Effect of Titanium Dioxide Nanoparticles on Hydrolytic and Antioxidant Enzymes during Seed Germination in Onion

Abstract

Surface sterilized onion seeds were germinated (25 seed per plate) with graded
concentrations (00, 10, 20, 30, 40 and 50 g mL-1) of TiO2 nanoparticles (NPs) in
petriplates lined with Whatman no. 1 filter paper. Similar experiment without
nanoparticles was conducted as control. TiO2 NPs at lower concentration enhanced
seed germination and seedlings growth in onion and however the germination and
growth showed inhibition at higher concentrations. The TiO2 NPs also induced
significant changes in activities of hydrolytic and antioxidant enzymes. Activities
of amylase and protease were enhanced in lower concentration, but showed
decrease at higher concentrations. The activity SOD showed concentration
dependent increase; however CAT and POD were found to be enhanced in the
presences of 10-30 μgml-1 NPs but showed decreased activities in 40 and 50 μgml-1
NPs concentrations.

Full text

Int.J.Curr.Microbiol.App.Sci
(201
4) 3(7)
749
-
760
749
Original Research Article
Effect of Titanium Dioxide Nanoparticles on Hydrolytic and Antioxidant
Enzymes during Seed Germination in Onion
S.
L.
Laware
*
and Shilpa Raskar
P.G. Department of Botany, Fergusson College, Pune
-
411004, University of Pun
e (MS), India
*Corresponding author
A B S T R A C T
Introduction
Increasing production and use of nano-sized
materials have raised concerns about their
possible impacts on environmental and
human health (Hood, 2004).
These
nanomaterials (NMs) are being produced,
sold and commercially used for various
purposes and even in some food products.
As NMs are being used on large scale they
might
have entered in ecosystems.
Such
NMs on entering in ecosystem might have
affected or effected seed germination
process, plant growth and metabolism.
Hence,
plants should be tested to establish
their response to nanomaterial stress
and
possible role of hydrolytic and antioxidant
enzymes to NMs during seed germi
nation
and early seedling growth.
Seeds enfold sufficient quantity of food
reserves which support the seed germination
and early seedling growth (
Zeeman
et al.,
2004). Germinating seeds generally exhibit
high amylase and protease activities. This is
be
cause these enzymes are synthesized
during seed germination to mobilize stored
food for the survival of the young plant until
it is capable of making its food by
photosynthesis (Schramm and Loyter,
1966).
Seed germination
is
regarded as a
series of steps which normally occur prior to
the emergence of the radicle from the seed
coat
(Mayerand and Shain, 1974).
The
water
intake
rate and seed reserve utilization are
important physiological and biochemical
processes
associated with
seed
germination.
ISSN: 2319
-7706
Volume
3
Number
7
(201
4
) pp.
749
-
760
http://
www.ijcmas.com
Keywords
TiO
2
nanoparticles,
amylase,
protease, SOD,
CAT, POD
Surface sterilized onion seeds were germinated (25 seed per plate) with graded
concentrations (00, 10, 20, 30, 40 and 50 g mL
-1
) of TiO
2
nanoparticles (NPs) in
petriplates lined with Whatman no. 1 filter paper. Similar experiment without
nanoparticles was conducted as control.
TiO
2
NPs at lower concentration enhanced
seed germination and seedlings growth in onion and however the germination and
growth showed inhibition at higher concentrations. The TiO
2
NPs also induced
significant changes in activities of hydrolytic and antioxidant enzymes. Activities
of amylase and protease were enhanced in lower concentration, but showed
decrease at higher concentrations. The activity SOD showed concentration
dependent increase; however CAT and POD were found to be enhanced in the
presences of 10
-
30
µgml
-1
NPs but showed decreased activities in 40 and 50
µgml
-1
NPs concentrations.

Int.J.Curr.Microbiol.App.Sci
(201
4) 3(7)
749
-
760
750
G
erminating
seeds, on imbibitions, consume
more oxygen for
activation
or hydration of
mitochondrial
enzymes, involved in the
Krebs cycle and electron transport chain
(Salisbury and Ross, 1991). Furthermore,
enzymes, like lipases, proteinases,
phosphatise and hydrolases (Bewley and
Black, 1985; Coccuni and Negrini, 1991;
Washio and Ishikawa, 1992; Bernier and
Ballance, 1993; Bernhardt et al., 1993) are
either activated or synthesized to avail
simpler substances for embryo growth.
These simpler substances produced from
stored food by the enzymatic actions are
carried from the endosperm or cotyledons to
the embryonic axis and are utilized in the
synthesis of new required material (Davies
and Slack, 1981; Mayer and Poljakoff
Mayber, 1989). Conversion of starch to
glucose i
s carried out by amylases (Schelgel,
2003); whereas proteases convert proteins
into amino acids. The enzyme most
frequently credited with the initial attack on
starch granules is -amylase, which initiates
starch mobilization in germinating seeds
(Trethewe
y and Smith, 2000; Fincher,
1989).
Reactive oxygen species generation (ROS)
and oxidative stress are proposed to explain
the toxicity of nanoparticles
(Nel
et al
.,
2006).
Plants are generally protected against
this oxidative stresses by a wide range of
radical scavenging systems such as
antioxidative enzymes like superoxide
dismutase (SOD), peroxidase (POD),
ascorbate peroxidase (APX), and catalase
(CAT), as well as non-
enzymatic
compounds like carotenoids (Cameron and
Reid, 2001; Zimmermann and Zentgraf,
2005).
Plants have special mechanisms to remove
or inactivate reactive oxygen species (ROS)
such as H
2
O
2
, OH
-
, and O
2
-
radicals that are
by products of naturally occurring reactions.
However, excess ROS can result in protein
breakdown,
lipid peroxidation
in membranes
and DNA injury (Choudhury and Panda,
2004). Previous studies have shown that
heavy metals increases the activity of the
antioxidant enzymes like catalase (CAT)
and ascorbate peroxidase (APX) in plants
(
Lopez
et al
., 2007
).
It has been proven that oxidative stress
represents a common mechanism for cell
damage induced by NPs (Pulskamp et al.,
2007), and the mechanism has been
validated in many NPs toxicity studies
(Yang
et al
.,
2009
).
Upon entering the cell,
particles may induce intracellular oxidative
stress by disturbing the balance between
oxidant and anti-oxidant processes.
Excessive oxidative stress may also modify
proteins, lipids, and nucleic acids, which
further stimulates the anti-oxidant defence
system or even leads to cell death.
Me
anwhile, with increased ROS production,
NPs can cause DNA damage and increase
gene expression of the death receptor
(Yang
et al., 2009). In addition, increased ROS
induced by NPs in lysosomes can cause
DNA point mutations or induce single or
double strand breaks (Singh et al
.,
2009).
Raskar and Laware (2014) observed various
chromosomal aberrations in onion root tip
cells treated with ZnO NPs. They ascribed
these to DNA lesions and chromosome
breakage.
Another major oxidative stress
response is intracellular Ca
2
+
release, which
leads to mitochondrial perturbation and cell
death (Xia et al., 2008). However, there is
lack of information on the effects of NPs
in
onion with respect to hydrolytic and
antioxidant enzymes during seed
germination and early seedling
growth.
Materials and Methods
Titanium dioxide (TiO
2
) P25 powder of
particle size 21 nm and purity > 99.5%
was obtained from the researchers in the

Int.J.Curr.Microbiol.App.Sci
(201
4) 3(7)
749
-
760
751
field of nanomaterial synthesis. These were
made of 80% anatase and 20% rutile. Seed
of local onion variety was procured from
NRC (National Research centre for onion
and garlic), Rajguruna
gar
(MS).
The graded concentrations (00, 10, 20, 30,
40 and 50 g mL
-1
) of TiO
2
nanoparticles
prepared in distilled water were added
aseptically to sterilized petriplates lined with
Whatman no. 1 filter paper. Surface
sterilized seed of onion were germinated (25
seed per plate) in each concentration of
nanoparticles. Similar experiment without
nanoparticles was conducted as control.
Seed germination and seedling growth in
terms of root length and shoot length was
determined
after 10 days of treatment. Th
e
crude enzyme extract was prepared from 7
day old seeding.
Extraction and assay of enzyme: T
reated
and control seedlings were collected and cut
into small pieces. Accurately 1
gm
o
f
sample
was homogenized
and extracted with
3ml of 0.1M phosphate buffer (pH 7.0) in
pre chilled mortar and pestl
e.
The extract
was centrifuged at 4
0
C in cooling centrifuge
at 15000 xg for 10 minutes and supernatant
was used as sources of enzyme
s.
Amylase
( -amylase EC. 3.2.1.1): The
Amylase activity was calculated by using
Jayaraman, (1981) method. Treated and
control seedlings were collected and cut into
small pieces. Accurately 1.0
gm
o
f
seedling
sample
was
hom
ogenized in pre chilled
mortar with pestle, in 5 ml of 0.1 M s
odium
acetate buffer (pH4.8) supplemented with
(10
mM
NaCl). The extract was centrifuged
at 4
0
C in cooling centrifuge at 15000xg for
10 minutes and supernatant was used as
sources of crude enzyme. Enzyme reaction
mixture contained 2.0 ml of 0.1M s
odium
acetate buffer (pH 4.7), 0.5ml of 1% s
tarch
and 0.5ml
en
zyme
extract
in total 3 ml
volume.
The reaction was initiated by
adding 0.5 ml of enzyme extract and
reaction mixture was incubated for 10
minutes at 37
O
C temperatures in water bath.
After 10 minutes of incubation 2ml of
Dinitrocalysilic acid (
DNS
) was
ad
ded and
reactions were heated in water bath at 100
o
C for 10 minutes. Same procedure was
followed for control but enzyme reaction
was terminated at zero minutes by adding
DNS
reagent. After heating reaction mixture
was diluted with distilled water to 10 ml and
OD was taken at 510
nm.
One unit of
amylase activity was defined as the amount
required for liberating 1 mg of maltose in 1
min at 37
o
C. The enzyme activity was
expressed as units g
-1
fresh tissue.
Protease:
The Protease activity was
calculated by using Issac and Gokhale
(1982)
method. Accurately 1.0
gm
o
f
seedling
sample
was homogenized in pre
chilled mortar
with
pestle, in 5ml of 0.1 M
phosphate buffer (pH 7.
4)
. The extract was
centrifuged at 4
o
C in cooling centrifuge at
15000xg for 10 minutes an
d supernatant was
use
d as sources of crude enzyme. Enzyme
reaction mixture contained 2.0 ml of 0.1M
(pH 7.5) phosphate buffer, 0.5ml casein and
0.5ml
plant extract in total 3 ml of volume.
The reaction was initiated by adding 0.5 ml
of enzyme extract and incubated for 10
minutes at 40
o
C temperatures in water bath.
The enzyme activity was stopped by adding
3ml of 5% H
2
SO
4
solution. The proteins
p
recipitat
ed in reaction mixture after 60
minutes of resting were separated by
centrifugation at 10,000 xg for 10
min
utes.
Exactly 2ml of supernatant was mixed
3ml
of
2%
Na
2
CO
3
and 1ml of folin phenol
reagent
.
The blue colour developed was read
at
660nm.
Similar procedure was followed
for control but enzyme reaction was
terminated at zero minutes by adding 3ml of
5%
H
2
SO
4
solution.
The protease activity
was measured by estimating the release of

Int.J.Curr.Microbiol.App.Sci
(201
4) 3(7)
749
-
760
752
tyrosine calculated from the standard curve
prepared with tyrosine. One unit of protease
activity was defined as the amount required
for liberating 1 mg of tyrosine in
1
min at
40
o
C.
The
protease
activity was
expressed as
units g
-1
fresh tissue.
Catalase
(CAT, EC. 1.11.1.6)
:
The CAT
activity was calculated by using
Maxwell
and Bateman (1967) method. The reaction
mixture contain
ed
2.9 ml of 0.06M
phosphate buffer, 10 mM H
2
O
2
and 0.1 ml
enzyme extract in final volume of 3 ml. The
absorbance of the reaction concoction was
measured at 240 nm compared to the
reaction mixture without an enzyme extract.
The reaction was initiated by adding 0.5 ml
of enzyme extract. The decrease in
absorbance was measured at 240 nm by
using UV visible spectrophotometer
(Shimadzu
-1700). The residual H
2
O
2
concentration was calculated using
extinction coefficient 0.036 µmole
-1
ml
-1
.
One unit of CAT activity (U g
-1
FW) was
defined as the amount of CAT, w
hich
decomposed 1 mol/L hydrogen peroxide in
1 min. The enzyme activity was expressed
as units g
-1
fresh weight.
Peroxidase
:
(POD,
EC
. 1.11.1.7) The POD
activity was assayed by Vidyasekharan and
Durairaj
(1973) method. The assay mixture
of 3 ml contained 1.7ml of 0.1M phosphate
buffer (pH 7.0), 1ml freshly prepare 10
mM
Guaiacol, 0.1 ml enzyme extract and 0.1 o
f
12.3 mM H
2
O
2
. Initial optical density was
read at 436 nm and then increase in optical
density was noted at the interval of 30
seconds on UV- visible spectrophotometer
(Shimadzu
-1700). By using the extinction
coefficient of guaiacol
dehydrogenase
product at 436 nm (i.e.6.36µmole
-1
ml
-
), the
enzyme activity was calculated as units g
-1
fresh Weight.
Superoxide Dismutase (
SOD
,
EC1.15.1.1
):
Superoxide Dismutase (SOD), a metal
containing enzyme plays a vital role in
scavenging superoxide radical. Superoxide
dismutase activity was calculated by using
Madamanchi
et al
(1994)
. Crude enzyme
extract 100µl was mixed with a 3ml reaction
cocktail: 50Mm potassium phosphate buffer
(pH7.8), 13mM methionine, 2µM riboflavin,
0.1mm
Ethylene
diamine
tetraacetic acid
(
EDTA
) and 75µM nitroblue tetrazolium
(
NBT
). Final volume of reaction mixture
was made equal by adding distilled water. A
blank was set without enzyme and NBT
to
calibrate the spectrophotometer and another
control was set with NBT but without
enzyme as reference control. The reaction
tubes were exposed to 400W bulbs (4x100
W bulbs) for 15 minutes and immediately
absorbance was taken at 560nm. The percent
inhibit
ion was calculated. The 50%
inhibition of the reaction between riboflavin
and NBT in the presences of mithionine is
taken as 1 unit of SOD activity and the
enzyme activity is expressed as units g-
¹
fresh tissue.
Results and Discussion
Effect of
different
concentrations of
TiO
2
NPs
on activity of hydrolytic enzymes like
amylase and protease as well as antioxidant
enzymes like SOD, CAT and POD was
evaluated
during seed germination in
Allium
cepa
and results are depicted in
Table
2 and
3.
Data pertaining to effect of TiO
2
NPs
treatment
on amylase activity indicate that
amylase
activity
was increased up to 40 µg
ml
-1
treatments and then decreased
over
control at 50 µgml
-
1
treatment. Maximum
increase in amylase (30.65%) was recorded
at
20 µg ml
-1
of
TiO
2
NPs
treatment.
Data
with respect to effect of TiO
2
NP treatment
s
on protease activity indicate that protease
activity
was increased up to 40 µg ml
-1

Int.J.Curr.Microbiol.App.Sci
(201
4) 3(7)
749
-
760
753
treatments and then decreased significantly
over control at 50 µgml
-
1
treatment.
Maximum increase in
protease
(11.68
%)
was recorded at 40 µg ml
-1
of
TiO
2
NPs
treatment.
The activit
ies
of antioxidant enzymes
like,
SOD, CAT and POD analysed from onion
seedlings after TiO
2
treatment clearly
indicate that activity of SOD increased with
increasing NPs co
ncentration.
Activity of
CAT increased up to 30 µgml
-
1
concentrations of TiO
2
NPs and then
decreased significantly in seedlings treated
with 40 and 50 µgml
-
1
TiO
2
NPs. Highest
increase i.e. 30.88% over control was
observed in seedlings treated with 20 µg ml
-
1
of TiO
2
NPs and then decreased in higher
concentrations. Significant increase in POD
activity was observed up to 30 µg
ml
-
1
concentrations of TiO
2
NPs and maximum
increase i.e. 10.
74%
over control was
recorded in 30 µg ml
-1
treatments of
TiO
2
NPs. At higher concentration POD activity
showed significant decrease over control.
The activit
ies
of key hydrolytic enzymes
like
amylase and protease in seedlings of
Allium
cepa that were grown in the presence
of
TiO
2
NPs were analyzed in present
investigation.
T
he results on e
nzyme activity
revealed that NPs at their lower
concentrations promoted enzyme activities,
however at higher concentrations inhibit
ed
activit
ies
of hydrolytic
enzym
es.
The increased activity of -amylase during
seed germination is probably due to the
de
novo
synthesis (Filner and Varner, 1967) of
this enzyme. According to
Wang
et al
(1988)
amylase activity increase
s
gradually
during initial days of germination and
conver
t
starch
to
soluble sugars needed for
growth of embryo axis. According to Tully
and Beevers (1978) proteins are hydrolysed
to
free amino acids, which support protein
synthesis in endosperm and embryo
.
Marambe
et al (1992) noted a high
significant correlation between the -
amylase activities; seed water uptake and
subs
equent percent seed germination as well
as linear correlation of amylase activity with
the starch degradation and increase in
sugar
content of the treated
sorghum
seeds
.
It was observed that in germinating b
eans,
the
proteolytic activity increases during
initial
7 days of germination and t
his
increase was partially dependent on the
embryonic axis (Gepstin and Han, 1980).
The combined action of various proteolytic
enzymes thus results in total degradation of
s
torage
proteins (Ikuko and Hiroshi, l980) to
free amino acids needed for protein
synthesis in embryonic axis. According to
Baron
(1979)
s
olutes
produced in seeds as a
result of
hydrolytic
enzyme activities during
the initial phases of germination
promote
the
water movement into the seeds that
contribute
to the seed osmotic potential. On
the other hand increased amino acid content
in
hypocotyls
and expanding leaves
significantly contributes to the water uptake
by tissues
(
Morgan,
1984)
.
Findings of
Navarro
et al (2008) indicated
that NPs can slowly penetrate into seeds and
affect their metabolism in vivo
.
Khodakovskaya
et al (2009) demonstrated
that
multiwalled carbon nanotubes
(MWCNTs)
can
penetrate through the coats
of tomato seeds after several days of co-
incubation
. It was further stated that once
nano
sized holes are created in the seed coat,
oxygen transfer and water uptake might
occur and drive the metabolic process for
plant growth. However, NPs in large
quantity are often observed in the
agglomerate
form and their interactions with
seeds are weak so uptake of water
/
oxygen is
limited and such conditions can
decrease
seed germination.

Int.J.Curr.Microbiol.App.Sci
(201
4) 3(7)
749
-
760
754
In the present study it was observed that
TiO
2
NPs treatment at the concentrations i.e.
10 to 40 µg ml
-1
in onion seed showed
enhanced activities of amylase and protease
and then there was decrease in activities of
these enzymes at higher concentration of
NPs.
The increase in seed germination and
better establishment of seedlings due to
lower concentrations of TiO
2
NPs might
be due to accelerated water and oxygen
uptake. Sufficient quantity of water and
oxygen in imbibed onion seed might have
accelerated the amylase and protease
activities and produced soluble sugars and
amino acids required for early se
ed
germination and seedling growth. However
on other hand higher concentrations of NPs
might have resulted in
agglomerat
ion of
particles which might have induced
moisture stress and
weak
ened
uptake of
water
and oxygen and hence decreased seed
g
ermination
(Raskar and Laware, 2013).
Antioxidant enzymes are known for ROS
scavenging and are more active in the
presence of biotic or abiotic stresses; hence,
in order to determine the probable
mechanism of toxicity of
TiO
2
NPs,
activities
of
antioxidant enzymes were
assessed in 7 day old onion seedlings. From
the results on enzyme activities it is clear
that the production of ROS due to NPs is
responsible for inducing the antioxidant
enzymes.
The activit
y
of key antioxidant enzyme i.e.
SOD
in Allium c
epa
seeds treated with NPs
and seedling grown in the presence of NPs
showed increase with increase
in
concentration of NPs.
Superoxide
dismutase
are
responsible to catalyze the
dismutation
of
superoxide
(O
2
)e. Hence,
SOD enzymes are considered as
important
antioxidant defence in nearly all
cells exposed to oxygen.
In a study on
wheat
seedlings treated with biogenic silver NPs a
significant increase in SOD activity was
reported by
Himakshi
et al (
2013).
Simi
larly,
Wang
et al
(2011)
observed
decrease in salt stress due to application of
silicon NPs in alfalfa plant and they
attributed
this decreased salt stress
to
elevated activities of SOD, POD and CAT
.
In other study on s
oybean
seed germination,
it was found that the seeds treated
with
a
mixture of Nano SiO
2
and Nano
TiO
2
exhibited
more germination and
higher
activit
ies
of nitrate reductase, superoxide
dismutase, catalase and peroxidase (
Lu
et
al.,
2002
).
Authors
concluded that
SiO
2
and
TiO
2
NPs
would be better for seed
germination and early seedling growth
in
soybean.
A report on SiO2
NPs
toxicity in
Arabidopsis thaliana clearly indicated that
SiO
2
NPs are less toxic than ZnO and Fe
3
O
4
NPs
(Lee
et al.,
2010).
Likewise
Lin
et al
(2009
) subjected
Arabidopsis
cells to
multiwalled carbon nanotubes (MWCNTs)
and reported significant decrease in the
superoxide dismutase activity (SOD) in
Arabidopsis
cells exposed to MWCNTs as
compared to control. However, Tan et al
(2009) observed significant time dependent
inducti
on in enzymes in rice cells treated
with MWCNTs at the concentration of 20
mgl
-1
.
Mohamed
et al (2014) studied effect
of nanoparticles on biological contamination
of
in vitro cultures of banana and reported
that NPs cause an oxidation-
reduction
reaction and produced superoxide ion
radical and hydroxide. According to them
these reactive oxygen species (ROS) can be
effective antimicrobial agents. Similar
results were reported by Helaly and Hanan
El
-
Hoseiny
(2011) on stressed in vitro
cultures of sweet orange. They observed
higher superoxide dismutase (SOD),
peroxidase (POX), ascorbic peroxidase
(A
PX
), Catalase (CAT) and glutathione
reductase (GR) activit
ies
.

Int.J.Curr.Microbiol.App.Sci
(201
4) 3(7)
749
-
760
755
In another study on rice, Tang and Cao
(2003
) confirmed
the
increased yield of rice
due to nano SiO
2
treatment
s; according to
them
such
increase in yield could be due to
increased strength and resistance to disease.
Toxicity of nanostructures such as flower
like ZnO capped with starch, spherical
uncapped ZnO and spherical CdS on aquatic
plant
Hydrilla verticillata was examined by
Mishra et al (2013). According to them
spherical CdS nanoparticles were more toxic
than the corresponding
ZnO
nanoparticles
since there was a decrease in
chlorophyll
content and increase in catalase
activity
.
The results with respect to shoot and
ra
dicle
(root)
growth depicted in table-1 clearly
indicate that root growth was significantly
affected due to TiO
2
NPs at higher
treatments as compared to shoots. It might
be due to the fact that after emergence;
growing radicles came in direct contact with
NPs and subjected to more ROS stress
exerted by NPs. This can be inferred by
scrutinizing increased values of SOD and
decrease values of CAT and POD in onion
.
It
has been identified that the increase in
SOD activity and generation of more H
2
O
2
are the indicators of damage to the root
growth
(Dong
et al
., 2002
).
It is well documented that
antioxidant
enzymes like catalases and peroxidases are
known to scavenge H
2
O
2
by breaking it
down into water and oxygen. The excessive
levels of H
2
O
2
are reported to be reduced
through the activities of catalase, POD and
APX
. Results on activities of CAT and
POD
enzymes responsible for elimination of H
2
O
2
are given
in table
-3.
It is clear from data the increase in CAT
activity due to NPs treatment at lower
concentrations was more as compared POD
activity. Higher activity of CAT in onion
seedlings treated with 10-30 µg
ml
-1
can be
attributed to generation of more H
2
O
2
and
the higher activity of SOD in respective
treatments
(
Fridovich,
1983
;
Bowler
et al
.,
1992
).
Himakshi
et al (2013) studied the
effect of biogenic silver NPs
in
wheat
seedling
and noted significant enhance
ment
in
activities of
CAT
and POD. They
ascribed these increased activities of
catalase and peroxidase to marked increase
in H
2
O
2
through enhanced activity of SOD
with
biogenic silver NPs
.
Comparatively low
er
increments in the
activity of POD in onion seedlings treated
with
TiO
2
NPs at lower concentrations can
be accredited to the remarkable activity of
CAT as a key enzyme for eliminating H
2
O
2
,
thereby regulating the activity of Peroxidase
.
Riahi
-
Madvar
et al (2012)
treated
wheat
seeds
with
nano
scale alumina and studied
the morphological characters of seedling
along with antioxidant enzymes. They
made
similar observations with respect to SO
D
and reported that excessive levels of H
2
O
2
might be reduced through the activities of
catalase and
APX.
Based on results of TiO
2
NP
s
effect on
onion seed germination and early seedling
growth it could be stated that NPs
might
have
helped the water absorption by the
seeds
(Zheng
et al., 2005), increased
abilities
of seed
for
absorbing and utilizing
water
efficiently, activated and promoted
hydrolytic enzymes seed antioxidant system
(Lu et al
., 2002).
The NPs might have reduced
ROS
stress
in
treated onion seedling by reducing H
2
O
2
,
superoxide radicals, and lipid peroxidation
products (
malonyldial
dehyde content) and
increasing activities of
superoxide
dismutase, ascorbate peroxidase, guaiacol
peroxidase, and catalase
enzymes
as
observed in spinach
(Lei
et al
., 2008).

Int.J.Curr.Microbiol.App.Sci
(201
4) 3(7)
749
-
760
756
Table
.1
Effect of TiO
2
on seed germination and early seedling growth in
Allium ce
pa
TiO
2
NPs
(
g ml
-1
)
% Seed
germinat
ion
PIOC/
PDOC
Radicle
Length
(cm)
PIOC/
PDOC
shoot
length
(cm)
PIOC/
PDOC
Radicle:
shoot
Ratio
Control
94.68 0.00 4.52 0.00 7.62 0.00 0.59
10
g ml
-1
95.25
0.60
4.58 1.33
7.84 2.89 0.58
20
g ml
-1
96.32
1.73
4.62 2.21
8.12 6.56 0.57
30
g ml
-1
97.68
3.17
4.56 0.88
8.38 9.97 0.54
40
g ml
-1
98.12
3.63
4.48 -0.88
8.46 11.02 0.53
50
g ml
-1
92.02
-2.81
4.18 -7.52
8.16 7.09 0.51
CD 5%
1.72 0.16 0.22 0.26
CD= critical difference; PIOC= percent increase over control; PDOC= percent decrease over control
Table
2
Effect of TiO
2
on hydrolytic enzymes during seed germination in
Allium cepa
TiO
2
NPs
(
g ml
-1
)
Amylase
(U g
-1
FW)
PIOC/PDOC
Pr
otease
(U g
-1
FW)
PIOC/PDOC
Control
42.28 0.00
42.81
0.00
10
g ml
-1
48.56 14.85
42.96
0.35
20
g ml
-1
52.45 24.05
45.24
5.68
30
g ml
-1
55.24 30.65
46.09
7.66
40
g ml
-1
46.28 9.46
47.81
11.68
50
g ml
-1
41.36 -2.18 41.56 -2.92
CD 5%
0.12 1.42
CD= critical difference; PIOC= percent increase over control; PDOC= percent decrease over control
Table
.3
Effect of TiO
2
on antioxidant enzymes during seed germination in
Allium cepa
TiO
2
NPs
(
g ml
-1
)
SOD
(U g
-1
FW)
PIOC/
PDOC
Catalase
(U g
-1
FW)
PIOC/
PDOC
Peroxidase
(U g
-1
FW)
PI
OC/
PDOC
Control
17.64 0.00 32.64 0.00 21.98 0.00
10
g ml
-1
21.22 20.29 38.52 18.01 24.06 9.46
20
g ml
-1
22.74 28.91 42.72 30.88 24.34 10.74
30
g ml
-1
23.04 30.61 40.16 23.04 23.98 9.10
40
g ml
-1
24.38 38.21 31.98 -2.02 20.12 -8.46
50
g ml
-1
25.26 43.20 31.14 -4.60 19.26 -12.37
CD 5%
2.52 2.14 2.58
CD= critical difference; PIOC= percent increase; PDOC= percent decrease

Int.J.Curr.Microbiol.App.Sci
(201
4) 3(7)
749
-
760
757
However, at higher doses of TiO
2
NPs
onion seed germination was reduced
significantly and even showed reduced
activity of amylase and proteases, this
might be due to agglomeration of TiO
2
NPs and
their
weak interactions with seed
or production maximum H
2
O
2
and
inhibition of CAT and POD at 40 and 50
µlml
-1
of TiO
2
NPs. PODs have a role in
very important physiological process
es
like control of growth by lignification,
cross
-linking of pectins and structural
proteins in the cell wall, and catabolism of
auxins (Gaspar et al., 1991). The reduced
radicle growth in onion seedlings at higher
concentrations might be due to lower
acti
vity of POD and limited lignifications
and
cross
-linking of pectins and structural
proteins
in the cell wall as well as
inadequate cell elongation.
TiO
2
NPs at lower concentration enhanced
seed germination and seedlings growth in
onion and inhibited germination growth at
higher concentrations. The TiO
2
NPs also
induced significant changes in hydrolytic
and antioxidant enzyme activities.
Amylase and protease activities showed
enhanced
values in lower concentrations,
but showed decrease at hig
her
concentrati
on. The activity SOD activity
show
ed
concentration dependent increase;
however
CAT
and
POD
were found to be
enhanced in the presences of 10-30
µgml
-1
NPs
but showed decreased activities in 40
and 50
µgml
-1
NPs
concentrations.
Acknowledgemen
t
Authors are thankful to The Principal
Fergusson College Pune (MS) for
providing necessary facilities and also
thankful to University of Pune (MS) for
financial assistance.
References
Baron, W.M.M. 1979: Organization in
Plants. Edward Arnold Limited,
London., pp. 176
-178.
Bernhardt, D., A. Trutwig and A. Barkhold,
1993. Synthesis of DNA and the
development of amylase and
phosphatase activities in cotyledons
and germinating seeds of
Vaccaria
pyramidata.
J. Exp.Bot
. 44,695
699.
Bernier, A.M. and Balance, G.M. 1993.
Induction and secretion of alpha-
amylase (1 3), (1
4) beta
glucanase
and (1
3)
beta
glucanase activities in
gibberellic acid and CaCl2 treated half
seeds and aleurones of wheat.
Cereal
Chem
. 70, 127 132.
Bewley, J.D. and Black, M. 1994.
Seed
s:
Physiology of Development and
Germination
. Plenum Pres, NewYork,
pp. 445.
Bhati
-Kushwaha H., Kaur, A. And Malik,
C.P. 2013. The synthesis and role of
biogenic nanoparticles in overcoming
chilling stress. Indian Journal of Plant
Sciences. 2 (4): 54
-62.
Bowler, C. M., Van Montague
and
D. Inze.
1992. 'Superoxide dismutase and stress
tolerance'.
Annlual Review of Plant
Molecular Biology. 43, 83
-116.
Cameron, A.C., Reid, M.S. (2001). 1-
MCP
blocks ethylene-induced petal
abscission of Pelargonium peltatum
but the effect is transient.Postharvest
Biol Technol. 22, 169
177.
Choudhury, S. and S.K. Panda, 2004.
Induction of oxidative stress and
ultrastructural changes in moss
Taxithelium nepalense (Schwaegr.)
both under lead (Pb) and arsenic (As)
phytotoxicity, Curr. Sci. 87, 34
2
348.
Cocucci, M. and Negrini, N. 1991. Calcium
calmodulin in germination of
Phacetia
tanacetifolia
seeds: Effects of light,
temperature, fusicoccin and calcium
calmodulin antagonist.
Physiol.
Plantarum. 82,143 149.

Int.J.Curr.Microbiol.App.Sci
(201
4) 3(7)
749
-
760
758
Davies, H.V. and Slack, P.T. 1981. The
control of food mobilization in seeds
of dicotyledonous plants.
New
Phytologist
. 88, 41 51.
Dong, B., W. L. Sang, X. Jiang, J. M. Zhou,
F. X. Kong, W. Hu
and
L. S. Wang.
2002. 'Effects of aluminum on
physiological metabolism and
antioxidant system of wheat (Triticum
aestivum
L) '.
Chemosphere
. 47, 87-
92.
Filner, P. and Varner J. E. 1967. A test for
de novo synthesis of enzymes by
density labeling with H2 18O of
barley -amylase induced by
gibberrellic acid. Proc. Nat. Acad. Sci.
USA. 58, 1520
- 1526.
Finche
r, G.B. 1989. Molecular and cell
biology associated with endosperm
mobilization in germinating cereal
grains.
Ann.
Rev. Plant Physiol. Plant
Mol. Biol
. 40, 305
-
346.
Fridovich, I. 1983. 'Superoxide radical: an
endogenous toxicant'. Annual Review
of Pharmacology and Toxicology. 23,
239-257.
Gaspar, Th., Penel, C., Hagege, D.,
and
H.
Greppin.
1991. Peroxidases in plant
growth, differentiation, and
developmental processes, In:
Biochemical, Molecular and
Physiological Aspects of Plant
Peroxidases
(Eds. J. Lobarzewski, H.
Greppin, C. Penel and Th.Gaspar),
249-280 Universite de Geneva Press,
Geneva.
Gepstin, S. and Han, I. 1980. Evidence for
the involvement of cytokinin in the
regulation of proteolytic activity in
cotyledons of germinating beans.
Plant
and Cell Phy
siol
. 21(1):57
-63.
Ghanati, F., A. Morita
and
H. Yokota. 2005.
'Effects of aluminum on the growth of
tea plant and activation of antioxidant
syste'.
Plant and Soil. 276, 133
-141.
Helaly, M.N., El-Hoseiny, H. 2011.
Combined effects between genotypes
and salinity on sweet orange during
the developments stages of its
micropropagation. Res J Bot. 6(2): 38-
67.
Hood, E. 2004. Nanotechnology, Looking as
we leap, Environ. Health Perspect. 112
,740 749
Ikuko, H. and Hiroshi, M. 1980. Pumpkin
(Cucurbita sps.) seed g
lobuline.VI.
Proteoiytic: activities appearing in
germinating cotelydons.
Plant and Cell
Physiol.
2 l (2): 233
-245.
Issac and Gokhale 1982. Autolysis: Journal
of Mycological Society. 78, 389
-
394.
Jayaraman, J. 1981. Laboratory Manual in
Biochemistry, Wiley Eastern Ltd. New
Delhi, India. 122
-123.
Khodakovskaya, M., Dervishi, E.,
Mahmood, M., Xu, Y., Li, Z.,
Watanabe, F. and Biris, A.S. 2009
Carbon nanotubes are able to penetrate
plant seed coat and dramatically affect
seed germination and plant growth.
ACS
Nano 3:3221 3227.
Lee, C.W. , S. Mahendra, K. Zodrow, D. Li,
Y. Tsai, J. Braam and P. J. J. Alvarez.
2010. Developmental phytotoxicity of
metal oxide nanoparticles to
Arabodopsis thaliana. Environ.
Toxicol. Chem. 29, 669
-675
Lei, Z., Mingyu, S., Xiao, W., Chao, L.,
Chunxiang, Q., Liang, C., Hao, H.,
Xiao
-qing, L., Fashui, H., 2008.
Antioxidant stress is promoted by
nano
-anatase in spinach chloroplasts
under UV-B radiation. Biological
Trace Element Research. 121, 69
79.
Lin, C., Y. Fugetsu, F. Watari, 2009
. Studies
on toxicity of multi-walled carbon
nanotubes on Arabidopsis T87
suspension cells, J. Hazard. Mater.
170, 578
583.
Lopez, M.L., J.R. Peralta-Videa, H. Castillo
Michel, A. Martinez-Martinez, M.
Duarte
-Gardea, J.L. Gardea-
Torresdey, 2007. Lead toxicity in
alfalfa plants exposed to
phytohormones and EDTA monitored
by peroxidase catalase and amylase
activities, Environ. Toxicol. Chem. 26.
2717
2723.

Int.J.Curr.Microbiol.App.Sci
(201
4) 3(7)
749
-
760
759
Lu, C.M., Zhang, C.Y., Wen, J.Q., Wa,
G.R., Tao, M.X. 2002. Research of the
effect of nanometer materials on
germination and growth enhancement
of
Glycine max and its mechanism.
Soybean Sci. 21, 168
-
172
Madamanchi, N. R., Donahue J.L., Cramer,
C.L., Alscher, R.G., and Pedersen, K.
1994. Differencial response of Cu.Zn
superoxide dismutase in two pea
cultivars
during a short term exposure
to sulpher dioxide. Plant Mol. Bio. 26,
95-103
Marambe, B., Tadao, A. and Kenji, K. 1992.
Alpha
-amylase and protease activities
and water relations in germinating
sorghum (Sorghum bicolor Moench.)
seeds as affected by animal-w
aste
composts. Soil Sci. Plant Nutr. 38
(1):123
-
131.
Maxwell, D.P. and Bateman D. F.
1967.Change in the activities of some
oxidases in extracts of Rhizoctonia
infected bean hypocotyles in relation
to lesion maturation. hytopathol. 57,
132-135.
Mayer, A. M. and Poljakoff-Mayber, A.
1975. The Germination of Seeds. 2
nd
Edition. Pergamon Press, London.
Mayerand, A.M. and Shain, Y. (1974)
Control of seed germination. Ann.
Rev. Plant Physiol
.
25, 167
-193.
Mishra, P., Shukla, V. K., Yadav, R. S. and
Pandey, A. C. 2013. Toxicity
Concerns of Semiconducting
Nanostructures on Aquatic Plant
Hydrilla verticillata. Journal of stress
physiology and biochemistry. 9(2):
287-298.
Mohamed Naser Helaly, Mohamed Ahmed
El
-Metwally, Hanan El-
Hoseiny,
Samar Abdelaziz Omar, Nabil I
brahim
El
-
Sheery5
2014. Effect of
nanoparticles on biological
contamination of in vitro cultures and
organogenic regeneration of banana
AJCS . 8(4):612
-624.
Morgan, J.M. 1984: Osmoregulation and
water stress in higher plants. Annu.
Rev. Plant Phvsiol. 35,
299
-319
Navarro, E., Baun, A., Behra, R., Hartmann,
N.B., Filser, J., Miao, A., Quigg, A.,
Santschi, P.H., Sigg, L., 2008.
Environmental behavior and
ecotoxicity of engineered
nanoparticles to algae, plants, and
fungi. Ecotoxicology. 17, 372
-386.
Nel, A., Xio, T., Mädler, L., Li, N. 2006.
Toxic potential of materials at the
nano level. Science. 311, 622
-627
Pulskamp, K.; Diabaté, S.; Krug, H.F. 2007.
Carbon nanotubes show no sign of
acute toxicity but induce intracellular
reactive oxygen species in depende
nce
on contaminants. Toxicol. Lett. 168,
58 74.
Raskar, S and Laware,S.L. 2013. Effect of
titanium dioxide nano particles on
seed germination and germination
indices in onion. Plant Sciences Feed,
3
(9): 103
-
107.
Raskar, S.V. and S. L. Laware. 2014. Effect
of zinc oxide nanoparticles on
cytology and seed germination in
onion.
Int.J.Curr.Microbiol.App.Sci
.
3(2): 467
-
473.
Riahi
-Madvar, A., F. Rezaee, and V. Jalili.
2012. Effects of alumina nanoparticles
on morphological properties and
antioxidant system of
Tr
iticum
aestivum
. Iranian Journal of Plant
Physiology, 3 (1): 595. 603
Salisbury, F.B. and Ross, C.W. 1991.
Plant
Physiology
. 4th Edition. Wadsworth
Publishing Company, Belmont,
California, USA.
Schelgel, H.G. 2003. Examining
carbohydrate contents of Asian
cassava. 5th edn, Cambridge,
Gen.
Biol. pp. 180
-185.
Schramm, M. and Loyter, A., 1966.
Purification of -Amylase by
Precipitation of Amylase-
Glycogen
Complexes.
Meth. Enzymol. 9, 533-
537.
Singh, N.; Manshian, B.; Jenkins, G.J.S.;
Griffiths, S.M.; Williams, P.M.;
Maffeis, T.G.G.; Wright, C.J.; Doak,

Int.J.Curr.Microbiol.App.Sci
(201
4) 3(7)
749
-
760
760
S.H.
2009.
NanoGenotoxicology: The
DNA damaging potential of
engineered nanomaterials.
Biomaterials. 30, 3891 3914.
Tan, X., C. Lin, B. Fugetsu, 2009. Studies
on toxicity of multi-walled carbon
nanotubes on suspension rice cells,
Carbon. 45, 3479
3487.
Tang, S.Y. and Cao, Y.P. 2003. Effects of
spraying different forms of silicon (Si)
on growth as stress resistance of rice
plants .Sci Fertil. 2,16
-22
Trethewey, R.N. and Smith, A.M. 2000.
Starch mobilization in leaves. In:
Advances in photosynthesis
(Eds.):
R.C. Leegood, T.D. Sharkey and S.
Von
-
Caemmerer.
Photosynthesis:
physiology and metabolism
.
Dordrecht, the Netherlands, Kluwer
Academic Publishers. 9, 205
-231.
Tully, R. E. and Beevers, H. 1978. Protease
and peptidases of castor bean
endosperm. Enzyme characterisation
and changes during germination.
Plant
Physiol
. 62, 726
-750.
Vidyasekharan, P. and Durairaj, P. 1973.
Shot hole syndrome in mango. Indian
Phytopath
.
.26, 49
-55.
Wang, X., Wei, Z. Liu, D. and Zhao, G.
2011. Effects of NaCl and silicon on
activities of antioxidative enzymes in
roots, shoots and leaves of alfalfa.
African Journal of Biotechnology. 10,
545-549.
Wang, X.D., Sun, C., Gao, S.X., Wang,
L.S., Han, K. 2001. Validation of
germination rate and root elongation
as indicator to assess phytotoxicity
with
Cucmis sativus. Chemosphere.
44, 1711-
1721
Wang, Y., Wang, D. M. and Liang, H.
G.1988. Eifect on plumular axis on the
amylase activity in cotyledons of
germinatin
g pea seeds.
Acta.
Phytophysiol. Sinica
. 14(3): 244
-249.
Washio, K. and Ishikawa, K. 1992. Structure
and expression during the germination
of rice seeds of the gene for a
carboxypeptidase.
Plant Molecular
Biol. 19,631 640.
Xia, T., Kovochich, M., Liong, M., Mädler,
L., Gilbert, B., Shi, H., Yeh, J.I., Zink,
J.I., Nel, A.E. 2008. Comparison of
the mechanism of toxicity of zinc
oxide and cerium oxide nanoparticles
based on dissolution and oxidative
stress properties. ACS Nano. 2, 2121
2134.
Yang, H., Liu, C., Yang, D.F., Zhang, H.S.,
Xi, Z.
2009.
Comparative study of
cytotoxicity, oxidative stress and
genotoxicity induced by four typical
nanomaterials: The role of particle
size, shape and composition. J. Appl.
Toxicol.
29
, 69 78.
Zeeman, S.C., D. Thorneycroft, N. Schupp,
A. Chapple, M. Weck, H. Dunstan, P.
Haldimann,N. Bechcold, A.M. Smith
and S.M. Smith. 2004. Plastidial -
glucan phosphorylase is not required
for in starch degradation in
Arabidopsis
leaves but has a role in
the tolerance of abiotic stress.
Plant
Physiol
. 135, 849
-858.
Zheng, L., Hong, F., Lu, S., and Liu, C.
2005. Effect of nano-
TiO
2
on strength
of naturally aged seeds and growth of
Spinach. Biol Trace Elem Res.105,
83-91.
Zimmermann, P., Zentgraf, U. 2005. The
correlation between oxidative stress
and leaf senescence during plant
development. Cell Mol Biol Lett. 10,
515 534.