*Correspondence to: Muhammad Jamil, Department of Biotechnology and Genetic Engineering , Kohat University

of Science and Technology, 26000 Kohat, Pakistan, E-mail: dr.jamil@kust.edu.pk

Journal of Bio-Molecular Sciences (JBMS) (2015) 3(1): 44-55.

Effects of Lead Toxicity on Plant Growth and Biochemical

Attributes of Different Rice (Oryza Sativa L.) Varieties

Sara Awan1, Maria Jabeen

1, Qari Muhammad Imran

1, Farman Ullah

1, Zaffer Mehmood

2,

Muhammad Jahngir3and Muhammad Jamil

1*

1Department of Biotechnology and Genetics Engineering, Kohat University of Science and

Technology, Kohat, Pakistan 2Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan

2Department of Agriculture, University of Haripur, Haripur, Pakistan

Received 17 Feb. 2015; Accepted 05 March 2015; Published 31 March 2015

Abstract: Rice (Oryza sativa) is one of the principal foods for a large part of the world's human

population, but its productivity is reduced by the toxic effects of heavy metals such as lead. In

the present study seeds of the rice cultivars KSK-133, NIAB-IR-9, Basmati-385 (B-385) and

Shaheen Basmati (SB) were treated with different concentrations of lead chloride (PbCl2) (0,

250, 500, 1000 and 2000 ppm) to evaluate the effect on germination, seedling growth and some

biochemical attributes. Lead had no effect on germination percentage, except cv. SB at 2000

ppm. Germination rate, root and shoot length and dry weight decreased with increasing lead

concentrations. A sand culture experiment was conducted to measure the ion contents (Na, K,

Ca), photosynthetic pigments, total protein and nitrogen contents. Chlorophyll a, chlorophyll b

and carotenoids decreased with the increasing concentration of lead. Three varieties (NIAB-IR-9,

KSK-133 and SB) had a decreasing trend in potassium and calcium ions and increasing in lead

ion concentration. There was an increase in sodium ion concentration with the increase in lead

concentration but B-385 showed contrary results. Results confirm that lead has an inhibitory

effect on plant growth and development.

Key words: Oryza sativa L., Lead stress, Germination, Plant growth

Introduction

Rice (Oryza sativa) is one of the

most important cereal crops, providing food

for nearly a half of the world population

(Panich-pat and Srinives, 2009) and

contributing with one fifth of the calories

consumed by human’s worldwide (Welch

and Graham, 2005). About90 percent of the

total rice is cultivated in Asia (Salimet al.,

2003). Rice is the second most important

food crop of Pakistan; not only because of

its local consumption but also in view of

large exports (Noor et al., 2005). Abiotic

stresses such as the presence of heavy

metals at toxic levels adversely affect

Awan et al. 45

growth and grain yield of crops (Salt et al.,

1995). Among the various heavy metals,

Lead (Pb) is commonly spread throughout

the environment, and reveals a

comparatively high reactivity to plant cells

(Lukaszeket al., 1998). Lead deposition

interferes with plantgrowth and

development, and can cause plant death

In Pakistan, only 172 billion cubic

meters of fresh water are available, which

are not adequate to meet the water

requirements of the crops (Ibrahim and

Salmon, 1992; Qadiret al., 1998). Therefore,

sewage waters are used to irrigate rice fields

which are usually mixed with irrigated

water; however, those are not treated

because of the lack of infrastructure and

facilities for sewage treatment (Nawaz et al.,

2006). Lead is the major component of those

nutritional elements by seedlings and plants

and causes deficiencies or adverse ion

distribution within the plant (Trivedi and

Erdei, 1992). Research about the effect of

heavy metals on plant growth has been well

documented (Qin et al., 2000; Kang et al.,

2002), including effects of lead pollution on

under extreme conditions (Laraque and

Trasande, 2005; Clemens, 2006). In

Triticum aestivum and Cucumis sativus, lead

toxicity decreases seed germination rate,

length and weight of fresh and dry mass of

roots and shoots (Munzuroglu and Geckil,

2002). These effects can be attributed to the

fact that lead obstructed the absorption of

seed germination and seedling growth in rice

(He, 1990; Caiet al., 2002). It has been

reported that lead deposition in leaves of

rice decreased the concentrations of

chlorophyll contents. Lead produced highly

significant effects on shoot, root lengths and

seedling dry biomass of Lythrumsalicaria

(Jusephet al., 2002).waste water, which is

used for irrigation. In such conditions there

is a strong need to investigate the effect of

lead on rice seed germination and seedling

and plant growth by evaluating various

physiological and biochemical attributes of

Oryza sativa. It was hypothesized that lead

toxicity might induce physiological and

biochemical changes in Oryza sativa. To

prove this hypothesis, the present study was

designed.

Materials and Methods

Plant material

Seeds of the rice varieties NIAB-IR-9,

KSK-133, B-385 and SB were obtained

from National Agricultural Research Centre

(NARC) Islamabad.

Seed germination

Prior to the tests, seeds were surface

sterilized in a 0.5 % sodium hypochlorite

(NaOCl) solution for 10 minutes to remove

dirt and fungal traces. The seeds were then

rinsed with distilled water. Ten seeds were

placed per Petri plate on Whatman No.2

filter paper discs. Afterwards, they were

moistened with 10 mL of either0, 250, 500,

1000, or 2000 ppm PbCl2 solution. There

were five replications per concentration and

variety. The plates were then covered and

kept in the dark at 30 ºC for 4 d in growth

chamber (K-HB 3015, Korea). The seeds

during that time, every seed that reached 2

mm radical growth were considered as

germinated. Seed germination in each set

was recorded every 12 h up to 4 d and

germination rate calculated as 1/t50 (where

t50 is the time to 50 % of germination). After

4 d of germination, the seeds were provided

with proper light and dark periods (16/8 hr)

and allowed to grow for 6 d more. After 10

d, the lengths of fresh shoots and roots of all

the seeds in all replicates were measured.

After the measurement, the roots and shoots

were excised from the seeds and their fresh

weights were taken using a digital balance

(ALS 120-4, Germany).After taking fresh

weights, the roots and shoots of all the seeds

were placed in an incubator (Memmert,

Model 100-800, Germany) in Petri plates at

Effects of Lead Toxicity on Plant Growth 46

80 oC for 48 h. After that, the dry weights of

roots and shoots were taken.

Sand culture

Seeds of four varieties were grown in

distill water. After 15 d, plants seedlings

were transferred to sand culture in clean pots

and were supplied with Hoagland's solution

for 14 d. The pH was maintained at 5.8.

After 14 d, a solution containing PbCl2 in

combination with Hoagland’s solution was

supplied to the plants for 7 d. Three

replicates of each variety for every

concentration (0, 250, 500, 1000, or 2000

ppm) were used. Twelve pots (three of each

variety) were supplied with Hoagland’s

solution only and were used as control. After

36 d, the plants were harvested, dried at 80 oC in an incubator and hand crushed for

biochemical tests.

Determination of Chlorophyll a, b and

Caroternoids

For the chlorophyll test, 25 mg of

crushed plant material from each replicate of

four varieties was taken in test tubes. To

protect chlorophyll from degradation by

light, 25 mg of magnesium oxide were

added to each tube. Then, 5 ml of methanol

were added and the tubes were placed on a

shaker at 200 g for 2 h. After that, the

material was centrifuged (5810R Eppendrof,

Germany) for one minute at 4000 g and the

absorbance of the liquid at three different

wavelengths (470, 653 and 666 nm) was

checked by using a spectrophotometer

(BMS UV-1900, Canada). From the values

of absorbance, chlorophyll a, chlorophyll b

and carotenoids contents were calculated by

the following formula proposed by

Lichtenthaler and Wellburn (1985):

Chlorophyll a = 15.65 × absorbance at 666

nm – 7.340 × absorbance at 653 nm

Chlorophyll b = 27.05 × absorbance at 653

nm – 11.21 × absorbance at 666 nm

Carotenoid (cx+c) = 1000 × absorbance at

470 nm – 2.86 × ch. a – 129.2 × ch. b/245

Determination of ions

For ion analysis, 25 mg of plant

material of both varieties (for each replicate)

was digested. For digestion, 2 mL of

sulphuric acid (H2SO4) were added to the

beaker and the beakers were placed on a hot

plate at 150 0C. When black slurry was

formed, one milliliter of H2O2 was added

and again heated until it all evaporated and

only a few drops were left. Then this was

dissolved in 30 mL of distilled water and

then filtered. Sodium, potassium and

calcium were analyzed in these samples

using a flame photometer while lead

concentration was determined using atomic

absorption (AAnalyst 400, Perkin Elmer,

USA).

Determination of nitrogen and protein

content

Protein content was determined by

using the BUCHI Distillation Unit k-350

micro-Kjeldahl apparatus. Dried plant

material (50 mg) was taken and transferred

to the digestion flask with 0.1 g of digestion

mixture (copper sulphate, iron sulphate and

potassium sulphate) and followed by the

addition of 2 mL of concentrated H2SO4.

The solution was heated until it became

clear. After cooling, it was diluted up to 10

mL with distilled water. The digest was

transferred to the distillation assembly and

10 mL of 50 % sodium hydroxide (NaOH)

solution were added to it. The distillation

was completed in 6 minutes, noticed by the

change of color of boric acid to yellow due

to the formation of ammonium borate

indicating the trapping of nitrogen in the

form of ammonium hydroxide. The boric

acid having the trapped ammonia was

titrated with 0.1 N sulphuricacid. Titration

was done by methyl red indicator for which

about 40 ml of boric acid were taken in a

volumetric flask and the methyl red

indicator was added, resulting in a pink

colored solution. During titration, the color

of boric acid having ammonia changed again

into pink. The protein content was

Awan et al. 47

calculated by first calculating the total

nitrogen and protein content using the

formula’s recommended by Pellett and

Young (1980).

Nitrogen (g/g) = (Titrant volume for sample

– Titrant volume for blank) × 1.4007 × 0.1

Dry weight of sample x 100

Where, 1.4007 = Nitrogen factor.

Now for protein, we used: Protein (g/g) =

Calculated nitrogen × 6.25

Where 6.25 is Protein factor

Statistical analysis

A completely randomized design with two

factors was used in the experiment. Analysis

of variance was performed by using MS-

Excel. Mean comparisons, where applicable,

were conducted using Tukey’s least

significant difference (Li, 1964).

Results and Discussion

Seed germination Germination of seeds started at 24

hours after planting in NIAB-IR-9, KSK-

133 and B-385 but in SB it started at 60

hours. Germination was higher in the control

and it was reduced at 2000 ppm of lead

concentration in SB. Lead chloride at

relatively low concentrations (250 ppm) had

no significant effect on seed germination of

all the varieties, since the germination was

almost the same as the control. Lead

chloride at 2000 ppm significantly reduced

germination but after three days all the seeds

germinated excepting SB (Figure 1).

Entrance of lead to seed caused delay in

germination because the seed membrane has

a selective permeability to lead ions

(Wierzbicka and Obidziska, 1998).It was

evident that increasing lead concentration

decreased the germination rate in all

varieties, and that the effect was more

drastic when concentrations were 500 or

1000 ppm (Figure 1B). The effect of lead

on germination percentage and rate was

found to be greater in SB than in NIAB-IR-

9, KSK-133 and B-385 (Figures 1 A and B).

This might be a situation of tolerance and

susceptibility to lead because all of these

varieties belong to different genetic

background. The decrease in seed

germination could be explained for what

was observed in Albezialebbeck where the

application of lead caused the rapid

breakdown of stored food material in seeds

(Farooqi et al., 2009). The effects of lead on

seedling growth seem to be different with

regard to plant species, cultivars and organs

(Sharma and Dubey, 2005). Lead toxicity

causes a decline in the seed germination

percentage and germination rate

(Munzuroglu and Geckil, 2002). Our result

coincides with Bhardwaj et al. (2009) who

also observed a decline in germination rate

of Phaseolus vulgaris with increasing

concentration of lead.

Root and shoot length

Root and shoot elongation of rice seedlings

of all the varieties was greatly inhibited by

PbCl2; the degree of inhibition increased as

the PbCl2level increased. Root growth was

more affected than shoot growth (Figs. 2A

and 2B). At 1000 and 2000 ppm of PbCl2

the root of several seeds of all the varieties

emerged through the seed coat, but failed to

elongate. In contrast, the respective shoots

were relatively longer at these

concentrations. This response had already

been observed by Sheng et al. (2005), who

stated that lead significantly reduces the root

length and shoot length of rice seedlings,

and that the degree of inhibition increased

with the increase of Pb concentration. The

primary effect of Pb toxicity in plants is a

rapid inhibition of root growth, probably due

to the inhibition of cell division in the root

tip (Eun et al., 2000).

Effects of Lead Toxicity on Plant Growth 48

Fig. 1. Effect of various concentrations of

lead chloride on seed germination (A) and

germination rate (B) of different varieties of

rice. Vertical bars are the means of five

replications±SD.

The justification for the behavior of root

and shoot growth to lead is not known, but it

might be due in part to a quicker deposition

of lead in the roots than in the shoots (Al-

Helal, 1995). Schulz-Baldes and Lewin

(1976) reported that Pb reduced cell division

and uptake of crucial elements as it

accumulate on the cell membrane and hence

inhibited seedling growth. Yang et al. (2000)

found that the oxalate content in the root and

root exudates decreases upon lead treatment

in the sensitive varieties, and proposed that

compounds such as oxalate secreted from

the root may decrease the bio-availability of

lead. The reduction in root growth by Pb

toxicity is most possibly from the result of a

non-selective suppression of both cell

division and cell elongation of the seedlings

(Ivanov et al., 1988; Eun et al., 2000). The

small and poorly developed root system of

rice seedlings at elevated Pb concentrations

might also be associated to a disorder of

metabolic processes (Dinev, 1988; Pahlsson,

1989; Obroucheva et al., 1998).

Fig. 2. Effect of various concentrations of

lead chloride on shoot length (A) and root

length (B) of different varieties of rice.

Vertical bars are the means of three

replications±SD.

Awan et al. 49

Fresh and dry weight of root and shoot

Lead toxicity strongly affected the

fresh and dry root weights in all the

varieties. The effect of Pb was more on roots

compared to shoots and weight was even

zero at higher concentrations of PbCl2

(Figures 2; A and B). Fresh and dry root and

shoot weights significantly decreased at

1000 and 2000 ppm Pb concentrations

(Figures 3). This is evident from these

results that lead stress predominantly affects

root growth. Consequently, Pb stored

primarily in the roots because shoots were

less affected in all varieties except at 1000

and 2000 ppm lead stress. Lead stress

negatively affected the fresh and dry weight

of shoots and roots with increasing Pb

application (Kibria et al., 2010). In the

present study, increasing concentration of

lead caused the decrease of the fresh and dry

weight of roots and shoots of all the four

varieties (Figure 3). The decreased shoot

and root biomass of rice seedlings might be

due to interference of Pb with the

physiological processes of the plant, as Lead

phytoxicity involves the decrease of enzyme

activities, disturbed mineral nutrition, water

imbalance, and alteration in hormonal status

and variation in membrane permeability

(Sharma and Dubey, 2005). The decline of

biomass by Pb toxicity could be the direct

consequences of the inhibition of

chlorophyll synthesis and photosynthesis

(Chatterjee et al., 2004).

Ion analysis

The Pb ion concentration

increased with increasing level of Pb (Figure

4). Similarly, concentration of Na ions of

NIAB-IR-9, KSK-133, and S.B. was

observed to be increased while that of B-385

decreased with the increasing concentration

of Pb (Figure 5). The concentration of K and

Ca ions decreased with the increasing

concentration of Pb but K ion concentration

of NIAB-IR-9 at 1000 and 2000 ppm

showed an increasing pattern and K ion

concentration of KSK-133 increased only at

2000 ppm. Bas-385 variety showed an

overall increasing pattern for K and Ca ion

content (Figures 5; A and B). Lead actually

blocks the entrance of many ions from

absorption sites of the roots (Goldbold and

Kettner, 1991). Our result showed the

increasing pattern of Na ion content in

NIAB-IR-9, KSK-133 and SB but irregular

pattern in B-385. A decrease in K and Ca

ion content with the increasing

concentration of Pb in NIAB-IR-9, KSK-

133 and SB is observed, except B-385 where

these ions increased (Figures 5). This might

be due to a direct competition between Pb

and other important nutrients for same site

(Chatterjee et al., 2004). It is presumed that

the increase of some nutrient ions in plant

tissues might be due to the synergistic

effects of Pb with those ions via diverse

mechanisms. Further research should be

done for the understanding (Kibria et al.,

2010). Similar results were found by Huang

and Cuningham (1996) with corn, where the

calcium concentration in corn shoots

decreased after Pb treatment. However, in

their study, the same Pb treatment did not

significantly affect Ca concentration in

shoots of ragweed. The results of Bas-385

(increase in K ion content) show similarity

with the work of Kibria et al. (2009). They

reported that lead application significantly

increased K concentrations in both shoots

and roots of Amaranthus oleracea.

Effects of Lead Toxicity on Plant Growth 49

Fig. 3. Effect of various concentrations of

lead chloride on fresh shoot weight (A) fresh

root weight (B) dry shoot weight (C) and dry

root weight (D) of different varieties of

rice.Vertical bars are the means of three

replications±SD.

.

Fig. 4. Effect of various concentrations of

lead chloride on lead content of different

varieties of rice.Vertical bars are the means

of three replications±SD.

Chlorophyll content

Chlorophyll a, chlorophyll b and

carotenoid content of all the varieties were

decreased with the increasing level of lead

as compared to control (Figure 6). It shows

that Pb negatively affects the chlorophyll

and carotenoids content and so decreases the

photosynthesis rate in contaminated plants.

The process of photosynthesis is badly

affected by Pb toxicity (Sharma and Dubey,

2005). Under the metal stress, the levels of

photosynthetic pigments, namely

Chlorophyll 'a' and Chlorophyll 'b' and

Carotenoids decrease as the concentrations

of Pb in soil increases. (Bhardwaj et al.,

2009). Our results show a decline in

chlorophyll a, b and carotenoid pigment

content in shoots of all the four varieties

under lead stress (Figures 6). Plants under

Pb stress show a reduction in photosynthetic

rate because of the distorted chloroplast

ultrastructure, restrained synthesis of

chlorophyll, plastoquinone and carotenoids,

obstructed electron transport, reduced

activities of Calvin cycle enzymes, as well

as deficiency of CO2 as a consequence of

Awan et al. 50

stomatal closure (Sharma and Dubey, 2005).

Inhibition of chlorophyll synthesis by Pb is

due to impaired uptake of important

elements such as Mg and Fe by plants

(Burzynski, 1987). An improvement of

chlorophyll degradation occurs in Pb-

stressed plants due to increased

chlorophyllase activity is also observed

(Drazkiewicz, 1994).

Fig. 5. Effect of various concentrations of

lead chloride on sodium (A) potassium (B)

and calcium (C) contents of different

varieties of rice. Vertical bars are the means

of three replications±SD.

Fig. 6. Effect of various concentrations of

lead chloride on chlorophyll a (A)

chlorophyll b (B) and carotene (C) contents

of different varieties of rice. Vertical bars

are the means of three replications±SD.

Nitrogen and protein content

Total nitrogen and protein content of

all the varieties showed a decreasing trend

with the increase in concentration of lead.

Nitrogen and protein content of NIAB-IR-9

was highest followed by KSK-133. At 2000

ppm PbCl2 concentration, B-385 and SB

Effects of Lead Toxicity on Plant Growth 51

showed lowest nitrogen and protein content

(Figure 7).Lead treatment results in decline

in total protein content (Neelofer et al.,

2010). Lead stress may restrain a synthesis

of some proteins and promote others with a

general trend of decrease in the overall

content (Ericson and Alfinito, 1984). Our

results showed a decreasing trend in total

protein and nitrogen content of all the

varieties with the increase in the

concentration of lead (Figures 7). The

reduction in protein content may be caused

by the increased protein degradation process

as a consequence of enhanced protease

activity that is found to increase under stress

condition (Palma et al., 2002). It is also

likely that lead may have induced

disintegration of proteins due to the toxic

effects of reactive oxygen species that led to

reduce the protein content (Davies et al.,

1987). Protein content under lead stress may

be affected due to enhanced protein

hydrolysis resulting in decreased

concentration of proteins (Melnichuk et al.,

1982) and catalytic activity of lead

(Bhattacharya and Choudhuri, 1997).

Conclusion

Lead stress negatively affected the vigor

of seedlings and biochemical attributes such

as ion, chlorophyll, nitrogen and protein

contents. All the varieties can tolerate low

concentrations of Pb but at higher

concentration of 500 and 1000 ppm, it

causes inhibitory effects. The effect of Pb is

different in each variety. SB is found to be

more sensitive at higher Pb concentrations.

Fig. 7.Effect of various concentrations of

lead chloride on protein (A) and nitrogen

(B) contents of different varieties of rice.

Vertical bars are the means of three

replications±SD.

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