toxic effect of copper on ten rice cultivars

Toxic effect of copper on ten rice cultivars

Keywords: Copper stress, Morphological parameters, growth, Oryza sativa L.

Abbreviations: Cu- copper, ROS - reactive oxygen species.

ABSTRACT: Copper is an essential metal for normal plant growth and development, although it is also potentially toxic. Copper participates in numerous physiological processes and is an essential cofactor for many metalloproteins, however, problems arise when excess copper is present in cells. Excess copper inhibits plant growth and impairs important cellular processes (i.e., photosynthetic electron transport).The mechanisms involved in the acquisition of this essential micronutrient have not been clearly defined although a number of genes have recently been identified which encode potential copper transporters. The present investigation is an attempt to understand of the copper toxicity and tolerance in rice cultivars, and to compare the ten rice cultivars of Karimganj district of Assam. Copper caused growth reduction in rice and among the screened cultivars Amu Sali seems to be a copper sensitive cultivar.

038-044 | JRPS | 2011 | Vol 1 | No 1

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Journal of Research in

Plant Sciences An International Scientific

Research Journal

Authors:

Upadhyaya H,

Bhattacharjee MK,

Deboshree Roy, Soumitra

Shome.

Institution:

Department of Botany and

Biotechnology, Karimganj

College , Karimganj-788710,

Assam, India

Corresponding author:

Upadhyaya H

Email:

[email protected]

Web Address: http://www.plantsciences.info documents/PS0011.pdf.

Dates: Received: 30 Nov 2011 /Accepted: 16 Dec 2011 /Published: 27 Dec 2011

Article Citation: Upadhyaya H, Bhattacharjee MK, Deboshree Roy, Soumitra Shome. Toxic effect of copper on ten rice cultivars. Journal of Research in Plant Sciences (2011) 1: 038-044

Original Research Paper

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INTRODUCTION Heavy metal ions play an essential roles in

many physiological processes. In trace amounts,

several of these ions are required for metabolism,

growth, and development. However,problems arise when cells are confronted with an excess of these

vital ions or with nonnutritional ions that lead to

cellular damage (Tiwari et al., 2006, Zhang et al., 2008,Panda, 2008, Britto et al., 2011). Heavy metal

toxicity comprises inactivation of biomolecules by

either blocking essential functional groups or by displacement of essential metal ions. In addition,

auto-oxidation of redox-active heavy metals and

production of reactive oxygen species (ROS) by the

Fenton reaction causes cellular injury (Cobbett, 2003, Choudhury and Panda, 2005, Azevedo and

Azevedo, 2006). Rice is the most important cereal

crops of developing countries like India and it is the major staple food for majority of world’s

population. Rice productivity has been greatly

affected by various abiotic stresses of which copper toxicity is also important like that of other heavy

metal (Cd, As etc., ) stress (Britto et al., 2011). This

study was conducted to determine the effects of Cu

on various rice cultivars of Karimganj district. Copper (Cu) is an essential element for plants,

being associated with proteins and enzymes

involved in electron transfer and redox reactions. Excess Cu is toxic to plants and affects a wide

range of biochemical and physiological processes,

such as photosynthesis, pigment synthesis, nitrogen

and protein metabolism, membrane integrity, and mineral uptake (Panda, 2008). The toxicity of Cu

can be considered as oxidative stress mediated by

reactive oxygen species (ROS; Luna et al. 1994; Panda 2008). These ROS react with lipids, proteins,

pigments, and nucleic acids, causing lipid

peroxidation, membrane damage and inactivation of enzymes, thus affecting cell viability. Parallel to

metalinduced growth inhibition, increased ROS

accumulation and lipid peroxidation by excess Cu

has been observed in plants ( Tewari et al. 2006; Panda 2008; Britto et al., 2011). In the present

study, Cu is selected to evaluate its effect on growth

responses on varoius rice cultivars. There is a need to investigate the influence of these metal treatment

on growth and physiological responses in crop

plants. The present investigation is an comparative account of growth changes in ten rice cultivars

under copper stress. The observation from the

present study will prove useful in understanding a

possible mechanism of copper toxicity and in rice during seedling development.

MATERIALS AND METHODS

Collection of seeds

Rice (Oryza sativa L.) seeds (about 26

cultivars) were procured from Regional

Agricultural Research Station, Akbarpur, Karimganj, Assam.

Seed viability test

Seed viability was carried out by the floatation method. The seeds obtained from

Regional Agricultural Research Station, Akbarpur,

Karimganj , were put in a beaker of water and allowed to stand for five to ten minutes. Seeds that

sank were considered viable.

Seed germination

Required amount of viable rice seeds of 26 different cultivars were taken and surface sterilized

with 0.1% HgCl2 solution for 3-5 minutes with

successive shaking. After this, HgCl2 solution was thrown, washed thoroughly in tap water for 3-5

minutes, rinsed with distilled water for 2-3 times

and decanted. Then the seeds were placed in petriplates containing moisten filter paper and

germinated at 28°C for three days. On the 3rd day

of incubation germination percentage was measured

for all the 26 cultivars and 10 best germinating cultivars were selected for copper tolerance study.

All the selected ten cultivars seeds were germinated

as mentioned above. The germinated seeds were grown in plastic cups .

Transfer of germinated seeds

After three day of incubation the healthy

germinated seeds with more or less equal height of shoots were transferred in the plastic cups (150ml)

containing half strength Hoagland nutrient medium.

The cups were labeled as per the treatment design and also date of transfer was marked. Then the cups

were put under tube light in growth chamber and

plants were grown for five days. After every two days the medium was changed for healthy growth.

On the 5th day plants were subjected to different

treatments.

Optimization of copper concentration Two best germinating cultivars were

selected for selecting the toxic concentration of

copper sulphate by growing the rice seedlings in a cup containing 0μM , 5μM, 10μM ,20 μM, 50 μM,

100 μM, 150 μM & 200μM of CuSO4 for 48h.

From the growth analysis data obtained after 48h 150 μM CuSO4 was selected for further analyzing

the copper toxicity response in selected 10 rice

cultivars.

Treatment On the 5th day from the day of transfer, the

Upadhyaya et al.,2011

039 Journal of Research in Plant Sciences (2011) 1: 038-044

solution of 0μM-cup was replaced with fresh Hoagland solution and kept as ‘Control’. The other

cups were replaced with 150μM CuSO4 solution.

Each cup contains at least ten plants for each

cultivar. All these cups were kept under 16h/8h light/dark cycle in the growth chamber at 22±30C.

Plants were sampled after 48h of treatments. Each

experiment was repeated three times and data presented are means of three independent repeats.

Germination Percentage

The appearance of the plumule at the filter paper surface was taken as germination.

Germination % was recorded after 72h of

incubation by counting the number of germinating

seeds out of total seed plated (25 numbers).

Root, shoot length and dry mass

After 48hs of treatment growing mung

seedlings at least 10 plants per treatment were sampled and root and shoot length were measured

using centimeter ruler and were separated into root

and shoot and then oven dried at 800C for 48h to estimate the drymass and expressed in g plant-1.

Root and shoot ratio was measured by dividing root

length by shoot length. Total dry mass of plant was

estimated by adding root and shoot dry mass.

Data analysis

Each experiment was repeated thrice with

each treatment sample containing ten individual plants and data presented are with mean ± standard

error (SE). The results were subjected to T test and

used for comparison between pairs of treatments.

The data analysis was carried out using MS excel 2003 and statistical package, SPSS 10.

RESULTS AND DISCUSSION

In the present investigation out of 26 different genotypes of rice (Oryza sativa L.)

procured from Regional Agricultural Research

Station, Akbarpur, Karimganj , Assam, 10 best germinating genotypes were used for copper

toxicity screening.

Morphological responses: Several studies have shown that Cu is an

essential element for plant metabolism and its

uptake by roots and transport to the upper part of

plants is a very rapid process (Burkhead et al., 2009). Although essential for the growth and

development of plant, copper can be toxic at higher

than the optimum concentration required by the plants. The toxic effect of copper may result

alterations in morphological, physiological,

biochemical and molecular responses in plants (Hansch and Mendel, 2009.). Growth inhibition is a

well known response of plants to toxic

concentration of heavy metals in general and copper

in particular. Our results with rice seedlings reveals that the upper part of plants was more sensitive to

toxic copper than the roots. As depicted in Fig. 1 &

2 a very few morphological changes was shown in response to copper stress by the growth analysis of

rice seedlings when subjected to 150µM CuSO4 for

48h. Although, comparative analysis of growth


Journal of Research in Plant Sciences (2011) 1: 038-044 040

Fig. 1. Phenotypic variation of rice (Oryza sativa L.) cultivars under control (C) and copper (Cu at 150µM) stress condition

inhibition by copper in different rice cultivars

showed varied response, morphologically visible

copper toxicity symptoms was evident in Amu Sali.

As depicted in Fig 1., decolorisation of leaves were visible in seedlings of Amu Sali grown for 48h of

copper stress, while the control showed no such

symptoms. Such loss of colour may be due to chlorophyll damage. A lower content of chlorophyll

and alterations of chloroplast structure and

thylakoid membrane composition was found in

leaves under such growth conditions reported elsewhere (Lidon and Henriques, 1991; 1993;

Quartacci et al., 2000). In particular, degradation of

grana stacking and stroma lamellae, increase in the number and size of plastoglobuli, and appearance of

intra-thylakoidal inclusions has also been reported.

It was proposed that Cu interferes with the biosynthesis of the photosynthetic machinery

modifying the pigment and protein composition of

photosynthetic membranes (Lidon and Henriques,

1991). As a consequence of such modifications along with decreased lipid content in thylakoid

membranes, alteration of PSII membrane fluidity

was reported (Quartacci et al., 2000).On the other hand, the decrease of the photochemical activity

caused by Cu is accompanied in vivo by an

alteration of the structure and composition of the thylakoid membranes, which can influence the

conformation and function of the photosystems

( Burkhead et al., 2009). The processes induced by

Cu could involve either the destruction of the oxygen-evolving complex polypeptide composition

or the interaction with ions necessary for proper

functioning of the complex such as Mn, Ca and Cl.

It is well known that transition metals like Cu

catalyze the formation of hydroxyl radicals(OH) from the non-enzymatic chemical reaction between

superoxide (O2-) and H2O2(Haber-Weiss reaction :

Halliwell and Gutteridge, 1984).

Copper induced changes in growth of rice

cultivars

The effect of copper in the growth of

growing seedlings of rice was significant. In the present experiment variation of copper tolerance

was tested among ten commonly growing rice

cultivars of Karimganj district of Assam. An exposure to Cu (150µm) caused significant changes

in root and shoot length in various tested cultivars

of rice (Fig. 3 & 4). There was a significant decrease in root length in all the tested cultivars

except the three cultivars [viz., Gagli Boro , Bishnu

Jyoti & TTB-176-12-3-1 ]. The root length

decreased by over 20.11, 1.71, 16.04, 3.20, 17.05, 34.55, 14.33, 2.43 % in Amusali, Gagli Boro,

Madahav Boro, Jum Kharang, Basmati T3, Agni

Sali, Boro-68, Kanaklata, & TTB-176-12-3-1 respectively in response to 150µM Cu. It clearly

showed that maximum decrease in root length was

observed in Boro 68 followed by AmuSali, Agni Sali , Basmati T3 , Kanaklata etc (Fig.3). In

contrast the changes in shoot length were

comparatively lesser in all the tested cultivars.

However, the cultivars like Gagili Boro, and Madhav Boro & Bishnu Jyoti showed increased



Fig. 2 Phenotypic variation of rice (Oryza sativa L.) cultivars under control (C) and copper (Cu at 150µM) stress condition

shoot length by 3.04%,4.26% & 0.69% respectively

in 150µM Cu treated plant relative to control plants. Copper induced reduction in shoot elongation was

evident in Amu Sali, Jum Kharang, Basmati T3,

Agni Sali, Boro-68, Kanaklata, & TTB-176-12-3-1 by 25.47, 8.55, 7.84, 31.71, 1.32, 6.49, & 12.26%

respectively when compared with control (Fig.4).

This was also evident from declining root-shoot

elongation ratio upon Cu treatment (as depicted in Table 1).Further as compared to control root dry

mass showed inconsistent results. Maximum

decrease in root dry mass was observed in Amu Sali and Agni Sali by 26.34 and 11.11 % respectively

(Fig. 5). The changes in shoot dry mass due to Cu

treatment also varies among rice cultivars. The

shoot dry mass decreased in Amu Sali, Gagili Boro, Madhav Boro, Jum Kharang, Basmati T3, Agni

Sali, Boro-68& TTB-176-12-3-1 by 14.63,

3.704,4.73, 17.98, 16.35, 34.81, 0.98, & 1.66% respectively , where as there was increase in shoot

dry mass in Bishnu Jyoti, & Kanaklata by over

14.28, & 9.19% respectively (Fig. 6). The changes in total biomass of rice due to Cu treatment also

varied among the tested cultivars (Table1). Some of

the cultivars showed little increase in total dry mass due to Cu treatment relative to control plants. Cu

induced increase in total dry mass was observed in

Gagili Boro, Bishnu Jyoti , Boro-68 , Kanaklata &

TTB-176-12-3-1 by ,0.84, 13.22, 0.78, 7.79 & 1.58 % respectively where as total dry mass decreased in

AmuSali, Madhav Boro, Jum Kharang, Basmati T3

& Agni Sali by 18.74, 4.6, 1.04, 10.1 &28.52 % respectively. The shoot root dry mass ratio was

increased in Amu Sali by15.89% where as it is

decreased in Gagili Boro, Madhav Boro, Jum

Kharang, Basmati T3, Agni Sali, Boro-68 & TTB-176-12-3-1 by over 12.87, 0.19, 42.80, 21.65,

26.64,7.81, & 10.59 % respectively.

Copper toxicity thresholds vary greatly



Fig. 3. Changes in root length in ten cultivars of rice

( Oryza sativa L.) under copper stress. Data presented

are mean±SE. Error bar superscript with different

letters within the cultivars represents mean

significant differences at p≤0.05 by T test.

Fig. 4. Changes in shoot length in ten cultivars of rice

( Oryza sativa L.) under copper stress. Data presented

are mean±SE. Error bar superscript with different

letters within the cultivars represents mean


Fig. 5. Changes in root dry mass in ten cultivars of

rice ( Oryza sativa L.) under copper stress. Data

presented are mean±SE. Error bar superscript with

different letters within the cultivars represents mean


Fig. 6. Changes in shoot dry mass in ten cultivars of

rice ( Oryza sativa L.) under copper stress. Data

presented are mean±SE. Error bar superscript with

different letters within the cultivars represents mean


between species of plants and affect tissues

differently depending on metabolic requirements.

Excess Cu concentrations in the medium tend to decrease root growth before shoot growth because

of preferential Cu accumulation in that organ as

was observed in the present work. The growth reduction in rice due to copper toxicity might be

attributed to its role in ROS generation in plants.

Copper has a particularly high affinity to dioxygen molecules that explains why copper is the catalytic

metal in many oxidases. The most prominent

member of this group is mitochondrial cytochrome

c oxidase as principal catalyst of the terminal oxidation. Copper is also found in electron carrier

proteins like plastocyanin that accounts for

about50% of the plastidic copper (Hansch and Mendel, 2009). More than half of the copper in

plants is found in chloroplasts and participates in

photosynthetic reactions. Hence, copper deficiency becomes first visible in young leaves and

reproductive organs, later consequences are stunted

growth of the whole plant and pale green leaves that

wither easily. Interestingly, copper metabolism is intimately linked to iron metabolism. Depending on

the bioavailability of copper and iron, plants

possess enzymes for the alternative use of copper

versus iron thus catalyzing the same biochemical reaction with completely different apoproteins , a

process that involves regulation by mRNAs

( Burkhead et al., 2009). Examples include Cu-nitrite versus heme-nitrite reductase, Cu/Zn-

superoxide dismutase versus Fe-superoxide

dismutase, and cytochrome oxidase versus diiron oxidase. The present study reveals that copper

tolerance varied among the rice cultivars can be

arranged in increasing order of toxicity as Amu Sali

>Agni Sali>Basmati-T3>Madhav Boro >Jum Kharang> Gagili Boro > Boro-68 >TTB-176-12-3-

1>Kanaklata>Bishnu Jyoti.

CONCLUSION

Hence it may be concluded, from the present

study that rice plant induced with toxic concentrations of copper reduced the growth

leading to the reduced productivity. But still

Konaklata and Bishnu Jyoti variety was found to be

tolerant while Amuli sali is sensitive to the copper stress when compared to the other varieties tested.



Table 1. Changes in total dry mass, shoot/root elongation and biomass ratio in ten cultivars of rice (Oryza sativa

L.) under copper stress. Data presented are mean±SE. Mean value superscript with different letters within the

cultivars represents mean significant differences at p≤0.05 by T test.

Thus this variety seemed to have low tolerance towards copper toxicity and may not be suitable to

grow in copper contaminated areas. Konaklata and

Bishnu Jyoti may be considered to have

comparatively higher potential for copper toxicity tolerance which can be further studied to analyze

copper transporter and other copper responsive

genes in rice.

ACKNOWLEDGEMENT

The authors thankfully acknowledge DBT, Government of India, New Delhi for financial

support for institutional Biotech Hub at Karimganj

College. Karimganj-788710, Assam India. Authors

are also grateful to the Chief Scientist, Regional Agricultural Research Station, Akbarpur,

Karimganj , Assam, for providing rice seeds.

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