PLANT SIGNALING & BEHAVIOR

2016, VOL. 11, NO. 5, e1150400 (9 pages)

http://dx.doi.org/10.1080/15592324.2016.1150400

RESEARCH PAPER

Ocimum sanctum leaf extract induces drought stress tolerance in rice

Veena Pandeya, M.W. Ansarib, Suresh Tulac, R.K. Sahooc, Gurdeep Bainsa, J. Kumard, Narendra Tutejae, and Alok Shuklaa
a
Department of Plant Physiology, G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India; bDepartment of Botany, Zakir
Husain Delhi College, Jawahar Lal Nehru Marg, New Delhi, India; cPlant Molecular Biology Group, International Center for Genetic Engineering and
Biotechnology, New Delhi, India; dDepartment of Plant Pathology, G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand, India;
e
Amity Institute of Microbial Technology, Amity University, Noida, UP, India

ABSTRACT ARTICLE HISTORY

Ocimum leaves are highly enriched in antioxidant components. Thus, its leaf extract, if applied in plants, is Received 18 December 2015
believed to efficiently scavenge ROS, thereby preventing oxidative damage under drought stress. Thus, Revised 29 January 2016
the present study was performed in kharif 2013 and rabi 2014 season to evaluate the effect of aqueous Accepted 29 January 2016
leaf extract of Ocimum sanctum against drought stress in 2 rice genotype under glass house conditions. KEYWORDS
Here we show that various morpho- physiological (chlorophyll fluorescence, leaf rolling score, leaf tip Aquaporin; chlorophyll
burn, number of senesced leaves and total dry matter) and biochemical parameters (proline, fluorescence; dehydrin;
malondialdehyde and superoxide dismutase content) were amended by Ocimum treatment in both the gene expression; leaf roll-
seasons. Application of Ocimum extract increased expression of dehydrin genes, while reducing ing; leaf senescence
expression of aquaporin genes in drought stressed rice plant. Thus, application of Ocimum leaf extract
under drought stress can be suggested as a promising strategy to mitigate drought stress in economical,
accessible and ecofriendly manner.

Abbreviations: DHN, Dehydrin; MDA, Malondialdehyde; ROS, Reactive oxygen species; SOD, Superoxide dismutase;
TDM, Total dry matter

Introduction
Ocimum is an aromatic plant in the family Lamiaceae. O. sanc-
tum L., O. gratissium, O. canum, O. basilicum, O. kilimand-
scharicum, O. ammericanum, O. camphora, O. minimum L., O.
tenuiflorum L. and O. micranthum are some of the commonly
known species of genus Ocimum which grow in different parts
of the world.1 Ocimum sanctum L. is found throughout India
and is widely cultivated in homes and temple gardens. Apart
from religious significance, it has a long history of medicinal
use and is mentioned in Charak Samhita, the ancient textbook
of Ayurveda.2 Fresh leaves and stems of Ocimum sanctum con-
tain cirsilineol, cirsimaritin, isothymusin, isothymonin, apige-
nin, rosmarinic acid and eugenol.3 The other main chemical
constituents of Ocimum include oleanolic acid, ursolic acid,
carvacrol, linalool and b-caryophyllene.4
Drought is a major challenge limiting rice production. A
common effect of drought stress is the generation of excess
reactive oxygen species (ROS), which causes peroxidation of
lipids, denaturation of proteins, mutation of DNA, disrupt cel-
lular hemeostasis and various types of cellular oxidative dam-
age, thereby affecting plant performance and yield.5 The ROS
can be scavenged by a complex antioxidant system comprising
of the non-enzymatic (ascorbate (AsA) and reduced glutathi-
one (GSH)) and enzymatic antioxidants (superoxide dismutase
(SOD), catalase (CAT), glutathione peroxidase (GPX), ascor-
bate peroxidase (APX), monodehydroascorbate reductase
(MDHAR), dehydroascorbate reductase (DHAR) and glutathi-
one reductase (GR)). Thus, applying exogenous antioxidants or
any strategy that can enhance antioxidant component in plants
can be considered as a potent strategy for reducing oxidative
stress and to ameliorate drought stress tolerance.
Several reports showed that Ocimum is a rich source of anti-
oxidants. The antioxidant property of any plant includes their
secondary metabolites such as: flavonoids and phenolic acids.
The methanolic leaf extract of Ocimum sanctum had high anti-
oxidant potential and free radical scavenging properties.6 It was
reported that the total phenolic content as well as radical scav-
enging activity was higher in leaf extract as compared to stem,
which was due to high amount of carotenoids and ascorbic acid
content.7 The antioxidant and free radical scavenging activity
of aqueous ethanolic (1:1) extract of Ocimum sanctum was
evaluated by means of DPPH radical, nitric oxide radical,
superoxide anion radical and hydroxyl radical scavenging
assays and it was suggested that some compounds in Ocimum
sanctum are both electron donors and could react with free rad-
icals to convert them into more stable products and to termi-
nate radical chain reactions.8 Thus they concluded that
Ocimum is a potential source of natural antioxidant. The anti-
oxidant activities and total phenolics of methanolic and etha-
nolic leaf extracts of Ocimum sanctum was evaluated and it was
observed that the ethanol: water extract had highest antioxidant
activity,9 while the methanol extract possesses higher amounts

CONTACT Veena Pandey veenapandey28@gmail.com

Supplemental data for this article can be accessed on the publisher’s website.
© 2016 Taylor & Francis Group, LLC
e1150400-2 V. PANDEY ET AL.
of plant phenol10 which are also known to be responsible for
the antioxidant activity of most of the plants. The antioxidant
activity8 and reducing power11 of aqueous extract of leaves was
found to be increased in a dose dependent manner. The total
antioxidant capacity of Ocimum sanctum leaf extracts was
investigated by measuring its ability to reduce the molybdate
(Mo) ion, thereby depicting their capacity to reduce oxidative
stress.12 Thus, our hypothesis was based on the fact that apply-
ing Ocimum leaf extract having well enriched antioxidant sys-
tem in drought-stressed rice, may efficiently scavenge ROS,
thereby preventing oxidative damage in rice and improving
plant performance under drought stress.
The methanolic and aqueous leaf extracts of O. gratissimum
contains tannins, steroids, phenolics, terpenoids, flavonoids
and cardiac glycosides which accounts for most of the antioxi-
dant activity of plants. Anthraquinones were detected only in
the aqueous extract while alkaloids were detected only in the
methanolic extract.13 Ocimum leaf extract is rich in polypheno-
lic compounds and scavenged the radicals in a concentration
dependent manner. The high concentrations of total phenolics,
flavonoids and appreciable amounts of flavonols were present
in the extract of O. americanum leaves and possess the ability
to inhibit oxidative stress by antioxidant mechanisms.14 O.
sanctum leaf extract possess free radical scavenging activity and
contain alkaloids, tannins, flavonoids, and saponins.15 Chemi-
cal composition analysis of essential oil isolated from the leaves
of Ocimum canum Sims. reported the presence of 36 com-
pounds, among them camphor (39.77%) was the major com-
pound. Several studies reported that high concentrations of
bioactive monoterpenes like camphor led to irreversible dam-
age of the whole plant, but low concentration or short term
fumigations with camphor can strengthen the plant fitness as
evidenced by altered gene expression of defence related genes
like MAP3 kinase, ABF4 and RD29B.60 It was suggested that
antioxidant activity of the O. canum essential oil might be due
to the presence of mono and sesquiterpenes.16 Thus, unravel-
ling the potential of Ocimum to induce drought stress tolerance
in rice could be of great importance under increasing drought
incidences. This might provide efficient ROS scavenging system
and prevent cell damage under stress. To the best of our knowl-
edge, there is no published literature reporting the efficacy of
Ocimum sanctum leaf extract to mitigate drought stress in
plants. So an attempt has been made to investigate the potential
of Ocimum leaf extract to induce morpho- physiological and
molecular changes in rice crop, leading to drought stress toler-
ance in rice.
Results
Morpho-physiological analysis
Ocimum treatment significantly amended various morpho-
physiological parameters like chlorophyll fluorescence (Fv/Fm),
leaf rolling score, leaf tip burn, number of senesced leaves and
total dry matter of drought-stressed rice leaves during both sea-
sons as presented inTable 1.
High chlorophyll fluorescence was maintained by Ocimum
treatment under drought stress in both the rice genotypes, as
compared to control. During kharif season, Ocimum treatment
resulted in highest fluorescence in PSD-17 (0.75), followed by
PB-1 (0.74). During rabi season also, highest fluorescence was
found in PSD-17 (0.72), followed by PB-1 in Ocimum treated
plants.
Leaf rolling was found to be more pronounced in treated
plants as compared to untreated control during both growing
seasons. During kharif season, Ocimum treatment resulted in
compactly rolled leaf in PSD-17 (4.33), followed by PB-1
(2.67). During rabi season, Ocimum treated leaves of PSD-17
and PB-1 were equally rolled (3.33).
Drought stress caused leaf tip burn in both rice genotypes
during both the seasons, while Ocimum treatment significantly
reduced incidence of tip burn except PSD-17 during kharif sea-
son where leaf tip burn was recorded more than control. PSD-
17 showed increase in leaf tip burn incidence under Ocimum
treatment (13.43%) as compared to untreated control (8.35%)
during kharif season; while during rabi season, tip burn was
significantly reduced from untreated control (17.97%) to Oci-
mum treated plants (12.28%). In PB-1, during kharif season,
untreated control showed 16.5% tip burn, which was reduced
to 10.39% by Ocimum treatment; while during rabi season,
untreated control showed 18.72% tip burn, which was reduced
to 14.23% by Ocimum treatment.
Ocimum treatment significantly decreased the number of
senesced leaves in both rice genotypes during both growing sea-
sons, compared with the control. In PSD-17, during kharif sea-
son, percent of senesced leaves per hill in untreated control and
Ocimum-treated plants were 74.51% and 33.68% respectively;
while during rabi season, it was 73.67% and 42.56% respec-
tively. In PB-1, during kharif season, percent of senesced leaves
per hill in untreated control and Ocimum-treated plants were
81.45% and 36.57% respectively; while during rabi season, it
was 77.32% and 39.01% respectively.
Total dry matter (TDM) per hill after harvesting was signifi-
cantly higher in Ocimum treated plants as compared to
untreated control in both the rice genotypes. During kharif sea-
son, Ocimum treatment resulted in highest TDM in PSD-17
(38.33g) followed by PB-1 (31.33g). During rabi season also,
Ocimum treatment resulted in highest TDM in PSD-17
(28.67g) followed by PB-1 (25.33g).
Biochemical and yield analysis
Table 2 shows the effects of Ocimum treatment on MDA, pro-
line and SOD activity in drought-stressed rice leaves during
kharif 2013 and rabi 2014 seasons.
Ocimum treatment significantly decreased the concentration
of MDA in all rice genotypes during both kharif and rabi grow-
ing seasons, compared with the control. The highest decrease
in MDA content in Ocimum treated rice leaf during kharif sea-
son was observed in PSD-17 (19.3%) followed by PB-1 (16.4%)
as compared to untreated control. During rabi season, the high-
est decrease in MDA content by Ocimum treatment was
observed in PSD-17 (23.3%) followed by PB-1 (21.4%), com-
pared with control. Seasonal variation was also evident from
the MDA contents as average MDA contents were compara-
tively higher in the kharif rice as compared with the rabi season
rice under drought stress.
 PLANT SIGNALING & BEHAVIOR e1150400-3

Table 1. Effect of Ocimum treatment on morpho-physiological attributes of 2 rice genotypes during kharif and rabi seasons.

Kharif season 2013

Chlorophyll fluorescence (Fv/Fm) Leaf rolling Leaf tip burn per hill (%) Senesced leaves per hill (%) TDM at maturity (g/hill)

Variety Treatment Control Treatment Control Treatment Control Treatment Control Treatment Control

PSD-17 0.75 § 0.002 0.70 § 0.019 4.33 § 0.33 2.67 § 0.33 13.43 § 0.65 8.35 § 0.54 33.68 § 1.36 74.51 § 3.11 38.33 § 1.45 22.33 § 1.33
PB-1 0.74 § 0.003 0.71 § 0.007 2.67 § 0.33 1.67 § 0.33 10.39 § 0.64 16.50 § 0.80 36.57 § 9.36 81.45 § 5.17 31.33 § 0.88 28.33 § 2.96
Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V)
S.Em. § 0.0058 0.0045 0.13 0.10 0.54 0.42 2.45 1.90 1.32 1.03
CD at 5% 0.0169 0.0131 0.38 0.29 1.57 1.21 7.12 5.51 3.85 2.98
Rabi season 2014

PSD-17 0.72 § 0.01 0.70 § 0.01 3.33 § 0.67 2.33 § 0.33 12.28 § 3.71 17.97 § 2.38 42.56 § 5.04 73.67 § 6.31 28.67 § 1.20 20.67 § 1.33
PB-1 0.71 § 0.00 0.69 § 0.01 3.33 § 0.33 2.00 § 0.00 14.23 § 1.98 18.72 § 3.71 39.01 § 4.09 77.32 § 6.90 25.33 § 2.33 24.00 § 2.65
Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V)
S.Em. § .004 .003 .227 .176 1.96 1.52 2.57 1.99 1.68 1.30
CD at 5% .014 .010 .657 .509 5.67 4.39 7.44 5.76 4.85 3.76

Ocimum treatment significantly reduced the proline content
for both the genotypes during both growing seasons, compared
with the control. The highest reduction in proline content by
Ocimum treatment during kharif season was observed in PB-1
(31.6%) followed by PSD-17 (22.7%), as compared to untreated
control. During rabi season, the highest reduction in proline
content in Ocimum treated rice leaf was observed in PSD-17
(53.1%), followed by PB-1 (35.8%), compared with control.
Among both the seasons, more proline under drought stress
was accumulated in control plants during rabi season as com-
pared to kharif season, indicating that the rabi season rice expe-
rienced more severe drought.
The Ocimum treatment significantly increased the activity of
SOD in rice flag leaves for both the genotypes during both
growing seasons, compared with the control. The highest
increase in SOD activity by Ocimum treatment was observed in
PB-1 (144.2%) followed by PSD-17 (97%) under kharif season,
as compared to control. During rabi season, the highest
increase in SOD activity in treated plants was observed in PSD-
17 (57.3%) followed by PB-1 (43%). Thus, among both the sea-
sons, higher increase in SOD activity in all the genotypes was
observed during kharif season.
Fetching greater harvestable yield is the ultimate purpose of
any strategy used to provide stress tolerance. In our study, Oci-
mum treatment significantly maintained higher number of
filled grains as compared to untreated control plants, under
drought stress during both the seasons. The highest increase in
number of grain by Ocimum treatment was observed in PSD-
17 (45.4%) followed by PB-1 (8.8%) during kharif season, as
compared to control. During rabi season, the highest increase
in number of grain in treated plants was observed in PSD-17
(43.2%) followed by PB-1 (22.8%). Genotypic variation was
also evident during both the seasons, under control as well as
Ocimum treatment.
Molecular analysis
In this study, expression of 4 out of the 8 dehydrins and 2 out
of the 37 aquaporin genes studied were significantly altered
during drought stress. All the rice genotypes showed a signifi-
cant increase in the transcript levels of dehydrins (DHN1,
DHN2, DHN3 and DHN4) and decrease in the transcript levels
of aquaporin genes (AQP1 and AQP2) under Ocimum treat-
ment, as presented in Figs. 1 and 2.
During both the seasons, in untreated control, there was no
significant difference in the expression of dehydrin genes
among the genotypes. But the extent of their increase under
Ocimum treatment differs considerably in different genotypes.
Highest normalized fold change in the expression of DHD1,
DHD3, DHD4 was observed in PB-1, while expression of

Table 2. Effect of Ocimum treatment on biochemical and yield attributes of 2 rice genotypes during kharif and rabi seasons.
Kharif season 2013

MDA (mg/g FW) Proline (mmol/g FW) SOD (U/mg protein) Number of filled grains per panicle

Variety Treatment Control Treatment Control Treatment Control Treatment Control

PSD-17 3.41 § 0.12 4.23 § 0.23 2.75 § 0.38 3.56 § 0.03 2.68 § 0.08 1.36 § 0.10 100.33 § 2.03 69.00 § 1.15
PB-1 3.75 § 0.05 4.49 § 0.13 2.74 § 0.21 4.01 § 0.09 2.98 § 0.19 1.22 § 0.10 86.33 § 3.48 79.33 § 2.67
Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V)
S.Em. § 0.073 0.057 0.097 0.075 0.094 0.072 2.448 1.896
CD at 5% 0.213 0.165 0.281 0.217 0.272 0.211 7.073 5.478
Rabi season 2014

PSD-17 3.08 § 0.05 4.02 § 0.04 2.19 § 0.02 4.67 § 0.02 2.80 § 0.03 1.78 § 0.15 96.00 § 6.03 67.00 § 0.58
PB-1 3.33 § 0.06 4.24 § 0.10 2.60 § 0.01 4.05 § 0.04 2.86 § 0.14 2.00 § 0.08 96.67.33 § 2.19 78.67 § 3.76
Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V) Treatment (T) Variety (V)
S.Em. § .058 .045 .077 .060 .070 .054 2.207 1.710
CD at 5% .169 .130 .223 .173 .204 .154 6.376 4.939
e1150400-4 V. PANDEY ET AL.

Figure 1. Effect of Ocimum treatment on expression of 4 dehydrin genes on drought stressed rice during (A) kharif and (B) rabi seasons.

Figure 2. Effect of Ocimum treatment on expression of 2 aquaporin genes on drought stressed rice during kharif and rabi seasons.

DHD2 was highest in PSD-17 (Fig. 1). Normalized fold change
in the expression of aquaporin genes decreased considerably
under Ocimum treatment in both the seasons, as compared to
control. Highest reduction in normalized fold change in the
expression of AQP1 and AQP2 was observed in PB-1 genotype,
in both the seasons. (Fig. 2).
Discussion
The use of plant extracts and phyto-products to alleviate various
stresses is nowadays gaining attention due to their availability,
cost effectiveness and biodegradability. In the present study, the
effect of Ocimum leaf extract on different drought related parame-
ters in 2 rice genotypes was evaluated. Previous reports showed
that drought stress reduces chlorophyll fluorescence (Fv/Fm) in
different rice cultivars.17-19 In our study, Ocimum treatment sig-
nificantly maintained the Fv/Fm values under drought stress,
while the fluorescence was found lowest in untreated control in
both rice genotypes, during both the seasons.
Leaf rolling is one of the acclimation responses of rice and
helps in maintaining internal plant water status.5 Ocimum
treatment resulted in compactly rolled leaf as compared to
untreated control plants. Ocimum treated PSD-17 genotype
showed highest rolling score during both the season, as com-
pared to PB-1, thus preventing large amount of transpirational
water loss under drought stress.
The present study indicated that drought tolerance of rice
genotypes was enhanced by Ocimum treatment as evidenced by
reduced leaf tip burn and drought-induced senescence. Percent
of senesced leaves per hill was lowest in Ocimum treated PSD-
17 during kharif season, while during rabi season, it was lowest
in Ocimum treated PB-1. Seasonal variation was also evident
from the percent of leaf tip burn and senesced leaf and these
parameters were comparatively higher in the rabi season rice as
compared to kharif season rice, indicating that negative impact
of drought stress was more severe during rabi season.
Reports revealed that total dry matter (TDM) production
decreased with decreasing soil moisture level.20,21 Ocimum
treatment significantly found to maintain higher TDM after
harvesting under drought stress, as compared to control. Sea-
sonal variation in TDM was also evident in both the varieties
and lower TDM was recorded during rabi season.
ROS production is inevitable consequence of drought stress
and severely damage cell component resulting in reduced per-
formance of plant. Ocimum leaves are well known for their
antioxidant potential and efficiently scavenge free radicals.6-9
Prominent antioxidant mechanism ensures low lipid peroxida-
tion resulting in low MDA content under drought stress. In our
study, Ocimum treatment significantly reduced MDA content
in flag leaf of drought stressed rice as compared to untreated
control in both the seasons. Lowest amount of MDA was found
in PSD-17 during kharif and rabi season in Ocimum treated
plant, as compared to control. Ursolic acid, a triterpene, iso-
lated from O. sanctum has been shown to be effective in pro-
tecting against lipid peroxidation.22 Leaf extract of O. sanctum
was found to inhibit lipid peroxidation in rats intoxicated with
arsenic23 and diazinon.24 Ethanolic leaf extract of O. basilicum
decreased the lipid peroxidation concentration in parasitized
mice.25
PLANT SIGNALING & BEHAVIOR e1150400-5
Changes in the concentration of proline have been observed
in many studies on drought stressed rice.26-28Proline accumula-
tion may be considered as an indicator of drought susceptibil-
ity. Rice plants which showed more tolerance under drought
stress, accumulated less proline than sensitive ones.29 Our
results showed that Ocimum treatment significantly reduced
proline content in drought stressed rice leaf. Lowest proline
content was found in PB-1 during kharif season, while during
rabi season, lowest proline was noted in PSD-17.
Superoxide dismutase (SOD) is an important antioxidant
enzyme which protects the plant with excessive
superoxide radicals formed in plants under stress. Ocimum
treatment significantly increased SOD activity in drought
stressed rice leaf as compared to untreated control, in both the
seasons. Ocimum treatment resulted in highest SOD activity in
PB-1 during kharif and rabi season. The O. sanctum leaves
were found to have antioxidant enzymes like SOD, CAT, GPX,
GSH and ascorbate and the extract was found to have potent
antioxidant activity.30 Administration of hydro-alcoholic
extract of O. sanctum leaves increased SOD activities in rats
intoxicated with arsenic.23 O. basilicum leaf extract increased
antioxidant enzymes namely SOD and CAT in rats.24 Ethanolic
leaf extract of O. basilicum increased the activities of SOD,
CAT and GPX in Plasmodium parasitized mice, indicating that
the extract scavenged superoxide radicals in mice.25 Study on
the antioxidant effect of O. basilicum aganist gibberllic acid and
auxin supplemention in broilers ration showed that antioxidant
activity (SOD, CAT, GSH, GPX) significantly increased when
O. basilicum is added.31 Foliar treatment of tomato plants with
30% aqueous extract of O. sanctum increased the antioxidant
enzymes.32
Drought triggers a variety of response at molecular level, by
modifying a range of genes expression. Among various genes,
aquaporins and dehydrins play a major role in providing
drought tolerance in plants. Aquaporins (AQP) are known to
facilitate water movement across membrane,33,34 while dehy-
drins (DHN) or group 2 LEA proteins are thought to be
involved in water binding molecules and stabilizing both mac-
romolecules and membranes.35 In our study, the gene expres-
sion of 4 dehydrin genes were found to be increased
significantly by Ocimum treatment while expression of 2 aqua-
porin genes were found to be reduced significantly by Ocimum
treatment. Moreover, we can say some elicitors must be present
in Ocimum extract which triggered signaling cascade in rice
plants that led to altered expression of aquaporins and
dehydrins.
Like our results, a positive correlation of dehydrin expres-
sion with drought tolerance has been reported in many stud-
ies.36-39 More recently, study in tomato reported that
overexpression of dehydrin tas14 gene helps in improving the
drought stress tolerance.40 The overexpression of OsDhn1 in
transgenic rice plants enhanced drought stress tolerance as
judged by chlorophyll fluorescence (Fv/Fm), fresh and dry
weight, water and chlorophyll content, and suggested
that OsDhn1 plays an important role in the stress tolerance via
scavenging reactive oxygen species (ROS).41
Several reports supporting the negative role of aquaporins
under drought stress are available. Over-expressing plasma
membrane aquaporin AtPIP1b in transgenic tobacco had a
e1150400-6 V. PANDEY ET AL.
negative impact during drought stress, with the transgenic
plants wilting more rapidly than the wild type plants.42 Thus it
was suggested that a general increase in water transport in
most plant tissues and cells is harmful under drought stress.
Transgenic tobacco and Arabidopsis plants over-expressing
PIP1;4 or PIP2;5 showed rapid water loss under dehydration
stress and it was speculated that enhanced water transport via
the plasma membranes of cells may be deleterious under water
stress.43 Constitutive over-expression of an AQP gene GoPIP1
from G. orientalis in Arabidopsis led to decreased drought
resistance and enhanced drought sensitivity in transgenic Ara-
bidopsis. The transgenic plants over-expressing GoPIP1 had
lower leaf water content and lower water potential and had
faster water loss through leaves to the atmosphere, indicating
that GoPIP1 might affect stomatal aperture thus modifying
water movement.44
In conclusion, the present study demonstrates the efficacy of
Ocimum leaf extract against drought stress in different rice gen-
otypes. It was observed that spraying Ocimum leaf extract
increased SOD activity and decreased accumulation of MDA
and proline in drought stressed flag leaves of rice, implying that
plants have had experienced less stress. Moreover, the percent
of senesced leaf and tip burn incidences were reduced by Oci-
mum treatment.
The application of O. sanctum leaf extract led to alteration in
gene expression of water channel proteins aquaporins and
defense related LEA proteins like dehydrins and amended the
biochemical processes in rice under drought stress, which ulti-
mately provided plant to tolerate stress for more period of
time. This alteration in gene expression might have occurred
Figure 3. Ocimum induced various responses in drought stressed rice plants and hypothetical signal transduction pathway. SOD- Superoxide dismutase, MDA- Malondial-
dehyde, TDM- Total dry matter.
due to certain elicitor molecules which are expected to be pres-
ent in Ocimum leaf extract and induced a series of signaling
cascade in rice, resulting better adaptation by rice under stress
(Fig. 3). Aqueous extracts of O. gratissimum leaves induced the
production of phytoalexins in soybean cotyledons and sorghum
mesocotyls and also induced systemic resistance in cucumber
as reflected by increase in chitinase production.45 Aqueous
extract of O. basilicum was found to induce the expression of
extracellular signal-regulated kinase 2 (ERK2), which is capable
of regulating several transcription factors, thus suggesting that
the extract can mediate the ERK2 MAP-kinase signal path-
way.46 Further experiments are necessary to determine the best
time of application and concentration of Ocimum leaf extract.
Of equal importance is the elucidation of the extract compo-
nents responsible and the signaling mechanism involved.
Materials and methods
Experimental site
Glass house experiments were conducted in the kharif season
(June to November) 2013 and repeated in the rabi season (Feb-
ruary to July) 2014 at Center of Advance Studies, Department
of Plant Pathology, College of Agriculture, G.B.P.U.A.T., Pan-
tnagar. Geographically, the site lies in Tarai plains about 30 km
southwards of foothills of Shivalik range of the Himalayas at
29 N latitude, 79 290 E longitude and at an altitude of 243.8
meter above the mean sea level. Treatments were arranged in a
completely randomized design with 4 replications.

Preparation of O. sanctum leaf extract
The leaves of O. sanctum were collected from local area at Pan-
tnagar, Uttarakhand. Fresh plant leaves of O. sanctum were
washed under running tap water, air dried and then homoge-
nized to fine powder. 20 gm. of air-dried powder of leaves was
added to 300 ml distilled water. It was then boiled on slow heat
for 24 h and then filtered. The filtrate concentrated to make the
final volume one by third of the original volume with the help
of water bath and this 20% (w/v) concentrated solution was
used as Ocimum extract.
Drought stress and Ocimum treatment
The seeds of rice genotypes, namely, Pusa Basmati 1(PB 1) and
Pant Sugandh Dhan 17 (PSD 17) were obtained from Seed Pro-
duction Center, G.B.P.U.A.T., Pantnagar. Seeds of both rice
genotypes were sown in nursery and transplanted after 21 days
in 5 kg capacity pots having sterilized soil. The pots were kept
in the well-ventilated glasshouse during the whole growing sea-
sons. Four replications were taken and the number of plants
per pot was maintained as four. Pots containing plants of each
genotype were divided into 2 groups. One set was given Oci-
mum treatment under drought stress, while other set was not
sprayed with Ocimum and maintained as control. Drought
stress treatment was induced at the time of flowering by irriga-
tion withholding for 5 subsequent days in all the pots. On sec-
ond day of drought application, aqueous extract of O. sanctum
was sprayed on all the pots by using hand held plastic spray
bottle, except control. The soil moisture content in pots on fifth
day was recorded as 11§2% and 8§2% during kharif 2013 and
rabi 2014 season respectively.
1.Morpho-physiological assay: Chlorophyll fluorescence
(Fv/Fm), leaf rolling score, tip burn (% per hill), number of
senesced leaves after drought (% per hill), total dry matter
(g)
Morpho-physiological parameters like chlorophyll fluorescence
and leaf rolling were observed on third day of stress while
observations on leaf tip burn, number of senesced leaves per
hill was taken on the fifth day of drought application.
Chlorophyll fluorescence of rice flag leaves was measured
with the help of Handy PEA (Hansatech, UK). Dark adaptation
of samples was achieved via lightweight plastic leaf clips, the
leaf clip shutter blade was closed to prevent the entry of light
and the clip left in place for 10 minutes to provide dark adapta-
tion. To perform a measurement, the Handy PEA sensor unit
was held over the clip and the shutter opened. A single button-
press immediately displayed automatically calculated fluores-
cence parameters F0, Fm, and Fv/Fm. All the observations were
recorded in the forenoon hrs (9–11 AM) to avoid
photoinhibition.
Leaf rolling may be a useful strategy to maintain leaf water
potential in rice under drought. It was scored visually after
2 days of the drought treatment using scales of 0–5 with 1 being
the first evidence of rolling and 5 being a closed cylinder. Leaf
tip burning and leaf senescence on fifth day of drought stress
was observed and calculated as the cumulative percentage of
PLANT SIGNALING & BEHAVIOR e1150400-7
leaf tip browning and leaf dying respectively, out of the total
leaf population.
The total plant dry matter per plant (g) was recorded at
maturity stage by uprooting the complete plant and then plac-
ing the plant sample in the oven at 65 C for 3 days.
Biochemical assay: Proline content, malondialdehyde
(MDA) content and superoxide dismutase (SOD) activity
All the biochemical analysis was performed on the fifth day of
drought application. Proline content was determined following
the method of Bates et al.47 Leaf tissue (0.2 g) was homogenized
in 4 ml of sulphosalicylic acid (3%) and centrifuged at 10,000 g
for 30 min. About 2 ml of the supernatant was taken in a test
tube and to it 2 ml of glacial acetic acid and 2 ml ninhydrin
reagent were added. The reaction mixture was boiled in water
bath at 100 C for 30 min. After cooling the reaction mixture,
4 ml of toluene was added and vortexed for 30s, the upper
phase containing proline was measured with spectrophotome-
ter at 520 nm using toluene as a blank.
Lipid peroxidation in terms of total MDA concentration
(mg/g FW) was estimated by the method of Heath and
Packer.48 The extent of lipid peroxidation was evaluated by the
thiobarbituric acid reaction. 0.1g of leaf was homogenized in
0.1% trichloroacetic acid (1:10, w:v) and centrifuged at 15,000 g
for 10 min. 0.5 ml of the supernatant was incubated with 1.5 ml
of 0.5% thiobarbituric acid diluted in 20% trichloroacetic acid
at 95 C for 25 min and then cooled in ice bath. Measure the
absorbance of the supernatant at 532 nm and 600 nm. The con-
centration of thiobarbituric acid reactive substances was calcu-
lated as malondialdehyde equivalent using the extinction
coefficient (155 mM¡1 cm¡1).
SOD activity was assayed by the method of Giannopolitis
and Ries.49 The reaction mixture contained 1.3 mM riboflavin,
13 mM L- methionine, 0.05 M Na2CO3, (pH 10.2), 63 mM p–
nitroblue tetrazolium chloride (NBT) and crude plant extract.
Reaction was carried out under illumination (75 mmol photon
m¡2 s¡1) from fluorescent lamp at 25 C. The initial rate of
reaction as measured by the difference in increase in absor-
bance at 560 nm in the presence and absence of extract was
proportional to the amount of enzyme.
Molecular assay
Sequence-retrieval and microarray-based expression study
of drought-related dehydrin and aquaporin genes
Sequence of drought-related dehydrin and aquaporin genes
(Table S1) were retrieved from Rice Genome Database
RGAP7 (http://rice.plantbiology.msu.edu/;50) using BLASTn.
The retrieved sequences were confirmed by evaluating the
annealing of primers used in the relevant studies by means of
Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-
blast/). Further, the microarray probe set ids were retrieved via
Rice Oligonucleotide Array Database (http://ricearray.org/;51).
Among the 8 dehydrin genes and 37 aquaporins genes reported
so far, specific probe set ids could be established for all. To
examine the expression profile, publicly accessible data for
single microarray platform – 51 K Affymetrix gene chip was
exercised (https://www.genevestigator.com/gv/plant.jsp;52,53).
e1150400-8 V. PANDEY ET AL.
Heat map with average linkage hierarchical clustering was pro-
duced with Multi Experiment Viewer software (http://www.
tm4.org/mev/;54) by means of Pearson correlation as the dis-
tance metric.
RNA isolation and quantitative real-time PCR (qRT-PCR)
Leaf samples of drought stressed rice plants were used for RT-
PCR. Total RNA was isolated via TriZOL LS reagent (Invitro-
gen, http://www.invitrogen.com) as per the manufacturer’s
instructions, and poly(A)-RNA was isolated. It was used for
making cDNA using the RevertAid H minus first-strand cDNA
synthesis kit (Fermentas, http://www.thermoscientificbio.com/
fermentas). Expression analysis of the dehydrin and aquaporin
genes was performed by qRT-PCR.55 The relative levels of the
transcripts accumulated for dehydrin and aquaporin genes
were normalized to a–tubulin (primers detailed in Table S2)
and the dehydrin and aquaporin genes expression in rice
plant56 using the 2–DDCt method from 3 independent experi-
ments.57 The PCR proficiency, which is reliant on the assay,
performance of the master mix and quality of the sample, was
calculated as efficiency D 10 (–1/slope) – 1 (3.6C slope C 3.1)
by the software itself (Applied Biosystems, http://www.applied
biosystems.com) ‘C’ is defined as threshold cycle.
Statistical analysis
Data presented are the averages of 3 replicates. The experiment
was performed in a factorial completely randomized design.
The data from the experiments were subjected to 2-way
ANOVA and followed by separation of means at P  0.05.
Standard error of each mean was calculated to represent that in
the tables.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
The first author is highly thankful to the Department of Science & Tech-
nology, New Delhi, India for providing financial support. The authors are
also grateful to All India Coordinated Rice Improvement Project, Indian
Council of Agricultural Research, New Delhi, India for providing necessary
facilities for conducting the present investigation.
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