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
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.plantsciences.info
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:
hkupbl_au@rediffmail.com










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
Journal of Research in Plant Sciences
J
o
u
r
n
a
l

o
f

R
e
s
e
a
r
c
h

i
n

P
l
a
n
t

S
c
i
e
n
c
e
s

An International Scientific Research Journal


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 worlds
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 28C 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 0M , 5M, 10M ,20 M, 50 M,
100 M, 150 M & 200M of CuSO4 for 48h.
From the growth analysis data obtained after 48h
150 M 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 0M-cup was replaced with fresh
Hoagland solution and kept as Control. The other
cups were replaced with 150M 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 2230C.
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 150M CuSO4 for
48h. Although, comparative analysis of growth
Upadhyaya et al.,2011
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 150M) 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 (150m) 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 150M 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
Upadhyaya et al.,2011
041 Journal of Research in Plant Sciences (2011) 1: 038-044
Fig. 2 Phenotypic variation of rice (Oryza sativa L.) cultivars under control (C) and copper (Cu at 150M) stress condition
shoot length by 3.04%,4.26% & 0.69% respectively
in 150M 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
Upadhyaya et al.,2011
Journal of Research in Plant Sciences (2011) 1: 038-044 042
Fig. 3. Changes in root length in ten cultivars of rice
( Oryza sativa L.) under copper stress. Data presented
are meanSE. Error bar superscript with different
letters within the cultivars represents mean
significant differences at p0.05 by T test.
Fig. 4. Changes in shoot length in ten cultivars of rice
( Oryza sativa L.) under copper stress. Data presented
are meanSE. Error bar superscript with different
letters within the cultivars represents mean
significant differences at p0.05 by T test.
Fig. 5. Changes in root dry mass in ten cultivars of
rice ( Oryza sativa L.) under copper stress. Data
presented are meanSE. Error bar superscript with
different letters within the cultivars represents mean
significant differences at p0.05 by T test.
Fig. 6. Changes in shoot dry mass in ten cultivars of
rice ( Oryza sativa L.) under copper stress. Data
presented are meanSE. Error bar superscript with
different letters within the cultivars represents mean
significant differences at p0.05 by T test.


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.
Upadhyaya et al.,2011
043 Journal of Research in Plant Sciences (2011) 1: 038-044
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 meanSE. Mean value superscript with different letters within the
cultivars represents mean significant differences at p0.05 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.

REFERENCES
Azevedo JA and Azevedo RA. 2006. Heavy
metals and oxidative stress: where do we go from
here? Commn Biometry Crop Sci., 1(2):135-138.

Britto R, Mary S R, Sebastian SR and Dharmar
K. 2011. Toxic effect of arsenic on ten rice
varieties. J Res Plant Sci.,1:007-012.

Burkhead JL, Kathryn A. Gogolin Reynolds,
Salah E. Abdel-Ghany, Christopher M. Cohu
and Marinus P. 2009. Copper homeostasis. New
Phytologist., 182:799-816.

Choudhury S and Panda SK. 2005. . Role of
salicylic acid in regulating cadmium induced
oxidative stress in Oryza sativa L roots. Bulg. J.
Plant Physiol., 30(3-4):95-110.

Cobbet CS, Hussain D and Haydon MJ. 2003.
Structural and functional relationships between type
1B heavy metal transporting P-type ATPases in
Arabidopsis. New Phytol,, 159:315-321.

Halliwell B and Gutteridge, JMC. 1984. Free
radicals in biology and medicine. Clarendon Press,
Oxford.ISBN.0-1985-5294-7.

Hansch R and Mendel RR. 2009. Physiological
functions of mineral micronutrients (Cu, Zn, Mn,
Fe, Ni, Mo, B, Cl). Curr Opinion Plant Biol.,
12:259-266.


Lidon FC and Henriques FS. 1991. Limiting step
in photosynthesis of riceplants treated with varying
copper levels. J. Plant Physiol., 138:115-118.

Lidon FC and Henriques FS. 1993. Changes in
the thylakoid membrane polypeptide patterns
triggered by excess Cu in rice. Photosynthetica
28:109-117.

Luna CM, Gonzlez CA and Trippi VS. 1994.
Oxidative damage caused by excess of copper in
oat leaves. Plant Cell Physiol., 35:11-15.

Panda S K. 2008. Impact of copper on reactive
oxygen species, lipid peroxidation and antioxidants
in Lemna minor. Biol Plant., 52(3):561-564.

Quartacci MF, Pinzino C, Sgherri CLM, Dalla
Vecchia F and Navari-Izzo F. 2000. Growth in
excess copper induces changes in the lipid
composition and fluidity of PSII-enriched
membranes in wheat. Physiol. Plant 108:87-93.

Tewari RK, Kumar P and Sharma PN. 2006.
Antioxidant responses to enhanced generation of
superoxide anion radical and hydrogen peroxide in
the copper-stressed mulberry plants. Planta
223:1145-1153.

Zhang H, Xia Y, Wang G and Shen Z. 2008.
Excess copper induces accumulation of hydrogen
peroxide and increases lipid peroxidation and total
activity of copperzinc superoxide dismutase in
roots of Elsholtzia haichowensis. Planta 227:465-
475.
Upadhyaya et al.,2011
Journal of Research in Plant Sciences (2011) 1: 038-044 044
Submit your articles online at www.plantsciences.info

Advantages
Easy online submission
Complete Peer review
Affordable Charges
Quick processing
Extensive indexing
You retain your copyright

submit@plantsciences.info

www.plantsciences.info/Submit.php.