Gold nanoparticle exposure induces growth and yield enhancement in

Arabidopsis thaliana
Vineet Kumar, Praveen Guleria, Vinay Kumar, Sudesh Kumar Yadav
Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur 176061, HP, India
H I G H L I G H T S
Exposure of 24nm GNPs enhanced the total seed yield of Arabidopsis thaliana.
GNP exposure improved growth and free radical scavenging activity of A. thaliana.
miR expression showed correlation with growth of A. thaliana on GNP exposure.
a b s t r a c t a r t i c l e i n f o
Article history:
Received 18 January 2013
Received in revised form 8 May 2013
Accepted 8 May 2013
Available online 7 June 2013
Editor: Charlotte Poschenrieder
Keywords:
Arabidopsis thaliana
Gold nanoparticles
microRNA
Plant growth
Seed yield
Nanotechnology has the potential to revolutionize agriculture eld. Towards this effort, carbon nanotubes
have recently been reported to induce growth enhancement of tobacco cells. In this study, exposure to
24 nm size gold nanoparticles (GNPs) at 10 g/ml concentration was found to enhance the total seed yield
of Arabidopsis thaliana by 3 times over the control. In addition, 24 nm size GNP exposure at both 10 and
80 g/ml concentrations has signicantly improved seed germination rate, vegetative growth and free radical
scavenging activity. A considerable correlation was found between expression of key plant regulatory molecules,
microRNAs (miRs) and seed germination, growth and antioxidant potential of A. thaliana on GNP exposure. This
is the rst report showing GNPs as a promising tool to enhance seed yield of plants.
2013 Elsevier B.V. All rights reserved.
1. Introduction
Nanotechnology comprises synthesis of nano-sized (1100 nm)
particles/materials and their manipulation to generate materials or
devices that can be used for various applications (Kumar and Yadav,
2009a; Biswas et al., 2012; Kumar et al., 2012). Nanomaterials
have become an important ingredient of analytical methodologies,
catalytic processes, DNA labels, biosensors, medicine, food industries/
nutraceutics and agriculture (Daniel and Astruc, 2004; Nel et al., 2006;
Mohanpuria et al., 2008; Sozer and Kokini, 2009). Application of
nanotechnology to improve agricultural outcomes is an emerging disci-
pline (Khodakovskaya et al., 2012). Enhanced efciency of nano-based
fertilizers, pesticides and other agricultural formulations can pave way
to sustainable agriculture (Perez-de-Luque and Rubiales, 2009;
Ghormade et al., 2011; Rai and Ingle, 2012). At the same time such
products should be economical and environment friendly. This depends
on the characteristics of NPs used for such applications. The properties
of NPs are determined by their chemical composition, size, surface
covering and most importantly, the dose at which they are used
(Nature Nanotechnology Editorial, 2011; Iravani, 2011; Khodakovskaya
et al., 2012).
The possibility of NP penetration into plant cells has already
been ruled out (Gonzales-Melendi et al., 2008; Liu et al., 2009;
Khodakovskaya et al., 2012). The dilemma that withstands is wheth-
er NPs have useful or harmful effects on plants. Reports demonstrat-
ing the effect of NPs, particularly carbon nanotubes on germination rate
and seedling growth enhancement are present (Barrena et al., 2009;
Seeger et al., 2009). In addition, studies have shown that NPs induce ox-
idative stress in plants (Barrena et al., 2009; Seeger et al., 2009;
Khodakovskaya et al., 2012). Several other workers have mentioned
the inuence of NPs on RUBISCO, photosynthetic activity and antioxi-
dant expression prole of plants (Lu et al., 2002; Lei et al., 2007,
2008). In order to decipher the role of nano-based products in agricul-
ture, an extensive and illustrative work is required. As far as gold
nanoparticles (GNPs) are concerned, reports documenting their effect
on plant physiology, development and metabolism are very limited.
Science of the Total Environment 461462 (2013) 462468
Corresponding author. Tel.: +91 9418050116.
E-mail addresses: skyt@rediffmail.com, sudeshkumar@ihbt.res.in (S.K. Yadav).
0048-9697/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.scitotenv.2013.05.018
Contents lists available at SciVerse ScienceDirect
Science of the Total Environment
j our nal homepage: www. el sevi er . com/ l ocat e/ sci t ot env
Nevertheless there is an enhanced interest in the application of GNPs as
efcient delivery system in plants to improve the agricultural outcome
(Torney et al., 2007).
GNPs are among the most commonly synthesized and studied
metallic NPs. They are being utilized for medical and staining purposes
since 16th century. The wide applications of GNPs have initiated a huge
research for their synthesis using various chemical, physical and biolog-
ical routes (Daniel and Astruc, 2004; Sengul et al., 2008; Kumar and
Yadav, 2009a; Lu et al., 2009). Since biological synthesis has been
found to be environment friendly, several groups are increasingly syn-
thesizing GNPs using plant extracts or microbes (Shankar et al., 2004;
Kumar and Yadav, 2009a; Iravani, 2011). Biologically synthesized
GNPs will be environment friendly and cost effective for various agricul-
tural applications. To evaluate this idea, we carried out exposure studies
of GNPs synthesized by Syzygiumcumini leaf extract on the model plant
Arabidopsis thaliana.
We exposed A. thaliana seeds to two different concentrations of
GNPs (10 and 80 g/ml) and studied the germination pattern, activity
of antioxidant enzymes and expression pattern of microRNAs (miRs).
miRs have emerged as the major regulatory molecule of plants and
animals (Szymanski et al., 2003). In plants, miRs are involved in many
aspects of plant physiology, including responses to abiotic stresses.
The expression pattern of miRs has been observed to be modulated
during stress conditions, thus inuencing plant growth and develop-
ment (Jones-Rhoades and Bartel, 2004; Sunkar and Zhu, 2004; Guleria
et al., 2011). Keeping these facts in mind, response of GNP exposure
on expression prole of miRs was also analyzed in A. thaliana.
2. Material and methods
2.1. NP exposure and seed germination
GNPs (24 nm) were synthesized by the same method reported
previously by us (Kumar and Yadav, 2012). Plants were exposed to
GNPs via germination media. The germination media was constituted
by standard Murashige and Skoog (MS) salts supplemented with MS
vitamins (1000), 3% sucrose and 0.7% agar (Murashige and Skoog,
1962). The pH of media was maintained at 5.85 with 1 N NaOH.
GNPs were added to media before autoclaving at a nal concentration
of 10 and 80 g/ml. Germination medium without GNPs was
employed as control. Seeds of A. thaliana were rinsed with ethanol,
followed by washings in autoclaved distilled water at least three
times. Seeds were surface sterilized by soaking in 0.04% mercuric
chloride for 5 min and rinsed with autoclaved distilled water. Equal
number of 20 sterilized seeds was placed on Petri plates containing
10 and 80 g/ml of GNPs. The number of germinated seeds was
observed till 15 days of germination and presented as percent germi-
nation rate. Relative water content and total fresh biomass of same
seedlings were also evaluated. Relative water content (RWC, %) was
calculated using the formula (FW DW) / (TW DW). Fresh weight
(FW) was obtained by harvesting and weighing freshly detached
rosette leaves of control as well as GNP treated plants. Turgid weight
(TW) was attained by incubating cut rosettes in de-ionized water for
16 h at room temperature. TW was estimated by weighing the plant
material after removing excess water by soaking with absorbent
paper. Rosette dry weight (DW) was weighed after drying the turgid
rosette leaves at 75 C in a dry oven (Bouchabke et al., 2013). The data
is presented as mean SD of mean value of three measurements.
2.2. Estimation of DPPH radical activity
DPPH free radical scavenging assay was performed as reported ear-
lier (Joshi et al., 2011). Briey, initial absorbance of DPPH in methanol
was measured by spectrophotometer at 517 nm until the absorbance
remained constant. A total of 50 l extract was added to 1950 l of
0.1 mM methanolic DPPH solution. The mixture was incubated at
room temperature for 30 min before the change in absorbance at
517 nm was observed and percent inhibition was calculated.
2.3. Estimation of antioxidant enzyme activity
The 100 mg of fresh leaf tissue was homogenized in a pre-cooled
mortar with buffer containing 2 mM EDTA, 1 mM DTT, 1 mM PMSF,
0.5% (v/v) Triton-X 100 and 10% (w/v) polyvinyl polypyrrolidone
(PVPP) in 50 mM phosphate buffer (pH 7.8) and centrifuged at
13,000 rpm for 20 min at 4 C. This enzyme extract was used for
determining the enzymes activity. SOD (EC: 1.15.1.1) activity was esti-
mated as function of inhibition of nitroblue tetrazolium (NBT) photo-
chemical reduction. The activity of SOD was determined as described
earlier (Gill et al., 2010). APx (EC: 1.11.1.11) activity was determined
following the earlier described method (Yadav et al., 2005). Total reac-
tion mixture of 1 cm
3
contained 50 mM sodium phosphate buffer
(pH 7.0), 0.5 mMascorbate, 0.1 mMEDTA and 1.2 mMH
2
O
2
. The reac-
tion was initiated by addition of enzyme extract and change in absor-
bance was recorded at 290 nm. The APx activity was calculated using
extinction coefcient of 2.8 mM
1
cm
1
. For CAT (EC: 1.11.1.6) activi-
ty (Jannat et al., 2011), total reaction mixture of 1 cm
3
contained
50 mM potassium phosphate buffer, 500 mM H
2
O
2
and enzyme
extract. Change in absorbance was measured at 240 nm. The extinction
coefcient of 39.4 mM
1
cm
1
was used to calculate the CAT activity.
GR (EC: 1.6.4.2) enzyme activity was determined as described earlier
(Kumar and Yadav, 2009b). Total reaction mixture contained 50 mM
potassiumphosphate buffer (pH 7.0), 0.8 mM EDTA, 0.5 mM GSSG,
0.2 mM NADPH and enzyme extract. Change in absorbance was read
at 340 nm and was used to calculate GR activity.
2.4. Small RNA isolation
Small RNA fraction was isolated from 15 day old control (no GNP
exposure) as well as 10 and 80 g/ml GNP exposed seedlings. Isolation
was carried out using Qiagen miRNeasy Plant Minikit as per the
manufacturer's instructions. The quality and quantity of isolated small
RNA samples were measured using Nanodrop ND-1000 (Nanodrop
Technologies, USA).
2.5. Stem-loop reverse transcription PCR
Stem-loop reverse transcription (SL-RT) primers were designed
manually for miR408, miR399, miR398, miR397, miR395, miR319,
miR169, miR167, miR164, and miR414 as described earlier
(Varkonyi-Gasic et al., 2007; Guleria and Yadav, 2011). The 50 ng
of small RNA fractions was used to synthesize miR-specic cDNA
by stem-loop reverse transcription. The 0.5 l of 10 mM dNTP mix
was added to RNA, incubated for 5 min at 65 C and then kept on ice
for 2 min. To the above mix, 2 l of 5 First Strand Buffer, 1 l of
0.1 M DTT and 0.5 l of Superscript III RT (100 units) were added.
Master mix of the above recipe was distributed equally in 10 tubes
and 1 l of each of miRNA specic stem-loop RT primers was added
separately. The reaction was conducted as follows: 16 C, 30 min for
1 cycle; 30 C, 30 s; 42 C, 30 s; and 50 C, 1 s for 60 cycles followed
by 85 C, 5 min.
2.6. microRNA expression analysis
Expression evaluation of miRs was performed by reverse
transcription-polymerase chain reaction (RT-PCR) with the above
synthesized cDNAs. For RT-PCR amplication, miR-specic forward
primer and a common reverse primer were used (Varkonyi-Gasic
et al., 2007; Guleria and Yadav, 2011). The 20 l reaction mix com-
prised of 1 l miR-specic cDNA, 0.4 l forward primer (10 M),
0.4 l reverse primer (10 M), 0.4 l of 10 mM dNTP mix, 2 l of
10 PCR Buffer and 0.4 l of Taq polymerase. The reaction was
463 V. Kumar et al. / Science of the Total Environment 461462 (2013) 462468
performed at 94 C, 3 min for 1 cycle; 94 C, 15 s; and 60 C, 1 min
for 25 cycles. The reaction products were visualized on 3.5% agarose
gel in 1 TAE and scanned using Alpha DigiDoc gel documentation
system (Alpha Innotech, USA). The reaction for each miR was repeated
three times for statistical analysis. Relative expression was calculated by
determining the integrated density values (IDV) of gel bands using
AD-1000 software. The synthesized cDNA was quantied using 18S
rRNA as equalizer.
3. Results
3.1. Effect of GNPs on seed germination and seedling growth
Seeds of A. thaliana were germinated on germination media
supplemented with 10 and 80 g/ml of GNPs. GNPs were added to
medium before autoclaving. The stability of GNPs was found to be
unaffected upon autoclaving (see Fig. S1 in the Supplementary
material). Seed germination was observed continuously for 15 days.
Higher percentage of seed germination was found in the GNP exposed
media plates. After 7 days, seed germination percentage was 90% on
medium supplemented with 10 g/ml of GNPs and 85% on medium
supplemented with 80 g/ml of GNPs. While the seeds on control
MS medium without GNPs showed the lowest germination rate of
75%. Similar fashion of relative seed germination percentage was
maintained till 15 days. Finally, the rate of seed germination was
92.5%onwhatever the GNP dose comparedto the 90%seed germination
rate on control medium (Fig. 1a).
Further, the effect of GNPs onthe growth of seedlings was evaluated.
A dramatic increase in seedling size was observed due to GNP exposure
(see Fig. S2 in the Supplementary material). Seedlings germinated on
GNPs containing medium possessed longer shoot and root system in
comparison to the control seedlings (Fig. 1b). The shoot length of seed-
lings exposed to 10 and 80 g/ml of GNPs was enhanced by 1.42 and
1.64 folds in comparison to control seedlings (Fig. 1c). Root length
was also found to follow the same trend. The increase in root length
was 1.75 and 2.45 times on exposure to 10 and 80 g/ml of GNPs com-
pared to control (Fig. 1c). Also, there was an increase in the number of
rosette leaves (Fig. 2a) and lateral roots (Fig. 2b) in the case of GNP
0
10
20
30
40
50
60
70
80
90
100
5 days 7 days 9 days 12 days 15 days
Control
Low
High
G
e
r
m
i
n
a
t
i
o
n

r
a
t
e

(
%
)
Control Low High
Germination period
a
b
0
0.5
1
1.5
2
2.5
3
3.5
Control
Shoot Root
L
e
n
g
t
h

(
c
m
)
Low High Control Low High
c
Fig. 1. Effect of GNPs on A. thaliana seed germination and seedling growth. (a) Time of
germination and germination rate of seeds in the presence and absence of GNPs for a
duration of 15 days, (b) 15 day old seedlings showing shoot and root morphology in
the presence and absence of GNPs and (c) length of shoot and root of A. thaliana seed-
lings grown on medium without GNPs, with 10 and 80 g/ml of GNPs. Results are
presented as average SD of three independent experiments.
86
88
90
92
94
Control Low
High
R
e
l
a
t
i
v
e

w
a
t
e
r

c
o
n
t
e
n
t

(
%
)
a
0
10
20
30
40
50
60
Control
Low High
T
o
t
a
l

f
r
e
s
h

w
e
i
g
h
t

(
m
g
)
c
High Low Control
Control Low
High
0
5
10
15
20
25
30
N
u
m
b
e
r

o
f

l
a
t
e
r
a
l

r
o
o
t
s
b
High Low Control
0
5
10
15
20
N
u
m
b
e
r

o
f

l
e
a
v
e
s
Control
Low High
d
Fig. 2. Evaluation of GNP exposure on growth and development of 15 day old A. thaliana. (a) Image shows rosette leaves (top) and bar diagram representing number of rosette leaves
(below) in the presence and absence of GNPs, (b) image shows lateral roots (top) and bar diagram representing number of lateral roots (below) on medium with and without GNPs,
(c) relative water content of A. thaliana seedlings grown in the presence and absence of GNPs and (d) total fresh weight of A. thaliana seedlings grown on medium with and without
GNPs. Results are presented as average SD of three independent experiments.
464 V. Kumar et al. / Science of the Total Environment 461462 (2013) 462468
treated seedlings. Additionally, the relative water content of GNP
treated seedlings was higher than control seedlings. The relative
water content of seedlings exposed to 10 g/ml of GNPs was
1.12 times higher than control (Fig. 2c). While seedlings exposed to
80 g/ml GNP dose showed an elevation of 1.33 fold in their relative
water content compared to control seedlings (Fig. 2c). Further, the
total fresh weight of A. thaliana seedlings was also increased by 3.68
and 6.3 times on their exposure to 10 and 80 g/ml of GNPs in compar-
ison to control (Fig. 2d).
3.2. Effect of GNPs on antioxidant potential of A. thaliana
Evaluation of various factors described in the previous section has
suggested the positive response of GNPs on the growth of A. thaliana
seedlings. To further investigate the inuence of GNPs on the growing
seedlings, we analyzed the antioxidant potential of control and
GNP-exposed seedlings. DPPH assay was conducted to analyze the
scavenging potential of control and GNP exposed seedlings. Exposure
of seedlings to 10 and 80 g/ml dose of GNPs led to 3.54 and 2.59
folds higher inhibition of reactive oxygen species, suggesting better
free radical scavenging activity upon GNP exposure as compared to
control (Fig. 3a).
To validate the improved free radical scavenging capacity, inuence
of GNP exposure was also analyzed on the activity of various antioxi-
dant enzymes like ascorbate peroxidase (APx), catalase (CAT), glutathi-
one reductase (GR) and superoxide dismutase (SOD). APx activity was
found to be 1.24 and 1.78 folds higher in seedlings exposed to 10 and
80 g/ml of GNPs, respectively than control (Fig. 3b). The increase in
CAT activity was 1.42 and 2.41 times in seedlings exposed to 10 and
80 g/ml of GNPs in comparison to control (Fig. 3c). Similarly, the in-
crease in GR activity of seedlings was 1.48 and 2.35 folds upon treat-
ment of 10 and 80 g/ml of GNPs, respectively (Fig. 3d). The activity
of SOD was also found to be higher by 1.83 and 2.83 times in seedlings
exposed to 10 and 80 g/ml of GNPs in contrast to control (Fig. 3e).
3.3. Effect of GNPs on microRNA expression pattern
microRNAs (miRs) have recently been identied as the major regu-
latory entities of plant growth and development during normal and ad-
verse conditions. Since GNP exposure inuences the growth and
development, effect of GNPs was assessed on the expression pattern
of miRs particularly of those whichare involved in growth and develop-
ment of A. thaliana. Hence, expression pattern of 10 miRs namely:
miR164, miR167, miR169, miR319, miR395, miR397, miR398, miR399,
miR408 and miR414 was analyzed. These miRs have been characterized
for their important regulatory role in seed germination and plant
physiology.
Expression of miR164, miR167, miR395, miR398, miR408 and
miR414 was found to be decreased in A. thaliana seedlings upon GNP
exposure compared to that of in unexposed control seedlings. Relative-
ly, the decrease in expression of these six miRs was signicantly higher
in seedlings exposed to 80 g/ml of GNPs than in the seedlings exposed
to 10 g/ml of GNPs (Fig. 4 and see Fig. S3 in the Supplementary mate-
rial). However, there was no considerable effect of 10 g/ml GNP dose
exposure on the expression of miR169 and miR319. While 80 g/ml of
GNP exposure has increased the expression level of miR169 and
miR319. The expression of miR397 was upregulated in the seedlings
by both the doses of GNPs. On the contrary, expression of miR399 was
not much affected in the seedlings by exposure to either of these two
GNP concentrations (Fig. 4 and see Fig. S3 in the Supplementary
material).
3.4. Effect of GNP exposure on plant seed yield
Plants were transferred to soil pots in order to notice the after effects
of GNP exposure. Plants were continuously observed till they complete
their life cycle. A signicant variation was observed in the life cycle of
plants exposed to GNPs. Plants exposed to 80 and 10 g/ml of GNPs
were observed to ower 15 and 5 days earlier than control plants
S
c
a
v
e
n
g
i
n
g

a
c
t
i
v
i
t
y
(
P
e
r
c
e
n
t
a
g
e

i
n
h
i
b
i
t
i
o
n
s
)
0
5
10
15
20
25
30
0
200
400
600
800
1000
1200
1400
n
m
o
l
e
s
/
m
i
n
/
m
g
0
2
4
6
8
10
12
14

m
o
l
e
s
/
m
i
n
/
m
g
0
5
10
15
20
U
n
i
t
s
/
m
g

p
r
o
t
e
i
n
0
0.1
0.2
0.3
0.4
0.5
0.6

m
o
l
e
s
/
m
i
n
/
m
g
a
d
b
e
c
Fig. 3. Comparison of antioxidant potential between control and GNP exposed A. thaliana seedlings. (a) DPPH radical estimation in 15 day old seedlings grown in control and GNP
supplemented medium. Exposure of GNPs leads to a signicant increase in inhibition of reactive oxygen species. Estimation of antioxidant enzyme activity in control and GNP
exposed seedlings: (b) ascorbate peroxidase, (c) catalase, (d) glutathione reductase and (e) superoxide dismutase. The activities of antioxidant enzymes were observed to be
increased on GNP exposure as compared to control seedlings. Error bars represent the SD of the mean of three measurements of independent experiments.
465 V. Kumar et al. / Science of the Total Environment 461462 (2013) 462468
(Fig. 5a, b). The pod length was higher in plants exposed to 10 g/ml
GNP concentration than exposed to 80 g/ml GNP concentration or
control plants (Fig. 5c). In 10 g/ml GNP exposed plants, the average
number of seeds per pod was increased to 57 3 in contrast to
43 3 seeds per pod of control plant. While, the number of seeds per
pod in plants exposed to 80 g/ml of GNPs was similar to control plants.
Total number of seeds per plant was enhanced to 15,104 803 and
4071 320 on exposure to 10 and 80 g/ml of GNPs respectively
than 3714 100 seeds per plant for control (Table 1). Hence, result
documents that exposure to 10 g/ml of GNPs has signicant inuence
on increasing the total seed yield of A. thaliana. The seed yield of
A. thaliana exposed to GNPs (24 nm) at a concentration 10 g/ml was
increased by 3 times of control, respectively.
4. Discussion
Gold nanoparticles (GNPs) are able to induce vegetative growth
and enhance seed yield of A. thaliana in a dose dependent manner.
Earlier, effects of various types of NPs on seed germination and
plant growth have been reported (Barrena et al., 2009; Seeger et al.,
2009). In a previous study, exposure to 62 g/ml of GNPs has been
documented to have a benecial effect on seed germination and
growth (Barrena et al., 2009) while, the exposure of 130 g/mg of
GNPs in soil for 11 days has been reported to negatively inuence
the root growth of lettuce seeds (Shah and Belozerova, 2009). The
mechanism of plant growth regulation on GNP exposure is yet to be
identied. However, very limited plant response studies have been
conducted with respect to GNPs. Similar studies have been conducted
by exposing plants to other types of NPs. Exposure to 100 and 500 g/ml
of silver NPs have earlier been reported to decrease plant biomass by 57
and 41% compared to control. However, dissolution of silver ions from
NPs was not completely causing the phytotoxicity. Hence, this study
has hinted for NP specic response in plant growth (Stampoulis et al.,
2009). Likewise, copper oxide NPs have been documented to inhibit
root growth in terrestrial plant models. The lowest used NP concentra-
tion 10 g/ml was found to inhibit root growth by 80%, followed by
94 and 100% inhibition of root growth on exposure to 500 and
1000 g/ml of copper oxide NPs, respectively (Atha et al., 2012). In
view of these various previous studies on different NPs, two concentra-
tions 10 and 80 g/ml of GNPs were used in the present study.
Seed germinationrate of A. thaliana was signicantly increasedupon
GNP exposure. Also, the fresh biomass and relative water content of
seedlings exposed to GNPs was higher compared to control. The data
suggests that GNP exposure has enhanced the water uptake capacity
of seeds and thereby, increased seed germination rate and fresh
biomass.
Roots are the primary tissue which comes in contact with NPs
after the seed germination. Analysis of root elongation has been
Control Low
High
Control Low High
High Low Control
a
b
c
Fig. 5. Effect of GNP exposure on A. thaliana seed yield. 15 day old GNP exposed and
control seedlings were transferred to soil pots and observed until they completed
their life cycle. (a) 80 g/ml GNP treated plants witnessed early owering than
10 g/ml treated or control plants, (b) GNP exposed plants showed early owering
and early seed set and (c) pods derived from control and GNP exposed plants. 10 g/ml
GNP treated plants possessed longer pods than 80 g/ml treated and control plants.
0
20
40
60
80
100
120
140
160
180
200
Control
Low
High
R
e
l
a
t
i
v
e

e
x
p
r
e
s
s
i
o
n
**
**
**
**
**
**
****
**
**
** **
*
*
Fig. 4. Analysis of expression pattern of microRNAs in 15 day old A. thaliana seedlings grown on control and GNP exposed medium by RT-PCR. Data is presented as mean of three
measurements SD. Expression of 18s RNA was utilized as equalizer. The '*' represents signicance at P b 0.05 and '**' represents signicance at P b 0.01.
466 V. Kumar et al. / Science of the Total Environment 461462 (2013) 462468
demonstrated as the easiest way to determine whether NPs have
useful/harmful or no effect on plant growth (Seeger et al., 2009; Ma
et al., 2010). Interestingly, GNP exposure in the present study was
found to enhance the root as well as shoot length compared to con-
trol. The root length was increased to a greater extent than shoot.
Overall, the shoot to root ratio was observed to decrease upon GNP
exposure. Earlier toxic effect of NPs has been documented by observ-
ing higher shoot to root ratio (Shah and Belozerova, 2009). Similarly,
24 h exposure to 15 g/ml of ZnO NPs has been reported to inhibit
50% root growth in garlic while, exposure to 50 g/ml of ZnO NPs
has completely blocked the root growth (Shaymurat et al., 2012).
These features collectively suggest the useful inuence of GNPs on
seedling growth in the present study.
This was further conrmed as GNP exposure improved the free
radical scavenging potential of A. thaliana seedlings. DPPH assay re-
sults depicted that total free radical scavenging activity was improved
in seedlings grown on GNP containing medium compared to control.
Since APx, CAT, GR and SOD are known to be important enzymes of
the defense system against reactive oxygen species in plants
(Dalton et al., 1986), the activity of these enzymes was determined
in GNP exposed and unexposed seedlings. The activity of these
enzymes was enhanced in seedling exposed to GNPs in a dose depen-
dent manner. Earlier, mixture of SiO
2
and TiO
2
NPs has been docu-
mented to profoundly increase in SOD and CAT activities of soybean
(Lu et al., 2002). Similarly, nano-anatase treatments have been
shown to enhance the activities of antioxidant enzymes like SOD,
CAT and APx in spinach. This also protects spinach chloroplast from
damage on exposure to UV-B radiations (Lei et al., 2008). Hence, im-
proved antioxidant potential of seedlings on GNP exposure signies
better performance.
To better understand the growth enhancing effect of GNPs, expres-
sion of microRNAs (miRs) was evaluated. To the best of our knowledge,
this is the rst report documenting the inuence of GNPs on miR
expression. miRs are known to be important regulatory molecules of
plants (Szymanski et al., 2003). The expression level of miRs actually
determines the fate of corresponding target genes (Jones-Rhoades and
Bartel, 2004; Sunkar and Zhu, 2004; Guleria et al., 2011). As a result,
alteration in the target gene can hamper the related morphological,
physiological or metabolic processes of the plant (Guleria et al., 2011).
Among various known families and types of miRs, the inuence of
GNPs on miRs having roles in plant growth and development was
analyzed. GNP treated seedlings have shown a signicant decrease in
the expression level of miR398 and miR408 in comparison to control
seedlings. Both miR398 and miR408 target the gene encoding antioxi-
dant enzyme SOD. Overexpression of miR398 and miR408 has been
reported to decrease the activity of SOD as well as CAT and APx (Feng
et al., 2010a,b). The decrease in expression level of miR398 and
miR408 in GNP exposed seedlings has supported the increase in SOD,
CAT, APx and GR enzymes activity. These two miRs are also known to
participate in the regulation of seed germination, seedling growth and
root length. Overexpression of miR398 and miR408 in transgenics has
been reported to inhibit seed germination, seedling growth and elonga-
tion of root length (Feng et al., 2010a,b). Hence, decrease in expression
level of miR398 and miR408 in GNP exposed seedlings was possibly
responsible for higher rate of seed germination, seedling growth and
root elongation compared to control seedlings.
miR164 is knownto target NAC1 gene whichencodes a transcription
factor. This transcription factor regulates lateral root emergence by
transducing auxin signals. Loss of function mutant of miR164 shows
higher accumulation of NAC1 mRNA and increases in emergence of
lateral roots (Guo et al., 2005). Decrease in level of miR164 expression
in GNP exposed seedlings may be responsible for the observed increase
in the number of lateral roots. While the role of miR169 has been docu-
mented in regulation of plant size (Todesco et al., 2010). Increase in the
level of miR169 expression of Arabidopsis upon GNP exposure could
be responsible for remarkable increase in the size of seedlings.
miR167 targets Auxin response factor 8 (ARF8) mRNA. Higher accumu-
lation of ARF8 due to downregulation of miR167 has been reported to
accelerate reproductive growth and maturity of plant (Ru et al., 2006;
Wu et al., 2006; Liu and Chen, 2009). GNP exposed plants were
observed to behave in the similar fashion. Decrease in expression level
of miR167 on GNP exposure might be responsible to induce early
owering and maturity of A. thaliana. In addition to these miRs, the
level of expression of other miRs: miR319, miR395, miR397, miR399
and miR414 was also altered in response to GNP exposure.
5. Conclusion
Taken together, the inuential responses of A. thaliana on GNP
exposure have been demonstrated in the form of model. The model
Table 1
Effect of GNP exposure on the seed yield of A. thaliana.
Control GNPs
(10 g/ml)
GNPs
(80 g/ml)
Seeds per pod 43 3 57 3 44 2
Total number of
seeds per plant
3714 100 15,104 803 4071 320
miR398
miR408
miR169
miR164
miR167
Seed germination
Seedling growth
Root length
Seedling size
Early flowering
Lateral roots
Antioxidant potential
Scavenging activity
Seed yield
GNPs exposure
Fig. 6. Schematic representation of effects of GNP exposure on A. thaliana. GNP exposure altered the expression of key regulatory molecules, microRNAs (miRs). The altered miRs
transcript expression modulated seed germination, seedling growth, morphological pattern and antioxidant potential of A. thaliana seedlings. The observed responses signicantly
enhanced the seed yield of A. thaliana.
467 V. Kumar et al. / Science of the Total Environment 461462 (2013) 462468
depicts that alteration of miR expression due to GNP exposure is
correlated with the growth and yield parameters of A. thaliana
(Fig. 6). GNP exposure has improved free radical scavenging potential
and antioxidant enzyme activity of A. thaliana. Also, GNP exposure
has altered levels of miR expression that are involved in the regula-
tion of various morphological, physiological and metabolic processes
in plants. These physiological and molecular modulations might be
responsible for ultimate improved seed yield by 3 times in 10 g/ml
GNP treated plants. Hence, treatment of seeds with 10 g/ml of
GNPs (24 nm) was recommended for better plant seed yield in lesser
time. For vegetative growth of plant, both GNP doses were effective
but 80 g/ml dose was observed for better response. So, 80 g/ml
dose of GNPs was recommended for better vegetative crop productivity
such as of fodder crops.
Acknowledgments
Authors thank the Director of CSIR-IHBT for his support in carrying
out this research. VK and PGwould like to thank the Council of Scientic
and Industrial Research (CSIR) for fellowship support in the formof SRF.
Funding fromOLP0029 andMLP0001 by CSIRis duly acknowledged. The
CSIR-IHBT communication number for this article is 3427.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.scitotenv.2013.05.018.
References
Atha DH, Wang H, Petersen EJ, Cleveland D, Holbrook RD, Jaruga P, et al. Copper oxide
nanoparticle mediated DNA damage in terrestrial plant models. Environ Sci
Technol 2012;46:181927.
Barrena R, Casals E, Colon J, Font X, Sanchez A, Puntes V. Evaluation of the ecotoxicity of
model nanoparticles. Chemosphere 2009;75:8507.
Biswas A, Bayer IS, Biris AS, Wang T, Dervishi E, Faupel F. Advances in top-down and
bottom-up surface nanofabrication: techniques, applications and future prospects.
Adv Colloid Interface Sci 2012;170:227.
Bouchabke O, Chang F, Simon M, Voisin R, Pelletier G, Durand-Tardif M. Natural variation
in Arabidopsis thaliana as a tool for highlighting differential drought responses. PLoS
One 2013;3:e1705.
Dalton D, Russel S, Hanus F, Pascoe G, Evans H. Enzymatic reactions of ascorbate and
glutathione that prevent peroxide damage in soybean root nodules. Proc Natl
Acad Sci USA 1986;83:38115.
Daniel MC, Astruc D. Gold nanoparticles: assembly, supramolecular chemistry,
quantum-size-related properties, and applications toward biology, catalysis and
nanotechnology. Chem Rev 2004;104:293346.
Feng XM, Qiao Y, Mao K, Hao YJ. Ectopic overexpression of Atmir398b gene in tobacco
inuences seed germination and seedling growth. Plant Cell Tissue Organ Cult
2010b;102:539.
Feng XM, Qiao Y, Mao K, Hao YJ, You CX. Overexpression of Arabidopsis ATMIR408 gene
in tobacco. Acta Biol Cracov Bot 2010a;52:2631.
Ghormade V, Deshpande MV, Paknikar KM. Perspectives for nano-biotechnology
enabled protection and nutrition of plants. Biotechnol Adv 2011;29:792803.
Gill T, Kumar S, Ahuja PS, Shreenivasulu Y. Overexpression of potentilla superoxide
dismutase improves salt stress tolerance during germination and growth in
Arabidopsis thaliana. J Plant Genet Transgen 2010;1:110.
Gonzales-Melendi P, Fernandez-Pacheco R, Coronado MJ, Corredor E, Testillano PS,
Risueno MC, et al. Nanoparticles as smart treatment-delivery systems in plants:
assessment of different techniques of microscopy for their visualization in plant
tissues. Ann Bot 2008;101:18795.
Guleria P, Yadav SK. Identication of miR414 and expression analysis of conserved
miRNAs from Stevia rebaudiana. Genomics Proteomics Bioinformatics 2011;9:
2117.
Guleria P, Mahajan M, Bhardwaj J, Yadav SK. Plant small RNAs: biogenesis, mode of
action and their role in abiotic stresses. Genomics Proteomics Bioinformatics
2011;9:18399.
Guo HS, Xie Q, Fei JF, Chua NH. MicroRNA directs mRNA cleavage of the transcription
factor NAC1 to downregulate auxin signals for Arabidopsis lateral root develop-
ment. Plant Cell 2005;17:137686.
Iravani S. Green synthesis of metal nanoparticles using plants. Green Chem 2011;13:
263850.
Jannat R, Uragi U, Morofuji M, Islam MM, Bloom RE, Nakamura Y, et al. Roles of
intercellular hydrogen peroxide accumulation in abscisic acid signaling in
Arabidopsis guard cells. J Plant Physiol 2011;168:191926.
Jones-Rhoades MW, Bartel DP. Computational identication of plant microRNAs and
their targets, including a stress-induced miRNA. Mol Cell 2004;14:78799.
Khodakovskaya MV, de Silva K, Biris AS, Dervishi E, Villagarcia H. Carbon nanotubes
induce growth enhancement of tobacco cells. ACS Nano 2012;6:212835.
Kumar V, Yadav SK. Plant mediated synthesis of silver and gold nanoparticles and their
applications. J Chem Technol Biotechnol 2009a;84:1517.
Kumar V, Yadav SK. Proline and betaine provide protection to antioxidant and
methylglyoxal detoxication systems during cold stress in Camellia sinensis (L.).
Acta Physiol Plant 2009b;31:2619.
Kumar V, Yadav SK. Characterization of gold nanoparticles synthesised by leaf and seed
extract of Syzygium cumini L. J Exp Nanosci 2012;7:44051.
Kumar V, Kumari A, Guleria P, Yadav SK. Evaluating the toxicity of selected types of
nanochemicals. Rev Environ Contam Toxicol 2012;215:3980.
Lei Z, Mingyu S, Chao L, Liang C, Hao H, Xiao W, et al. Effects of nanoanatase TiO
2
on
photosynthesis of spinach chloroplasts under different light illumination. Biol
Trace Elem Res 2007;119:6876.
Lei Z, Mingyu S, Xiao W, Chao L, Chunxiang Q, Liang C, et al. Antioxidant stress is
promoted by nano-anatase in spinach chloroplasts under UV-B radiation. Biol
Trace Elem Res 2008;121:6979.
Liu Q, Chen Y-Q. Insights into the mechanism of plant development: interactions of
miRNAs pathway with phytohormone response. Biochem Biophys Res Commun
2009;384:15.
Liu Q, Chen B, Wang Q, Shi X, Xiao Z, Lin J, et al. Carbon nanotubes as molecular trans-
porters for walled plant cells. Nano Lett 2009;9:100710.
Lu CM, Zhang CY, Wen JQ, Wu GR. Effects of nano material on germination and growth
of soybean. Soybean Sci 2002;21:16871.
Lu X, Rycenga M, Skrabalak SE, Wiley B, Xia Y. Chemical synthesis of novel plasmonic
nanoparticles. Annu Rev Phys Chem 2009;60:16792.
Ma Y, Kuang L, He X, Bai W, Ding Y, Zhang Z, et al. Effects of rare earth oxide
nanoparticles on root elongation of plants. Chemosphere 2010;78:2739.
Mohanpuria P, Rana NK, Yadav SK. Biosynthesis of nanoparticles: technological
concepts and future applications. J Nanopart Res 2008;10:50717.
Murashige T, Skoog F. A revised medium for rapid growth and bio-assays with tobacco
tissue cultures. Physiol Plant 1962;15:47397.
Nature Nanotechnology Editorial. The dose makes the poison. Nat Nanotechnol
2011;6:329.
Nel A, Xia T, Madler L, Li N. Toxic potential of materials at the nanolevel. Science 2006;311:
6227.
Perez-de-Luque A, Rubiales D. Nanotechnology for parasitic plant control. Pest Manag
Sci 2009;65:5405.
Joshi R, Poonam, Gulati A. Biochemical attributes of tea owers (Camellia sinensis) at dif-
ferent developmental stages in the Kangra region of India. Sci Hortic 2011;130:
26674.
Rai M, Ingle A. Role of nanotechnology in agriculture with special reference to manage-
ment of insect pests. Appl Microbiol Biotechnol 2012;94:28793.
Ru P, Xu L, Ma H, Huang H. Plant fertility defects induced by the enhanced expression of
microRNA167. Cell Res 2006;16:45765.
Seeger EM, Baun A, Kastner M, Trapp S. Insignicant acute toxicity of TiO
2
nanoparticles to willow trees. J Soils Sediments 2009;9:4653.
Sengul H, Theis TL, Ghosh S. Toward sustainable nanoproducts: an overview of
nano-manufacturing methods. J Ind Ecol 2008;12:32959.
Shah V, Belozerova I. Inuence of metal nanoparticles on the soil microbial community
and germination of lettuce seeds. Water Air Soil Pollut 2009;197:1438.
Shankar SS, Rai A, Ankamwar B, Singh A, Ahmad A, Sastry M. Biological synthesis of
triangular gold nanoprisms. Nat Mater 2004;3:4828.
Shaymurat T, Gu J, Xu C, Yang Z, Zhao Q, Liu Y, et al. Phytotoxic and genotoxic effects of
ZnO nanoparticles on garlic (Allium sativum L.): a morphological study.
Nanotoxicology 2012;6:2418.
Sozer N, Kokini JL. Nanotechnology and its applications in the food sector. Trends
Biotechnol 2009;27:829.
Stampoulis D, Sinha SK, White JC. Assay-dependent phytotoxicity of nanoparticles to
plants. Environ Sci Technol 2009;43:94739.
Sunkar R, Zhu JK. Novel and stress-regulated microRNAs and other small RNAs from
Arabidopsis. Plant Cell 2004;16:200119.
Szymanski M, Barciszewska MZ, Zywicki M, Barciszewski J. Noncoding RNA transcripts.
J Appl Genet 2003;44:119.
Todesco M, Rubio-Somoza I, Paz-Ares J, Weigel D. A collection of target mimics for
comprehensive analysis of microRNA function in Arabidopsis thaliana. PLoS Genet
2010;6:e1001031.
Torney F, Trewyn BG, Lin VS, Wang K. Mesoporous silica nanoparticles deliver DNA and
chemicals into plants. Nat Nanotechnol 2007;2:295300.
Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP. Protocol: a highly sensitive
RT-PCR method for detection and quantication of microRNAs. Plant Methods
2007;3:12.
Wu MF, Tian Q, Reed JW. Arabidopsis microRNA167 controls patterns of ARF6 and ARF8
expression and regulates both female and male reproduction. Development
2006;133:42118.
Yadav SK, Singla-Pareek SL, Reddy MK, Sopory SK. Transgenic tobacco plants
overexpressing glyoxalase enzymes resist an increase in methylglyoxal and maintain
higher reduced glutathione levels under salinity stress. FEBS Lett 2005;579:626571.
468 V. Kumar et al. / Science of the Total Environment 461462 (2013) 462468