parameters from agronomical point of view. The main process that is badly affected (Shanker et al., 2005) and reduced by heavy metals is the seed germination (An, 2004). Chun et al. (2007) reported that at very low Pb concentration (0 to 0.5 mg/kg of soil), germination index and germination energy increased. Similarly root and shoot growth also increased at low Pb concentration (Ma and Hong, 1998). But concentration above 5 mg/Kg of soil is detrimental and decreases the growth of root, shoot and germination (Hartley-Whitaker et al., 2001). Root gets more affected then other parts of the plant body (Wang et al., 2007) because roots are the first contact point for toxic metal species (Abedin and Meharg, 2002). According to Gupta et al. (1999) excess heavy metals ( including Pb) put plant in another stress by the generation of reactive oxygen species (ROS) in mitochondria and chloroplast, like superoxide (O-2), hydroxyl radicals (OH-) and hydrogen peroxide (H2O2). ROS are very reactive and Immediate after synthesis attack all biomolecules such as lipids, nucleic acid, amino acids and proteins, which leads to irreparable metabolic defects and cell death (Dat et al., 1998; Mittler 2002).

The results of our study are in accordance with that of Singh et al. (2004). Gopal and Rizvi (2008) also reported that leaf area, root length, root girth, root fresh weight and root dry weight decrease with the increase in Pb concentration. The detrimental effect of Pb was not apparent during early age of plants but as the age increase the deleterious effect increase. This may be due to the more uptake and accumulation with the age. Tomar et al. (2000) concluded that roots developments were poor in the presence of excess Pb in soil. This poor root development was substantiated by reduced root weight at all stages of determinations (Bekiaroglou and Karataglis, 2002). Similarly Kastori at al. (1998) reported reduction in leaf area in sunflower. Among other reasons of reduction in biomass, reduction in photosynthesis and nitrogen metabolism is the major one (Fargasova, 2001). According to Gisbert et al. (2006) decrease in shoot fresh weight not only depends on the concentration of Pb but also to the species. Decrease in shoot fresh weight was in the following order, B. carinata ; B. oleracea I ; B. juncea. Some plants are resistant to Pb contamination and are not affected by the presence of Pb in soil. Kenaf (Hibiscus cannabinus L.) biomass showed no reduction and thus can be used as phyto-remediation (HO et al., 2008).

Today farmers prefer those varieties that give more yields. Scientists are also involved in developing new improved verities (through genetic engineering), for more yield. Present study demonstrated that Pb has sever negative effect on the yield of wheat. Mensah et al. (2008) reported the yield reduction of different vegetables (carrot, cabbage and lettuce), when irrigated with contaminated water. Yield reduction in cabbage, lettuce and carrot were 16%, 61% and 53%, respectively. These results were obtained under the effect of 0.5mg/l of Pb. yield reduction was due to the poor development and growth of seed. Pb accumulation in seed disturbs the enzymatic activities which result in poor yield. Similarly, Zheljazkov and Nielsen (1996) demonstrated that, 400m far air pollution source (emitting Pb) may reduce the yield of corn mint by 9-16%. Essential oils were found to be decreased by 14% due to (Pb) air pollution (Lavado et al. (2001).

Zeng at al. (2006) applied lead acetate in the range of 0-900 mg/kg of soil at six different levels. Their results revealed an initial consistent trend of increase in chlorophyll contents and then gradually decrease with the increase in concentration of Pb. Pb induce proline accumulation and cause oxidative stress in rice plants. Excess Pb disturbs the protein metabolism. This is supported by the finding of Tomar et al. (2000), who reported increase in the activity of acid phosphatases, ribonuclease and peroxidase and decrease in the activity of catalase. Under excess Pb condition free radicals are formed. These free radicals oxidize biomolecules. Peroxidase and catalase decompose them and protect the plant from oxidative stress (Geebelen et al., 2002).

Accordint to Gwozdz et al. (1997), at higher Pb concentration (0.1-0.5mM) catalase antioxidant activity decreases. This implies that antioxidant system response is dose dependent. At higher Pb concentration free radical production is more, which is beyond the capacities of antioxidant enzymes. These free radicals in turn decrease enzyme activities.To protect from oxidative stress (induced by free radicals) plants have developed non-enzymatic molecular species as cysteine, nonprotein thiol and ascorbic acid, etc. (Singh et al., 2004; Sinha and Singh 2005; Sinha and Saxena, 2006). Cysteine (–SH containing amino acid) is a major constituent of phytochelatins and has an important role in metal detoxification. Cysteine content increase with the increase in metal concentration in leaves and root of plants (mustard, Zea mays and wheat) (Nussbaum et al., 1988). Tolerance against metal toxicity increases with the rise in cysteine content, whereas decreased cysteine content might be due to decreased activity of enzymes (ATP sulfurylase, sulfate reduction, sulfo-transferase and adenosine-5-phosphosulfate) (Nussbaum, 1988). Glutathione and ascorbic acid both plays prominent role in scavenging free radicals (Smith et al., 1989). The maximum ascorbic acid contents were observed in roots after 90 days (of growth period). Similarly, Singh and Sinha (2005) reported increased ascorbic acid level in B. juncea (L.).

Pb toxicity adversely affects the process of photosynthesis by damaging ultra-structure of chloroplast, decreased synthesis of chlorophyll, carotenoids, abstraction in electron transport chain, decreased activities of enzymes involved in Calvin cycle, CO2 deficiencies (due to stomatal closure), changes in thylokoid membrane (Stefanov et al., 1995; Ahmed and Tajmir, 1993). Drazkiewicz, (1994) reported that the decrease in chlorophyll content under the influence of Pb toxicity is because of the increased activity of chlorophyllase. Chlorophyll a get less affected by Pb treatment then chlorophyll b (Vodnik et al., 1999).

Lead concentration in soil has a marked influence on its uptake by root. There exist a strong positive correlation between Pb in root and Pb in soil (extractable). Other factors that effect Pb adsorption are: microbial activity (Wang et al., 2002), type of fertilizer (Stoltz and Greger, 2002), increasing pH (Lee et al., 1998), presence of phosphate and carbonate precipitates (Blaylock et al., 1997) and extractable lead in soil (Wai et al., 2008). Liu et al. (2003) reported that Pb concentration decrease significantly as the distance from the roots decreases, thus roots accumulate more Pb then shoot and seeds. The bioaccumulation and translocation of heavy metals in crop tissues are major factors that determine its role in phyto-remediation. kenaf root can accumulate 85% of total Pb present in plant (Ho et al., 2008), Thlaspi praecox Wulf. can retain up to 80% Pb (Vogel-Mikus et al., 2005) and Brassica junce (Indian mustard) has the ability of accumulating over 95% of total Pb present in plant boby (Liu et al., 2003). These metal containing roots after decomposing release Pb into the environment again (Weis and Weis, 2004). Liu et al. (2003) worked on different rice cultivars and found that their exist a great differences in amount of Pb uptake and translocation. Different crops have different properties that favour Pb uptake and its distribution to aerial parts. Root-accumulator store Pb in roots and allow small amount to be transported to above ground parts. On the other hand shoot-accumulators store more Pb in shoots then in roots (Shallari et al., 1998). Root exudates, root surface area, mycorrhisation and transpiration rate affect the Pb uptake by plants (Davies, 1995), for that reason different crops uptake Pb amount significantly different (Stoltz and Greger, 2002). Difference in Pb translocation, may possible due to difference in Pb forms that exist in plants. Eltrop et al. (1991) reported that low-molecules complexes and ions of Pb are mobile in crop plants. Although the root absorb more Pb but its translocation and distribution to aerial parts is restricted.

Conclusion
This study demonstrated that lead nitrate is detrimental to wheat plant and it affects severely, the germination, growth and biomass. Present study probed the causes of damaging effects of Pb on wheat growth and development by monitoring the physiological and biochemical parameters. It came to know that negative effects of Pb were due to decreased photosynthesis and other related processes like stomatal conductance and transpiration rate. High concentration of Pb also adversely affected many metabolic processes like total chlorophyll, carotenoids, proteins, nitrogen, potassium and phosphorous uptake. Pb accumulated in wheat parts (roots, shoots and seeds) above threshold value. This Pb contaminated wheat may be very detrimental for animals an
d humans, if consumed. Through slow bioaccumulation this can cause serious illness and functional disorder (Jianjie et al., 2008). Even the small dose of Pb over a long period of time may cause cancer.

Acknowledgments
We are thankful to field/research staff of SDSC and Botanic Garden, Government College University, Lahore. We are also grateful to PCSIR Labs, Lahore for their technical assistance.

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