Toxic effect of arsenic on ten rice varieties

Keywords: Arsenic stress, Morphological parameters, SDS-PAGE, Molecular variation, Oryza sativa L. Abbreviations: SDS PAGE- Sodium Dodecyl Sulphate Poly Acrylamide Gel Electrophoresis.

ABSTRACT: Rice is one of the most important cereal crops of developing countries and the staple food of about 65% of the world’s population. The rice crops have been greatly disturbed by the heavy metals. The present study deals with the toxic effect of sodium arsenate on morphological and molecular variation through SDS-PAGE in 10 rice (Oryza sativa L.) varieties. Ten varieties of rice were grown under different concentration (25 ppm, 50 ppm and 100 ppm) of sodium arsenate against control. Morphological parameters like shoot length, root length, leaf area and biomass showed marked differences among ten rice varieties. The proteins were separated through SDS-PAGE gel electrophoresis and calculated their molecular weight. The morphological and molecular variations induced in rice varieties by arsenic stress provide a new insight leading to a better understanding of the heavy metal response in plants.

011-016 | JRA | 2011 | Vol 1 | No 1

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.

www.jagri.info

Journal of Research in

Agriculture An International Scientific

Research Journal

Authors:

John De Britto R, Mary

Sujin R, Steena Roshan

Sebastian and Dharmar K.

Institution:

Plant Molecular Biology

Research unit, St. Xavier's

College (Autonomous),

Palayamkottai, 627 002,

Tamilnadu, India.

Corresponding author:

John De Britto A

Email:

bjohnde@yahoo.co.in

Web Address:

http://www.jagri.info

documents/AG0003.pdf.

Dates: Received: 12 Oct 2011 Accepted: 19 Oct 2011 Published: 25 Oct 2011

Article Citation: John De Britto R, Mary Sujin R, Steena Roshan Sebastian and Dharmar K. Toxic effect of arsenic on ten rice varieties. Journal of Research in Agriculture (2011) 1: 011-016

Original Research

Journal of Research in Agriculture

Jou

rn

al of R

esearch

in

A

gricu

ltu

re

An International Scientific Research Journal

INTRODUCTION

Heavy metals constitute a heterogeneous

group of elements widely varied in their chemical

properties and biological functions (Holleman and

Wiberg, 1985). Heavy metals are kept under

environmental pollutant category due to their toxic

effects in plants, human and food. Some of the

heavy metals are cumulative poison especially

Arsenic (As), Cadmium (Cd), Lead (Pb) and

Mercury (Hg) (Mildvan, 1970). Metal toxicity to

plants varies with plants species, specific metals,

concentration, chemical form, soil composition and

pH (Mukesh et al., 2008). Off many, arsenic is one

among that drastically affects the crops.

The increased level of arsenic has been

mainly resulted from mining, industrial, agricultural

and geochemical processes (Flora et al., 2007).

Food crops such as rice and vegetables grown on

arsenic contaminated soil can accumulate high

levels of arsenic in roots, shoots and seeds (Zhu et

al., 2008). Thus arsenic uptake by crop plants plays

an important role in the transfer of this toxic

element into the food chain. Additionally, inorganic

arsenic species are phytotoxic and the elevated

concentration of arsenic in the soil causes a

significant reduction in crop yield (Meharg, 2004).

This stress may induce genetic changes in the crop

plants.

Rice is one the world's most important food

crops and grown mostly in tropical and subtropical

countries (Singh, 1993). The demand for rice is

increasing tremendously as the population in rice

growing areas is increasing at an alarming rate.

However, increasing rice production is becoming

more difficult because of biotic and abiotic stresses

(Hert, 1991).

In the present study, analysis of morphology

and protein variability of the ten rice varieties

induced with arsenic stress through SDS-PAGE has

been carried out.

MATERIALS AND METHODS

Materials The seeds of rice varieties were procured

from seed testing centre, Palayamkottai. Ten

popular, medium duration varieties namely IR 32,

IR 36, IR 45, IR 46, IR 50, ADT 32, ADT 39, CO

45, Ambai 16 and Singapore were selected for the

present study.

Methodology A pot culture experiment was conducted in

Plant Molecular Biology Research Unit, St.

Xavier’s College, Tirunelveli. The seeds were

surface sterilized and soaked overnight in double

distilled water (ddw) and then kept in dark for 24 hr

for germination. Then these were sown in pots

(paper cup), which were watered adequately. After

10 days the samples were exposed to 25 ppm, 50

ppm and 100 ppm of arsenic solution (Na2HAsO4),

and a control was maintained.

Morphometric analysis

After 15, 20, 25 & 30th day, the shoot length,

leaf area were measured using a metric scale. On

the 30th day, the plants were harvested and

separated in to root and shoot. Length of root and

their fresh and dry weights were measured.

Protein profiling The collected shoots were taken for protein

isolation. The separation of protein was carried out

at -50°C at 100V thereafter for 3-5 hours. SDS-

PAGE electrophoresis preparation was followed by

(Laemmli, 1970).

2.4. Data analysis:

The obtained data was tabulated, the

molecular weight of protein was determined and

their banding patterns were analyzed.

RESULTS AND DISCUSSION

The effect of sodium arsenate metal on

morphological and molecular parameters in ten rice

varieties were carried out.

The plants were treated with different

concentrations of sodium arsenate (25, 50 and 100

ppm), which caused a reduced growth in shoot

length, leaf area, root length, fresh and dry weights

(Table 1). Regarding shoot and root length there

were significant difference in the height of the plant

treated with different sodium arsenate concentration

when compared to the control. The fresh and dry

weight of stressed plants reduced when compared to

control in response to arsenic stress.

In most of the varieties, the shoot length at

highest concentration remained the same for 20th,

25th and 30th days; whereas, the low concentrations

showed normal growth with an increase in shoot

length. Decrease in shoot length may be due to the

inhibition of cell division and cell enlargement. The

IR45 variety with shoot length 12.6 cm was more

tolerant to the arsenic stress, whereas ADT39

variety with shoot length 9 cm was less tolerant

among the ten rice varieties chosen.

Muhammad et al. (2008) reasoned that the

reduced shoot and root length in metal treated

seedlings could be due to the reduction in the

meristematic cells present in this region and some

enzymes contained in the cotyledon and endosperm

012 Journal of Research in Agriculture (2011) 1: 011-016

Britto et al.,2011

cells become active and begin to digest the stored

food, which is converted into soluble form and

transported to the radicle and plumule tips. Hence,

when activities of hydrolytic enzyme are affected,

the food does not reach the radicle and plumule

thereby affecting the seedling length.

Leaves of metal treated plants showed

damage symptoms such as chlorosis, necrosis, leaf

burn and senescence as well as leaf area reduction.

High concentration (100 ppm) of metal treatment

strongly reduced the leaf area of the IR46 variety at

30th day. The root length measured at 30th day

showed decrease in growth with respect to

increased metal concentration. Prasad (1995)

reasoned that the increased phytotoxicity with

increase in the concentration of heavy metals was

due to the accumulation of heavy metals in the root

which reduced the mitotic division of the

meristematic zone and also due to the reduction in

both new cell formation and cell elongation in the

extension region of the root.

Remarkable reductions of fresh and dry

weight were observed in shoot part in response to

arsenic. Similar results were observed by Jian et al.

(2008), when arsenic accumulates in root and of

rice grain which results in reduced yield;

Germination of two rice (Oryza sativa L.) cultivars

(Ratna and LR 36) in the presence of 10 mu M

PbCl2 and 10 mu M HgCl2 decreased germination

percentage, germination index, shoot/root length,

tolerance index and dry mass of shoots and roots

(Mishra and Choudhari, 1998) The effects were

more pronounced in tolerant cultivar IR 36 than in

the relatively susceptible cultivar Ratna.

The arsenic contamination lead to the

change of morphological characters of Salsola kali

was reported by Guadalupe et al. (1996);

Accumulation and distribution of arsenic

interrupted the physiological activities of tea plant

(Yuan, 2008); Azizur et al. (2007) reported that

arsenic toxicity affects the photosynthesis which

ultimately results in the reduction of rice growth

and yield ; Arsenic accumulation in rice inhibited

the metabolism of rice for which he suggested to

develop crop varieties to overcome the arsenic

contamination (Rakesh et al., 2010); Two varieties

of Cicer arietinum L. were tolerant to arsenic

accumulation in the root and shoot and detoxified

arsenic through chelation with GSH (Gupta et al.,

2008).

In an attempt to understand the molecular

basis of stress proteins, produced by the plants

under stress was detected using SDS-PAGE. The

protein was isolated from 30th day old plants.

Comparing the profiles of control and metal treated

rice plants with different concentrations of sodium

arsenate 25, 50 and 100 ppm, induced significant

changes in the patterns of proteins. These results

revealed that the proteins were expressed in specific

regions of rice plants adapted to metal stress (Fig.

1).

Out of 10 rice varieties, a total of 76, 68 and

50 protein bands were observed for 25, 50 and 100

ppm respectively, whereas control produced 86

protein bands with molecular weight ranging from

36.30 to 218.7 KD. In 25 ppm, proteins with

different molecular weight (165.95-27.54 KD) were

observed in each variety. In 50 ppm, protein bands

decreased when compared with control and 25 ppm,

and their molecular weight ranged from 218.77 to

Journal of Research in Agriculture (2011) 1: 011-016 013

Britto et al.,2011

23.98 KD. In 100 ppm, protein bands highly

decreased when compared with all other

concentration and control, and their molecular

weight ranged from 218.77 to 18.11 KD.

Proteins which are up regulated by stress

conditions (stress proteins) have been observed in

response to high and low temperatures, salinity,

droughts and several other stress factors (Pareek et

al., 1997). Large amount of protein observed in low

concentration may be ascribed to the inhibition of

protein synthesis under increasing concentration of

stress treatment. The number of polypeptides is

reduced in each concentration when compared to

control. Similar work was done in arsenic stress-

induced rice leaves in which differentially

expressed proteins were observed by Nagib Ahsan

et al. (2010) A total of 14 protein spots showed

reproducible changes in expression of at least 1.5-

fold when compared to the control and showed a

similar expression pattern in 50m and 100 µM

treatments. In the present study, 68 protein bands in

50 ppm concentration and 50 bands in 100 ppm

concentrations were observed.

The rate of protein biosynthesis shows a

general decline during stress conditions. Despite

overall reduction in protein synthesis activity, it is

interesting to note that cell preferentially synthesize

stress protein. In certain cases stress proteins play a

crucial role in assisting the cell to carry out their

metabolic activities during adverse conditions

(Grover et al., 1993; Viswanathan and Khanna,

1996). The synthesis and accumulation of most of

the polypeptides in arsenic stress, in the present

study suggests major mechanisms that underlie

adaptation or tolerance to arsenic stress. It is

generally assumed that stress induced proteins play

a role in tolerance, but direct evidence is still

lacking and the function of many stress responsive

genes are unknown (Ingram and Bartels, 1996).

Earlier, Mani et al. (2010) identified a

dehydration responsive nuclear protein in rice and

Huang et al. (2009) cloned a Zinc finger protein

gene from rice and designated as SRZ1 (Stress

repressive Zinc finger protein 1). Stress associated

proteins are either synthesized de novo in response

to stress or present constitutively at low level and

their expression increases in response to stress (Arti

and Aruna, 2004)

CONCLUSION

From the present study it may be concluded

that rice plant induced with different concentrations

of arsenic reduced the growth leading to the

reduced productivity. But still IR45 variety was

found to be tolerant to the arsenic stress when

compared to the other varieties chosen. Thus this

variety seemed to have high tolerance towards

arsenic toxicity and may be grown in arsenic

contaminated areas without any major risk of

significant accumulation of arsenic in aerial parts.

ACKNOWLEDGEMENT

The authors are grateful to the Indian

Council of Medical Research, Government of India,

New Delhi (Ref: 59/12/2006/BMS/TRM dt.

26.03.2009) for financial support.

REFERENCES

Arti Chandra and Aruna Tyagi. 2004. Protein

profiles of two rice varieties by 2-D gel

electrophoresis under moisture stress. Ind J

Biochem Biophy., 41:191-194.

Azizur Rahman M, Hasegawa H, Mahfuzur

Rahman M, Nazrul Islam M, Majid Miah MA

and Tasmen A. 2007. Effect of arsenic on

photosynthesis, growth and yield of five widely

cultivated rice (Oryza sativa L.) varieties in

Bangladesh. Chemosphere 67(6):1072-1079.

Flora SJS, Smrati Bhadauria GM, Kannan and

Nutan Singh. 2007. Arsenic induced oxidative

stress and the role of antioxidant supplementation

during chelation: A review. J Environ Biol., 28:333

-347.

Grover A, Pareek A, Singla SL, Minhas D,

Katiyar S, Ghawana S, Dubey H, Agarwal M,

Rao GU, Rathee J and Grover A. 1998. Engineering crops for tolerance against abiotic

stresses through gene manipulation. Curr. Sci.,

75:689-696.

Guadalupe de la Rosa, Jason Parsons G,

Alejandro Martinez-Martinez, Jose Peralta-

Videa R and Jorge Gardea-Torresdey L. 1996. A spectroscopic study of the impact of arsenic

speciation on arsenic/phosphorous uptake and plant

growth in tumbleweed (Salsola kali). Environ Sci

Technol., 40(6):1991-1996.

Gupta DK, Tripathi RD, Mishra S, Srivastava S,

Dwivedi S and Rai UN. 2008. Arsenic accumulation in root and shoot vis-a-vis its effects on growth and level of phytochelatins in seedlings of

Cicer arietinum L. J Environ Biol., 29(3):281-286.

014 Journal of Research in Agriculture (2011) 1: 011-016

Britto et al.,2011

Hert RW. 1991. Research priorities for rice

biotechnology. In: G.S. Khush and G.H.

Toenniessen (eds) Rice Biotechnology, CAB

Intemational, UK. 19-54.

Holleman AF and Wiberg E. 1985. Lehebuch du

Anoranischen chemie. Water de Gruyter, Berlin,

868.

Huang J, Wang, MM, Jian Y, Wang QH, Huang

X and Zhang HS. 2009. Stress repressive

expression of rice SRZ1 and characterization of

plant SRZ gene family. Mol plant 2(2):357-367.

Ingram J, and Bartels D. 1996. The molecular

basis of dehydration tolerance in plants. Annu Rev

Plant Physiol Mol Biol., 47:377-403.

Jian Feng Ma, Naoki Yamaji, Namiki Mitani,

Xiao-Yan Xu, Yu-Hong Su, Steve McGrath P

and Fang-Jie Zhao. 2008. Transporters of arsenite

in rice and their role in arsenic accumulation in rice

grain. P.N.A.S. 105(29):9931-9935.

Laemmli UK. 1970. Cleavage of structural proteins

during the assembly of head of bacteriophage T4.

Nature 227:680-685.

Mani Kant Choudhary, Debarati Basu, Asis

Datta, Niranjan Chakraborty and Subhra

Chakraborty. 2010. Dehydration-responsive

Nuclear Proteome of Rice (Oryza sativa L.)

Illustrates Protein Network, Novel Regulators of

Cellular Adaptation, and Evolutionary Perspective.

Mol Cell Proteomics 9:2019-2033.

Meharg AA. 2004. Arsenic in rice: understanding a

new disaster for South- East Asia. Trends Plant

Sci., 9:415-417.

Mildvan AS. 1970. Metals in enzymes catalysis.

In: The enzymes, Vol. II (Ed: D.D.Boyer). London:

Academic Press 445-536.

Mishra A and Choudhuri MA. 2004.

Amelioration of lead and mercury effects on

germination and rice seedling growth by

antioxidants. Biol Plantarum 41(3):469-473.

Muhammad Shafiq M, Iqbal Zafar and

Mohammad Athar. 2008. Effect of lead and

cadmium on germination and seedling growth of

Leucaena leucocephala. J Appl Sci Environ

Manage 12(2):61-66.

Mukesh K, Raikwar, Puneet Kumar, Manoj

Singh and Anand Singh. 2008. Toxic effect of

heavy metals in livestock health. Veterinary World

1(1):28-30.

Nagib Ahsan, Dong-Gi Lee, Kyung-Hee Kim,

Iftekhar Alam, Sang-Hoon Lee and Ki-Won Lee.

2010. Analysis of arsenic stress-induced

differentially expressed proteins in rice leaves by

two-dimensional gel electrophoresis coupled with

mass spectrometry. Comptes Rendus Biologies

3:224-231.

Pareek A, Singla SL and Grover A. 1997. In

strategies for improvement salt tolerance in higher

plants (Jaiwal PK, Singh RB & Gulati A,eds).

Oxford and IBH, New Delhi. 365-391.

Prasad MNV. 1995. Cadmium toxicity and

tolerance in vascular plants. Environ. Exper. Bot.,

35:525-545.

Rakesh Tuli, Debasis Chakrabarty, Prabodh

Kumar Trivedi and Rudra Deo Tripathi. 2010. Recent advances in arsenic accumulation and

metabolism in rice. Mol breeding 26(2):307-323.

Singh RB. 1993. Research and developmental

strategies for intensification of rice production in

the Asia-Pacific region. In: New Frontiers in Rice

Research (Edited by K. Muralidharan and E. A.

Siddiq), Directorate of Rice Research, Hyderabad.

25-44.

Viswanathan C and Khanna-Chopra R. 1996. Heat shock proteins - Role in thermotolerance of

crop plants. Curr. Sci., 71:275-284.

Yuan zhi shi, Jian-vun Ruan, Li-feng Ma, Wen-

van Han, Fang Wang. 2008. Accumulation and

distribution of arsenic and cadmium by tea plants. J

Zhejiang Univ Sci B., 9(3):265-270.

Zhu YG, Williams PN and Meharg A. 2008. Exposure to inorganic arsenic from rice: a global

health issue. Environ Pollut., 154:169-171.

Journal of Research in Agriculture (2011) 1: 011-016 015

Britto et al.,2011

016 Journal of Research in Agriculture (2011) 1: 011-016

Britto et al.,2011

Sample

Conc. of

Arsenic

(ppm)

Shoot length (cm) Leaf area (cm2) Root

length

(cm)

Fresh

wt (g)

Dry

wt (g) 15th

day

20th

Day

25th

Day

30th

day

15th

day

20th

day

25th

day

30th

day

IR 32 Control 9.2 11 12.3 14 1.2 1.76 2.2 2.4 5.5 1.410 0.638

25 8.5 9.0 9.5 10 1.72 1.68 1.35 0.92 5.5 1.512 0.412

50 8.5 8.8 9 9 1.35 0.97 0.83 0.47 5.1 1.420 0.828

100 9.4 9.5 9.5 9.5 1.12 1.01 0.96 0.48 4.8 1.528 0.292

IR 36 Control 10.3 12 13.2 15 1.17 1.68 1.80 2.45 4.5 1.431 0.670

25 9.5 10.2 10.5 11 1.64 1.72 1.38 0.92 5.3 1.448 0.412

50 9.5 9.7 9.9 10 1.42 1.23 0.55 0.43 4.9 1.397 0.350

100 11.4 11.5 11.6 11.6 1.14 0.78 0.42 0.39 4.7 1.448 0.312

IR 45 Control 13.3 14.5 11.1 18 1.84 1.84 2.5 2.75 4.4 1.528 0.618

25 12.5 14.1 14.5 15 1.38 1.47 1.13 1.06 5.1 1.510 0.432

50 12.6 12.8 13 13 1.47 1.41 1.32 0.98 4.9 1.423 0.398

100 12.4 12.5 12.5 12.6 2.15 1.72 1.32 0.88 4.6 1.391 0.360

IR 46 Control 12.8 14.8 15.4 17 1.84 1.84 2.08 2.35 4.3 1.632 0.712

25 12.5 13.5 13.8 14 1.88 1.88 1.5 1.41 4.9 1.574 0.412

50 13.8 14.9 14.9 15 1.26 1.26 0.88 0.43 4.6 1.549 0.398

100 13.0 13.5 13.5 13.5 1.12 0.90 0.68 0.41 4.7 1.519 0.352

IR 50 Control 10.5 11.2 12.1 13 1.20 1.60 2.25 2.60 4.7 1.502 0.643

25 9.8 10.2 10.5 10.8 1.68 1.54 1.38 0.93 5.3 1.439 0.433

50 10.2 10.5 10.5 10.5 1.54 1.23 0.86 0.48 5.1 1.397 0.339

100 9.0 9.2 9.2 9.2 1.17 1.02 0.78 0.46 4.8 1.358 0.298

ADT32 Control 12.8 13.8 14.3 16 1.30 1.60 1.76 2.30 4.8 1.328 0.518

25 12.4 13.2 13.5 14.5 1.83 1.62 1.47 1.38 5.5 1.382 0.419

50 13.0 13.5 14 14 1.74 1.57 1.44 0.80 5.2 1.405 0.314

100 10.7 10.8 11 11 1.60 1.38 0.92 0.48 4.6 1.492 0.293

ADT 39 Control 10.0 11.1 12 13 1.17 1.56 1.76 1.88 4.7 1.650 0.712

25 9.5 10.0 10.5 11 1.56 1.43 1.38 1.35 5.2 1.682 0.687

50 9.2 9.6 9.9 9.8 1.48 1.14 0.98 0.52 5.1 1.593 0.428

100 8.7 8.9 9 9 1.32 1.09 0.90 0.46 4.4 1.584 0.390

CO45 Control 10.4 11.3 12.1 13 1.64 1.83 2.25 2.60 5.0 1.238 0.512

25 7.5 8.2 8.5 8.5 1.68 1.35 1.26 1.00 5.5 1.259 0.403

50 9.0 9.5 10 10 1.59 1.26 0.94 0.53 5.2 1.205 0.321

100 9.4 9.5 9.5 9.5 1.35 1.17 0.82 0.47 5.1 1.329 0.282

Ambai 16 Control 12.0 13.5 14.5 16 1.29 1.72 2.28 2.52 4.7 1.325 0.502

25 11.3 12.0 12.2 13 1.32 1.21 1.13 0.98 5.1 1.438 0.418

50 10.2 10.4 11.5 11.5 1.17 0.98 0.94 0.57 5.0 1.392 0.321

100 11.4 11.5 11.5 11.6 1.08 0.92 0.72 0.43 4.9 1.486 0.243

Singapore Control 11.6 12.8 13.7 15 1.64 1.92 2.05 2.5 4.8 1.486 0.670

25 11.0 12.0 12.6 13 1.35 1.29 1.11 1.0 4.8 1.592 0.412

50 10.1 10.3 10.3 10.3 1.21 0.96 0.90 0.49 4.9 1.436 0.350

100 9.4 9.5 9.5 9.5 1.17 0.93 0.86 0.43 5.0 1.588 0.292

Table 1. Effect of arsenic stress on shoot length, Leaf area, Root length, Fresh and Dry weight of the ten rice varieties