Microsoft Word - 6-Agra_23461
709
Original Article
Biosci. J., Uberlândia, v. 31, n. 3, p. 709-718, May/June. 2015
PHYSIOLOGICAL AND MOLECULAR CHARACTERIZATION OF
DROUGHT RESPONSES AND SCREENING OF DROUGHT TOLERANT
RICE VARIETIES
CARACTERIZAÇÃO FISIOLÓGICA, MOLECULAR E SELEÇÃO DA RESPOSTA A
TOLERÂNCIA À SECA EM VARIEDADES DE ARROZ
Loo Lean FEN
1
; Mohd Razi ISMAIL
1
; B. ZULKARAMI
1
;
Mohammad SHUKRI Abdul RAHMAN
1
; M. Robiul ISLAM
1, 2
1. Laboratory of Food Crops,
Institute of Tropical Agriculture Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia; 2.
Department of Agronomy and Agricultural Extension, Rajshahi University, Rajshahi 6205, Bangladesh. razi@putra.upm.edu.my
ABSTRACT: Drought stress has now become a severe threat to ensure food security in the developing world as
well as in Malaysia. To dertermine physiological and molecular determinants of drought stress and screening of drought
tolerant rice varieties, an experiment was conducted in a greenhouse at Universiti Putra Malaysia using eleven rice
varieties and two irrigation regimes (well irrigated and water stressed). The present study indicated that traditional rice
variety Puteh Perak and Siam is superior drought tolerant while IRRI 2011- IRLON Plot no: 064, MR 220 and BRRI
Dhan 56 are moderately drought tolerant, and IRRI 2011- IRLON Plot no: 050 and MR 84 are drought sensitive rice
variety. Drought tolerance of those varieties were measured based on rate of tiller reduction, leaf rolling score and drought
score during water stress condition. Leaf rolling score was positively correlated to drought score, chlorophyll content and
proline accumulation. Significant increase in the proline accumulation and antioxidant enzyme activities (peroxidase and
catalase) were also observed under drought stress in all the rice varieties except Siam and Puteh Perak. All the rice
varieties including drought tolerant and sensitive showed the existence of OsLEA 30 genes.
KEYWORDS: Drought stress. Leaf rolling score. Drought score. Oryza sativa L.. OsLEA 30 gene
INTRODUCTION
Rice (Oryza sativa L.) is one of the three
major food crops of the world. Being grown world
wide, it is the staple food for more than half of the
world’s population. It is a nutritious cereal crop,
provides 20% calories and 15% protein
requirements of world population. Besides being the
cheapest source of carbohydrate and protein in Asia,
it is also a source of minerals and fibre. Rice straw
and bran are important animal feed in many
countries. About 92% of the world's rice is produced
and consumed in Asia. A majour part of asian rice
grown under flooded irrigation and water is the
main limiting factor for increased production of rice
(AKINBILE et al., 2011).
The lower productivity of Asian rice in most
of the cases is attributed to various abiotic stresses
including drought. Drought is defined as water
stress mainly due to lack of rain during crop
growing period. Shortage of water is the main
obstacle for rice production in rainfed ecosystems
since most of the rice varieties are susceptible to
water stress (MOSTAJERAN; RAHIMI-EICHI,
2009). Drought stress has now become a severe
threat to ensure food security in the developing
world including Malaysia. Although water is
required all over the growth periods of rice plants,
there are some critical growth stages when drought
stress impacts seriously and create a massive
reduction in quality and quantity of yield (ISLAM et
al., 2011). The effect of drought on agriculture is
extensive as it limiting crop growth and yield. Other
than that, drought stress also involved with many
biochemical, molecular, and physiological changes
that influence various cellular and whole plant
processes and reduce quality and quantity of yield
(PRASAD; STAGGENBORG, 2008). Crop
responses to drought stress and its tollerence level
can be measured by monitoring different phyto-
physiological changes at increasing drought period
(ISLAM et al. 2011).
Developing rice plants resistance to drought
is one of the famous methods to increase crop
productivity (XOCONOSTLE-CAZARES et al.,
2010). Nevertheless, this approach requires an
understanding of physiological mechanisms at
different developmental stage and duration of
drought period. Hence, the aim of the present study
is screening of drought tolerant rice varieties and to
evaluate morphological, physiological, biochemical
and molecular aspects of stress tolerance in different
varieties of rice.
MATERIAL AND METHODS
Plant materials and growth condition
Received: 27/08/13
Accepted: 10/10/14
710
Physiological and molecular characterization… FEN, L. L et al.
Biosci. J., Uberlândia, v. 31, n. 3, p. 709-718, May/June. 2015
This study was conducted in a greenhouse at
Universiti Putra Malaysia using eleven rice varieties
(Oryza sativa cv. MR4, MR9, MR84, MR219,
MR220, Dular, Siam, Puteh Perak, BRRI Dhan 56,
IRRI 2011-IRLON Plot No: 50 and IRRI 2011-
IRLON Plot No: 64) collected from Gene bank
MARDI Research Station, Malaysia and Bangladesh
Rice Research Institute (BRRI), Bangladesh. Pre-
germinated seeds of each variety were grown in a
pot containing clay loom soil. Water stress was
imposed by withholding irrigation at 21 days after
sowing. Water level in well watered treatment
(control) was maintained at 5cm above the surface
of the soil. The experiment was arranged with
completely randomized design (CRD) with three
replications.
Soil moisture measurement
Soil moisture was measured by using soil
moisture meter (HH2) at 3, 6, 9, 12 and 15 days
after stress.
Chlorophyll content (SPAD value)
Chlorophyll content of leaf was determined
by using SPAD meter at 11 days after stress.
Leaf rolling score
Leaf rolling was recorded mid-day at 15
days after stress using the scale describe in Figure 1,
according to O'Toole and Cruz, 1979.
Figure 1. Flat (1) and tightly rolled (5) leaf and their rolling score.
Drought score
Data’s were taken on 15 days after drought
imposed and drought score was observed visually
using scale according to De Datta et al. (1988) as
mentioned in the Table 1.
Table 1. Leaf symptoms and corresponding drought score according to De Datta et al. (1988)
Drought score Description
1 No symptoms of stress effects
2 Slight tip drying
3 Tip drying extended to ¼ length in 25% of all leaves (normally the older leaves)
4 Tip drying extended to ¼ length in at most 50% of all leaves
5 Tip drying extended to ¼ length or more in 50% of all leaves with 25% of leaves
fully dried
6 50% of all leaves fully dried
7 More than 50% but less than 70% of all leaves fully dried
8 70% of all leaves fully dried
9 More than 70% of all leaves fully dried
10 All plants apparently dead
711
Physiological and molecular characterization… FEN, L. L et al.
Biosci. J., Uberlândia, v. 31, n. 3, p. 709-718, May/June. 2015
Proline determination
Free proline content was estimated
following the procedure described by BATES et al.
(1973). Youngest fully expanded leaves was frozen
in the liquid nitrogen and stored in a refrigerator at -
70°C. 0.5g of frozen sample was ground with liquid
nitrogen using pestle and mortar and homogenized
with 10 ml of sulfosalicylic acid (3%). The
homogenate was filtered using filter paper. 2 ml of
the filtrate was added with 2 ml of glacial acetic
acid and 2 ml acid ninhydrin in a test tube for one
hour in a water bath at 95°C. The reaction mixture
was then cooled in an ice bath for 10-15 minutes.
After that, 4 ml of toluene was added to the reaction
mixture and mixed vigorously with a test tube stirrer
for 20 seconds. Supernatant layer was put into a
quartz cuvette and measured at 520 nm using
spectrophotometer to get the absorbance data.
Toluene was used as blank. Proline standard curve
was used to determine the concentration of proline
in the samples.
Peroxidase determination
Enzyme extraction was determined using
the procedure derived by Aebi (1984). 0.5g of
frozen leaf sample was crushed to a fine powder
with the present of liquid nitrogen. The mixture was
then centrifuged at 10,000g for 10 minutes at 4°C.
The supernatant was placed into a 2.5 ml centrifuge
tube. Enzyme extraction was stored in a refrigerator
at -80°C for long term uses. For peroxidase
measurement, 1.8 ml of reaction mixture and 200 µl
distilled water as a blank was placed in a quartz
cuvette. 1200 µ l of 71.4mM potassium phosphate
buffer, 200 µ l of 1 mM EDTA, 200 µl of 1 mM
H2O2 and 200 µ l of 1 mM guaiacol were added into
the cuvette for sample measurement. The reaction
was started by added 200 µ l of enzyme extraction at
420 nm for 30 seconds using spectrophotometer to
get the absorbance data. Protein absorbance of each
sample was measured by 40 µ l of enzyme extraction
and 2 ml of Bradford reagent at 595 nm using
spectrophotometer. Protein standard curve was used
to determine the concentration of peroxidase in the
samples.
Catalase determination
Catalase activity (CAT) was measured with
modified procedure of AEBI (1983). 120µl enzyme
extract was added to reaction mixture consisted
2.8ml of 50mM phosphate buffer (pH=7.0) and 80µl
of H2O2. Catalase activity was determined by
measuring the increase in absorbance of H2O2
during 1min at 240nm in a spectrophotometer with
3ml of total reaction mixture. CAT activity was
expressed as change in absorbance by mmolmin
-
1
mg
-1
protein.
Detection of OsLEA30 genes
Plant material and RNA isolation: Fresh
leaf samples were used for genomic RNA extraction
and the RNA isolation was done with The
NucleoSpin
®
RNA/Protein kit. 0.1g of leaf sample
was homogenized carefully with mortar and pestle
in presence of liquid nitrogen. Cell lysis began when
the extraction transferred to a test tube containing
350µ l Buffer PL2 and 3.5µl β-mercaptoethanol. The
mixture was vortex thoroughly. Lysate was filtrated
through violet ring (NucleoSpin
®
Filter) and
centrifuged for 1 min at 11,000g. The violet ring
was dissolved with 350µ l ethanol (70%) and added
into the homogenized lysate. The sample was loaded
into NucleoSpin
®
RNA/ Protein Column (light blue
ring) and centrifuged for 30s at 11,000g. 350µ l
MDB was added to the sample and centrifuged at
11,000g for 1 min to dry the membrane. 95µ l
rDNase reaction mixture was applied directly onto
the center of the silica membrane of the column and
incubated at room temperature for 15 min. First
wash was done by added 200µ l Buffer RA2 to the
light blue ring and centrifuged for 30s at 11,000g.
The light blue ring was placed into a new collection
tube. During second wash, 600µl of Buffer RA3
was added to the light blue ring and centrifuged for
30s at 11,000g. The flow through was discarded.
250µ l Buffer RA3 was added at the third wash. The
mixture was centrifuged for 2 min at 11,000 g in
order to dry the silica membrane completely. The
light blue ring was placed into a new RNase-free
collection tube. The RNA was eluted in 60µ l
RNase-free H2O2 and centrifuged for 1 min at
11,000g. The nucleic acid concentration and purity
of nucleic acid samples were measured by using
NanoDrop 1000 Spectrophotometer.
sqRT-PCR amplification: The RNA
samples were used directly for sqRT-PCR with
OsLEA30 primers (Wang et al., 2007). The sqRT-
PCR amplification was performed in a volume of
20µ l containing 10µ l of KAPA SYBR
®
FAST qPCR
Master Mix (2X), 0.2µ l of 0.2µ M of OsLEA 30
forward and OsLEA30 reverse primer, 0.5µ l of
dUTP and KAPA RT Mix (50X), 4µ l of RNA
template and 4.6µl of nuclease-free water. The step
of cDNA synthesis was run together with
conventional PCR amplification where the initial
step of 42°C for 15 min was cDNA synthesis. The
following step was inactivate RT which was set at
95°C for 5 min. The conventional PCR
amplification was 95°C for 5 min, followed by 32
712
Physiological and molecular characterization… FEN, L. L et al.
Biosci. J., Uberlândia, v. 31, n. 3, p. 709-718, May/June. 2015
cycles of 95°C for 30 s, 59°C for 30 s and 72°C for
1 min and 1 cycle of 5 min at 72°C for the final
extension.
Agarose gel electrophoresis of PCR products
Amplified PCR product was analyzed by
1.5% agarose gel electrophoresis. About 0.375g of
agarose was melted in 25ml of 1X TBE buffer. The
gel mixture was poured into a gel tray with comb at
one end. After solidification, the agarose gel was
removed carefully and kept in gel electrophoresis
unit containing 1X TBE running buffer. 20µl of
PCR product was loaded into the wells. The
electrophoresis was carried out at 75 volt for about
40 minutes.
Statistical analysis
All data were analyzed using SAS software.
Each treatment was analyzed in three replications.
When ANOVA showed significant treatment and
variety effects, the Least significant Difference
(LSD) range test was applied to compare the means
at P<0.05.
RESULTS
Soil moisture content: Soil moisture in
well watered condition was always higher than
water stress condition as shown in Figure 2. Soil
moisture in well watered condition was maintained
at about 90% volumetric throughout the period of
treatment imposed while soil moisture in water
stress treatment decrease from 90% to 10% at 15
days after withholding water.
Figure 2. Soil moisture contents under well watered and water stress condition
Leaf rolling score
Water stress significantly affected the leaf
movement of rice. Response of leaf rolling score to
soil water stress was shown in Figure 3. According
to this experiment, Puteh Perak obtained the lowest
leaf rolling score among the varieties while MR
219, Dular and IRRI 2011- IRLON Plot no: 050
showed the highest leaf rolling score. Significant
differences were noted between the varieties and
leaf rolling score under water stress treatment. Leaf
rolling score was positively correlated with drought
score (r=0.82162), chlorophyll content (r=0.24426)
and proline activity (r=0.93852) (Table 4).
Drought score
Water stress significantly increased leaf
drying and increased drought score as describe in
Figure 3. Significant differences were obtained
between variety and drought score during water
stress condition. MR 84, MR 220 and IRRI 2011-
IRLON Plot no: 064 showed the lowest drought
score while IRRI 2011- IRLON Plot no: 050
showed the highest. Drought score was positively
correlated with chlorophyll content (r = 0.28539)
and proline activity (r = 0.85520) as shown in Table
4.
Number of tiller per plant
Number of tiller per plant under water stress
was found significantly lower than control (Table
2). MR 4 showed the highest number of tiller per
plant while Siam obtained the lowest number of
tiller during well watered treatment (control). MR 4,
MR 220, MR84 and IRRI 2011- IRLON Plot no:
050 recorded higher number of tiller per plant
during water stress. Number of tiller per plant was
negatively correlated with leaf rolling score (r = -
0.44778), drought score (r = -0.49797) and proline
activity (r = -0.45311) as shown in Table 4.
0
20
40
60
80
100
0 3 6 9 12 15
S
o
il
m
o
is
tu
re
(
v
o
l.
%
)
Days after withholding irrigation
w…
713
Physiological and molecular characterization… FEN, L. L et al.
Biosci. J., Uberlândia, v. 31, n. 3, p. 709-718, May/June. 2015
Figure 3. Leaf rolling score and drought score of eleven rice varieties during water stress; bars with different
letters indicate significant differences (P<0.05) according to Least Significant Difference test.
Table 2. Number of tiller per plant and chlorophyll contents in rice varieties under well watered and water
stressed condition.
Varieties Number of tiller per plant Chlorophyll content
(SPAD value)
Well watered Water stressed Well watered Water stressed
MR 4 20.00 a 9.33 a 43.50 a 44.20 a
MR 9 11.33 cd 7.33 ab 35.56 bc 38.56 ab
MR 84 16.66 ab 8.00 a 40.13 ab 42.36 a
MR 219 13.33 bc 6.66 ab 36.33 bc 41.53 ab
MR 220 10.00 de 9.00 a 32.90 c 34.80 c
Dular 8.33 ef 4.66 bc 38.10 ab 36.16 bc
Siam 5.00 g 4.33 c 37.33 bc 42.40 a
Puteh Perak 6.33 fg 4.66 bc 40.16 ab 40.10 ab
BRRI Dhan 56 10.33 de 7.33 ab 36.23 bc 40.26 ab
IRRI 2011- IRLON Plot no:050 15.00 bc 8.00 a 40.13 ab 43.33 a
IRRI 2011- IRLON Plot no:064 10.00 de 6.66 ab 37.40 bc 39.73 ab
Mean 11.48 6.90 37.97 40.31
Means with different letters within a column indicate significant differences (P < 0.05) according to Least Significant Difference test
Table 4. The correlation coefficients of the experimental traits
Tiller Lrs Drs Chl Pro POD
Lrs -0.44778*
Drs
-0.49797* 0.82162*
Chl 0.06925
0.24426*
0.28539*
Pro -0.45311* 0.93852*
0.85520*
0.20715
POD 0.18298
-0.10423
0.03579
0.21503
0.02499
CAT 0.15899
-0.07504
-0.12080
-0.17913
-0.04018
0.40727*
*represent the significant levels of P ≤ 0.05; Lrs – Leaf rolling score; Drs – Drought score; Chl – Chlorophyll content; Pro – Proline
content; POD – Peroxidase activity; CAT – Catalase activity
714
Physiological and molecular characterization… FEN, L. L et al.
Biosci. J., Uberlândia, v. 31, n. 3, p. 709-718, May/June. 2015
Chlorophyll content (SPAD value): All
the varieties showed higher chlorophyll content
during well-watered condition compared with water
stress except Puteh Perak and Dular (Table 2). MR
4, MR 84, Siam and IRRI 2011- IRLON Plot no:
050 showed the highest chlorophyll content while
MR 220 showed the lowest chlorophyll content
during water stress. However, there was no
significant difference between variety and
chlorophyll contents were observed in both watering
treatments.
Proline contents: The proline accumulation
was higher under water stress condition compared
with well watered condition in all the rice varieties
(Table 3). Among the rice varieties, MR 219
showed the highest increase in proline accumulation
(389.05%), followed by MR 9, BRRI Dhan 56, IRRI
2011- IRLON Plot no: 050, MR 4, Dular, MR 220,
MR 84, IRRI 2011- IRLON Plot no: 064, Puteh
Perak and Siam. Puteh Perak showed the lowest
value of proline activity during water stress.
Peroxidase activity
The peroxidase activities in leaves increased
for water stress and MR 4 and MR 220 showed the
higher peroxidase activity during well watered
condition (Table 3). MR 84 showed the highest
peroxidase activity during water stress condition
while MR 219 showed the lowest. All the varieties
showed higher peroxidase activity during water
stress condition except Siam, MR 4, MR 219 and
MR 220. These four varieties showed reduction in
peroxidase activity compared to well-watered
condition.
Catalase activity
The catalase activity was not affected by
water supply treatments but it varied within the rice
varieties (Table 3). MR 220 and IRRI 2011- IRLON
Plot no: 050 were the only two varieties that showed
significant difference under different irrigation
treatments. Generally, catalase activity decrease
during water stress. MR 220 and MR 9 showed
higher catalase activity during well watered
condition while MR 84 and MR 9 showed higher
catalase activity under water stress.
OsLEA 30 gene Detection
The OsLEA 30 gene detection of selected
rice varieties under water stress and well watered
was shown in Figure 4. All the varieties included
drought tolerant (BRRI Dhan 56) and sensitive (MR
84) variety showed the existence of OsLEA 30 gene
in both water stress and well watered condition. The
base pair of product was about 200 base pairs
according to the Figure 4.
715
Physiological and molecular characterization… FEN, L. L et al.
Biosci. J., Uberlândia, v. 31, n. 3, p. 709-718, May/June. 2015
Table 3. Proline content, Peroxidase and catalase activity in rice varieties under well watered and water stressed condition.
Varieties Proline content
Mg/g FW
Peroxidase activity
µ mol/mg protein/sec
Catalase activity
µ mol/mg protein/min
Well watered Water stressed Well watered Water stressed Well watered Water stressed
MR 4 5.20 bc 22.01 a 10.00 a 8.37 ab 2278 bc 193 cd
MR 9 4.78 d 23.18 a 8.01 ab 8.67 ab 3244 a 276 ab
MR 84 4.85 cd 15.84 bc 8.47 ab 9.80 a 2875 ab 304 a
MR 219 4.78 d 23.39 a 3.14 bc 2.30 c 777 e 107 ef
MR 220 4.86 cd 19.67 ab 8.36 ab 3.99 bc 3404 a 256 ab
Dular 5.48 ab 23.18 a 6.58 ab 7.17 ab 2221 bc 262 ab
Siam 5.92 a 12.06 c 5.91 ab 4.68 bc 1992 c 222 bc
Puteh Perak 5.31 bc 11.93 c 2.49 c 8.43 ab 1091 de 91 f
BRRI Dhan 56 4.96 cd 22.44 a 4.97 ab 7.37 ab 2298 bc 194 c d
IRRI 2011- IRLON Plot no:050 4.88 cd 20.96 a 5.48 ab 6.05 ab 1930 c 176 de
IRRI 2011- IRLON Plot no:064 5.09 cd 16.13b c 5.60 ab 8.62 ab 1665 cd 206 cd
Mean 5.10 19.16 6.27 6.85 2161.3 207.9
Means with different letters within a column indicate significant differences (P < 0.05) according to Least Significant Difference test
716
Physiological and molecular characterization… FEN, L. L et al.
Biosci. J., Uberlândia, v. 31, n. 3, p. 709-718, May/June. 2015
Figure 4. Detection of OsLEA 30 gene in selected rice varieties under both irrigation treatments by sqRT-PCR;
NTC- Non template control; T1- water stress; T2- well watered; V1- MR4; V9- BRRI Dhan 56; V4-
MR219; V8- Puteh Perak; V3- MR84; V6- Dular
DISCUSSION
The sensitivity of rice varieties to water
stress was noted during our experiment. MR 219,
Dular and IRRI 2011- IRLON Plot no: 050 were the
most sensitive variety to water stress while Puteh
Perak was less sensitive. Leaf rolling score was
positively correlated with drought score, chlorophyll
content and proline accumulation. According to
Chutia and Borah (2012), traditional rice varieties
(such as Dular, Siam and Puteh Perak) had long and
droopy leaves with larger leaf angle, are more
susceptible to rolling due to their ability to
conserved water in plant tissue. Reduction of
transpiration rate by creating microclimate is one of
the benefits of leaf rolling (KADIOGLU; TERZI,
2007; KADIOGLU et al., 2012). It has been
reported that greater leaf rolling may be an
important indicator linked to drought tolerance and
may have a positive impact on crop yield under
water stress conditions. Drought score is an
alternative method to determine drought stress
tolerance. Drought score of all the varieties were
ranged between 2 to 4, which indicates there nature
as moderately resistant variety to drought stress.
Siam, Puteh Perak, MR 4, MR 9 and IRRI 2011-
IRLON Plot no: 050 showed higher drought score,
and this result was similar to the findings of
Henderson et al., 1993, that the plants with higher
leaf area or with a larger number of plants per pot
showed higher drought score under water stress
condition. The number of tiller per plant decreased
with decreasing in soil moisture content. The
number of tiller was also associated with leaf rolling
score, drought score and proline accumulation.
Drought stress reduce turgor pressure in the cell and
therefore rusticated cell elongation and expansion
which ultimately caused reduction in plant height,
leaf area and crop growth (FAROOQ et al., 2009;).
The closing of stomatal aperture and inhibition of
photosynthesis during water stress declines internal
carbon dioxide concentration. Drought tolerance
varieties have the potentials to survive under such
unfavourable condition. Therefore, lower production
of tiller during water stress is also a determinant of
drought tolerance since traditional low tillaring
variety variety Siam and Puteh Perak showed higher
tolerance to drought stress.
Proline contents in the leaf tissues increased
significantly in all rice varieties except Siam and
Puteh Perak (Table 3). Proline acts as an important
osmolyte that widely produced by plants to stabilize
membranes and maintain the conformation of
proteins at low leaf water potentials. Free proline
accumulation is related to drought tolerance. Many
drought tolerant plant has high accumulation of
proline (KADIOGLU; TERZI, 2007).
Antioxidant enzyme activities (Peroxidase
and Catalase activity) were not affected either by
watering treatments or rice variety. Puteh Perak was
the only one variety, showed significantly increase
in peroxidase activity during water stress while MR
220 and IRRI 2011- IRLON Plot no: 050 showed
significantly decrease in peroxidase activity. Abedi
and Pakniyat (2010) reported increased in
peroxidase activity but decrease in catalase activity
in oilseed rape. Increased in both enzyme activity
during drought stress also reported in chickpea
(MAFAKHERI et al., 2010), soybean (XUE et al.,
2011) and rice. Decrease in peroxidase activity
under severe drought stress may reflect the low ROS
scavenging capacity and increased damage in cells
(ABEDI; PAKNIYAT, 2010). Reduction of
catalase activity is probably due to the inhibition of
717
Physiological and molecular characterization… FEN, L. L et al.
Biosci. J., Uberlândia, v. 31, n. 3, p. 709-718, May/June. 2015
enzyme synthesis or change in the assembly of
enzyme subunits under stress conditions (ABEDI;
PAKNIYAT, 2010).
LEA genes are a gene family plays
important role in protection of water stress. OsLEA
30 gene is one of the members in LEA gene family.
According to Wang et al. (2007), OsLEA 30 gene
usually expressed under normal condition.
However, this gene was expressed in all the selected
variety and thus conclusion on drought tolerance
variety was not able to make from this result alone.
This is due to the limitation of end point detection of
reverse transcriptase PCR, which do not expressed
the results as numbers. It is hard to differentiate
between the different fold changes on the agarose
gel. Hence, real time PCR is recommended for
further experiments on similar study.
The present study indicated Puteh Perak and
Siam is superior drought tollarent, IRRI 2011-
IRLON Plot no: 064, MR 220 and BRRI Dhan 56
are moderately drought tolerant variety while IRRI
2011- IRLON Plot no: 050 and MR 84 were drought
sensitive rice variety. Drought tolerant variety is
selected based on rate of reduction of number of
tiller, leaf rolling score and drought score. These
traits may have greater relevance and benefit to
future breeding program, particularly for screening
drought tolerance at early stage.
ANDACKNOWLEDGEMENT
This research is supported by Long term
Research Grant Scheme (LRGS), Food Security
Project, Ministry of Higher Education, Malaysia.
RESUMO: Tolerância à seca tornou-se um importante tema da segurança alimentar nos países em
desenvolvimento, bem como na Malásia. Com o objetivo de determinar os fatores fisiológicos e moleculares da tolerância
à seca em variedades de arroz foi conduzido um experimento em casa de vegetação na Universidade Putra da Malásia
usando 11 genótipos e dois sistemas de irrigação (vaso contendo plantas irrigadas e estressadas). O estudo mostrou que a
variedade Puteh Perak e Siam foram superiores em condições de estresse hídrico, enquanto IRRI 2011-IRLON – PLOT
064 e MR 220 e BRRI Dhan 56 foram de tolerância moderada e IRRI 2011-IRLON 050 foram genótipos que apresentaram
sensibilidade à seca. A tolerância à seca foi quantificada pela taxa de crescimento, nota visual para o enrolamento das
folhas durante e nível de estresse durante o tempo de estresse hídrico. Enrolamento das folhas foi correlacionado com o
nível de estresse hídrico, teor de clorofila e acúmulo de prolina. Aumento significativo no acúmulo de prolina e na
atividade enzimática anti-oxidante (peroxidase e catalase) foram também observadas sobre estresse hídrico em todas as
variedades de arroz tanto tolerantes quanto as sensíveis que esteve associado a existência de genes OsLEA 30.
PALAVRAS-CHAVE: Tolerância à seca. Enrolamento de folhas. Oryza sativa L. Gene OsLEA 30.
REFERENCES
ABEDI, T.; PAKNIYAT, H. Antioxidant enzyme changes in response to drought stress in ten cultivars of
Oilseed Rape (Brassica napus L.) Czech Journal of Genetics and Plant Breeding, v. 46, n. 1, p. 27-3, 2010.
AEBI, H. Catalase. In: Methods of Enzymatic Analysis (Vol. 3). Verlag Chemie, Weinheim, Florida. 1983.
AEBI, H. Catalase in vitro. Methods Enzymol, v. 105, p. 121-126, 1984. http://dx.doi.org/10.1016/S0076-
6879(84)05016-3
AKINBILE, C. O.; El-LATIF, K. M.; ABDULLAH, R.; YUSOFF, M. S. Rice Production and Water use
Efficiency for Self-Sufficiency in Malaysia: A Review. Trends in Applied Sciences Research, v. 6, n. 10, p.
1127-1140, 2011. http://dx.doi.org/10.3923/tasr.2011.1127.1140
BATES, L. S.; WALDREN, R. P.; TEARE, L. D. Rapid determination of free proline in water stress studies.
Plant and Soil, v. 39, n. 1, p. 205-207, 1973. http://dx.doi.org/10.1007/BF00018060
CHUTIA, J.; BORAH, S. P. Water stress efffects on leaf growth and chlorophyll content but not the grain yield
in traditional rice (Oryza sativa L.) genotypes of Assam, India ll. Protein and proline status in seedlings under
PEG induced water stress. American Journal of Plant Sciences, v. 3, n. 7, p. 971-980, 2012.
http://dx.doi.org/10.4236/ajps.2012.37115
718
Physiological and molecular characterization… FEN, L. L et al.
Biosci. J., Uberlândia, v. 31, n. 3, p. 709-718, May/June. 2015
De DATTA, S. K.; MALABUYOC, J. A.; ARAGON, E. L. A Field Screening Technique for Evaluating Rice
Germplasm for drought tolerance during the vegetative stage. Field Crops Research, v. 19, n. 2, p. 123-134,
1988. http://dx.doi.org/10.1016/0378-4290(88)90050-0
FAROOQ, M.; WAHID, A.; LEE, D. J.; ITO, O.; SIDDIQUE, K. H. M. Advances in Drought Resistance of
Rice. Critical Reviews in Plant Sciences, v. 28, p. 199-217, 2009.
http://dx.doi.org/10.1080/07352680902952173
HENDERSON, S. A., FUKAI, S., LILLEY, J. M., GEORGE, D. L., COOPER, M., WAMALA, M.
H.,WATIKI, J. M., VILLARICIENCIO, J. N. CHINYAMAKOBVU, E., UAIENE, R., LUDLOW, M. M.
Influence of water stress on leaf death among rice lines:comparison between glasshouse and field.
Proceedings of the 7th Australian Agronomy Conference. 1993.
ISLAM, M. R.; XUE, X.; MAO, S.; REN, C.; ENEJI, A. E.; HU, Y. Effects of water-saving superabsorbent
polymer on antioxidant enzyme activities and lipid peroxidation in oat (Avena sativa L.) under drought stress.
Journal of the Science of Food and Agriculture, v. 91, n. 4, p. 680-686, 2011.
http://dx.doi.org/10.1002/jsfa.4234
KADIOGLU, A.; TERZI, R. A dehydration avoidance mechanism: leaf rolling. The Botanical Review, v. 73,
n. 4, p. 290-302, 2007. http://dx.doi.org/10.1663/0006-8101(2007)73[290:ADAMLR]2.0.CO;2
KADIOGLU, A.; TERZI, R.; SARUHAN, N.; SAGLAM, A. Current advances in the investigation of leaf
rolling caused by biotic and abiotic stress factors. Plant Science, v. 182, p. 42-48, 2012.
http://dx.doi.org/10.1016/j.plantsci.2011.01.013
MAFAKHERI, A.; SIOSEMARDEH, A.; BAHRAMNEGAD, B.; STRUIK, P. C.; SOHRABI, Y. Effect of
drought stress on yield, proline and chlorophyll contents in three chickpea cultivars. Australian Journal of
Crop Science, v. 4, n. 8, p. 580-585, 2010.
MOSTAJERAN, A.; RAHIMI-EICHI, V. Effects of Drought Stress on Growth and Yield of Rice (Oryza sativa
L.) Cultivars and Accumulation of Proline and Soluble Sugars in Sheath and Blades of Their Different Ages
Leaves. American-Eurasian Journal of Agricultural and Environmental Science, v. 5, n. 2, p. 264-272,
2009.
O'TOOLE, J. C.; CRUZ, R. T. Leaf rolling and traspiration. Plant Science Letters, v. 16, n. 1, p. 111-114,
1979. http://dx.doi.org/10.1016/0304-4211(79)90015-4
PRASAD, P. V. V.; STAGGENBORG, S. A. Impacts of Drought and/or Heat stress on physiological,
developmental, growth, and yield processes of crop plants. In Ahuja, L. R.; Reddy, V. R.; Saseendran S. A.;
Yu, Q. (Eds.), Response of Crops to Limited Water: Understanding and Modeling water stress effects on plant
growth processes: American Society of Agronomy,Inc., Crop Science Society of America, Inc., Soil Science
Society of America, Inc. 2008.
WANG, X.; ZHU, H.; JIN, H.; WU, W.; ZHU, J. Genome-scale identification and analysis of LEA genes in
rice (Oryza sativa L.). Plant Science, v. 172, p. 414-420, 2007. http://dx.doi.org/10.1016/j.plantsci.2006.10.004
XOCONOSTLE-CAZARES, B.; RAMIREZ-ORTEGA, R. A.; FLORES-ELENES, L.; RUIZ-MEDRANO, R.
Drought Tolerance in Crop Plants. American Journal of Plant Physiology, v. 5, P. 241- 256, 2010.
http://dx.doi.org/10.3923/ajpp.2010.241.256
XUE, L.; ANJUM, S. A.; WANG, L.; SALEEM, M. F.; LIU, X.; IJAZ, M. F.; BILAL, M. F. Influence of straw
mulch on yield, chlorophyll contents, lipid peroxidation and antioxidant enzymes activities of soybean under
drought stress. Journal of Food, Agriculture & Environment, v. 9, n. 2, p. 699-704, 2011.