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Research Article
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Exogenous Abscisic Acid Enhances Sugar Accumulation in Rice (Oryza sativa L.) under Drought Stress |
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Wattana Pattanagul
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ABSTRACT
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The objective of this study was to determine the effects of exogenous ABA on growth and carbohydrate metabolism in rice seedlings cv. Khao Dawk Mali 105 under drought stress. Rice seedlings were grown for 10 days and subjected to water restriction for 7 days. Drought stress caused significant reduction in shoot as well as root growth and biomass. Leaf relative water content and chlorophyll contents were reduced by drought stress. Application of exogenous ABA helps the plants by improving leaf relative water content. Drought stress also caused significant increases in sugar accumulation, accompanied by increases in sucrose phosphate synthase activities. Application of exogenous ABA enhanced sugar accumulation but decreased starch content in the leaf. The results indicated that sugar accumulation is associated with drought stress and exogenous abscisic acid can improve plant water status as well as enhances sugar accumulation under drought stress.
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Received: February 28, 2011;
Accepted: May 22, 2011;
Published: July 28, 2011
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INTRODUCTION
Water deficit is one of the crucial environmental factors limiting plant growth
and productivity and is expected to become increasingly important in many regions
because of the ongoing climate change (Xoconostle-Cazares
et al., 2010). It is well established that drought stress impairs
numerous physiological and biochemical processes in plants. Photosynthetic rate
is heavily reduced under drought condition due to stomatal closure, so it is
assumed to be responsible for decreased dry matter production (Sepehri
and Modarres Sanavy, 2003; Lawlor and Tezara, 2009).
The production of Reactive Oxygen Species (ROS) can seriously disrupt normal
metabolic process during stress through chlorophyll loss, membrane lipid peroxidation,
protein carbonylation, inactivating the -SH containing enzymes and ultimately
cell death (Farooq et al., 2009b; Basu
et al., 2010). Therefore, by limiting plant growth, production and
consumption of photoassimilates will inevitably be altered at both the leaf
and the whole plants levels (Praxedes et al., 2006).
Plants can response and tolerate water stress by altering their cellular metabolism
and evoking various defense mechanisms including stomatal closure, accumulation
of solutes, cell wall hardening and production of proteins and enzymes involved
in cellular protection and ROS scavenging (Guo et al.,
2006; Esfandiari et al., 2008; Omidi,
2010).
Abscisic Acid (ABA) has been shown to mediate many physiological and developmental
processes throughout the life cycle of plants including responses of plants
to environmental stresses. Its level increase as a result of stresses including
drought, salinity and cold stresses that involved cellular water stress (Khadri
et al., 2006). ABA is known to act as a major signaling molecule
involved in the response of plants to drought stress. Stress-related responses
induced by ABA often occur earlier than the change of plant water status during
soil drying and thereby constitute the first line of defense as soil water deficits
are encountered (Liu et al., 2005). The hormone
triggers stomatal closure to limit water loss through transpiration, as well
as mobilizes a battery of genes that presumably serve to protect the cells from
the ensuing oxidative damage in prolonged stress (Wasilewska
et al., 2008). These physiological and biochemical changes have been
proposed to reduce the deleterious effects induced by water stress. Many studies
have shown that ABA is able to induce changes including synthesis of stress
proteins, proline, sugar alcohols, soluble carbohydrates and glycine betaine
in which may involve in stress tolerance (Bagniewska-Zadworna
et al., 2007).
Carbohydrate metabolism is strongly affected by drought stress. Water stressed
plants often accumulate sugars and their derivatives, such as polyols and raffinose
family oligosaccharides (Valliyodan and Nguyen, 2006;
Toldi et al., 2009). Accumulation of these osmolytes
may help plants to tolerate dehydration by improving their ability to maintain
osmotic balance within the cell (Choluj et al., 2008;
Costa et al., 2008). Additional benefits of these
solutes have been described, including buffering cellular redox potential, protecting
the cell from dehydration by stabilizing membrane and protein structures and
providing possible energy source under severe stress (Hasegawa
et al., 2000). Furthermore, alteration in photoassimilate partitioning
between source and sink tissues may also contribute to the accumulation of these
solutes (Hare et al., 1998). The aim of this study
was to investigate the effects of exogenous ABA on growth and carbohydrate metabolism
in rice seedling under drought stress. The results could be important for better
understanding the mechanism of drought tolerance in rice.
MATERIALS AND METHODS Plant material, growth conditions and treatment: Rice (Oryza sativa L.) seeds, cvs. Khao Dawk Mali 105 was obtained from Rice Research Station, Khon Kaen, Thailand. Seeds were soaked in 5% sodium hypochlorite for 15 min and rinsed thrice with distilled water. Rice seedlings were germinated by placing the seeds on the moistened filter paper for 3 days. Approximately 10 seedlings were then potted in a plastic pot containing soil:sand:peat mix (1:1:2, v/v) and kept in the greenhouse at the Department of Biology, Faculty of Sciences, Khon Kaen University, Thailand under natural illumination and temperature conditions. The seedlings were watered daily with 100 mL half strength Hoagland solution and allowed to grow for 10 days. The seedlings were then separated into 4 treatments containing 5 pots per treatment. The control plants were sprayed to runoff with distilled water and watered daily with 100 mL distilled water. For the drought stress treatment, the plants were sprayed to runoff with distilled water, one day prior to drought treatment and water was withheld for a period of 7 days. For exogenous ABA treatment, the seedlings were sprayed to runoff with an aqueous solution of ABA (20 mg L-1) and watered daily with 100 mL distilled water. For exogenous ABA and drought treatment, the seedlings were sprayed to runoff with an aqueous solution of ABA (20 mg L-1), one day prior to drought treatment and water was withheld for a period of 7 days thereafter.
Plant growth, chlorophyll and relative water content measurements: After
drought treatment, the seedlings were randomly sampled from each pot. Root and
shoot lengths of 5 plants from each treatment were determined. Fresh weights
of shoot and root tissues were recorded and then the tissues were oven-dried
at 80°C for 3 days in order to determine dry weight. Five additional plants
from each treatment were randomly selected to determine chlorophyll and leaf
relative water contents. About 0.1 g leaf sample was used for chlorophyll determination.
Leaf samples were extracted with 5 mL of acetone and chlorophyll contents were
determined spectrophotometrically at 645 and 663 nm (Arnon,
1949). For leaf relative water content determination, about 0.1 g leaf sample
was cut into smaller pieces and weighed to determine fresh weight. The leaf
sample was floated in freshly de-ionized water for 12 h and weighed thereafter
to determine fully turgid weight. The leaf sample was oven-dried at 80°C
for 3 days and the dry weight was obtained. The Relative Water Content (RWC)
was determined using the following formula:
where, FW is fresh weight, DW is dry weight and tW is Turgid weight (Turner,
1981).
Carbohydrate extraction and analysis: Fully mature leaves were randomly
selected from each treatment and frozen on dry ice to transfer to the laboratory.
Approximately 0.1 g leaf tissue was used for carbohydrate analysis. Soluble
sugars were extracted from the tissues in hot 80% (v/v) ethanol and determined
as previously described (Pattanagul and Thitisaksakul, 2008).
Starch in the leaf residue was digested with amyloglucosidase overnight and
released glucose was quantified enzymically using hexokinase/glucose-6-P dehydrogenase.
Enzyme extraction and assay: About 1 g leaf tissue was collected during
midday for enzyme extraction and determination of sucrose phosphate synthase
and invertase activities. The leaf sample was ground on ice using mortar and
pestle in 5 mL grinding buffer containing 50 mM HEPES, 10 mM MgCl2,
1 mM EDTA, 0.25% BSA and 5 mM dithiothreitol, pH 7.5. The extract was filtered
through 4 layers of cheesecloth, then centrifuge for 1 min at 10000 g. Crude
extract was desalted on 2 mL Sephadex G25 columns equilibrated with the grinding
buffer. The amount of protein in the enzyme extract was determined by Bradford
method (Bradford, 1976).
SPS activity was determined by measuring sucrose-6-phosphate produced from
the substrates, UDP-glucose and fructose-6-phosphate. Approximately 80 μL
desalted enzyme was incubated in a reaction mixture containing 25 mM UDP-glucose,
8 mM fructose-6-phosphate, 5 mM MgCl2 at 25°C for 1 h and terminated
by adding 100 μL of 1 N NaOH. Unreacted fructose-6-phosphate and fructose
were destroyed by boiling the tube in a boiling water bath for 10 min. Sucrose-6-phosphate
formed during the reaction was determined by reacting with 0.25 mL resorcinol
solution and quantified by a spectrophotometer at 520 mM (Robbins
and Pharr, 1987).
Invertase activities were determined under acidic (pH 4.0) and alkaline (pH 7.6) conditions. Approximately 50 μL of desalted enzyme was incubated with 125 mM sucrose (w/v) in the extraction buffer at the appropriate pH. Assays were run at 30°C for 30 min and stopped by boiling for 1 min. The glucose content of 10 μL aliquots of the assay mixture was determined spectrophotometrically at 340 nm using hexokinase/glucose-6-P dehydrogenase enzymes. Data analysis: Statistical analyses were carried out by ANOVA tests with the SPSS program version 17.0 (SPSS Inc., Chicago, IL). Significant differences were determined by post-hoc comparison (Duncans multiple range test) at p<0.05. RESULTS
Plant growth, chlorophyll and relative water contents: Drought stress
caused significant reduction in shoot growth. Shoot length was reduced from
40.20 cm plant-1 in the control plants to 31.40 cm plant-1
in the droughted plants (Fig. 1a). Shoot fresh and dry weights
were also reduced from 0.34 and 57.28 mg plant-1 in the control plants
to 0.11 and 35.2 mg plant-1 in the drought plants (Fig.
1c, e). Shoot fresh weights were heavily reduced by drought
stress accompanied by the significance reduction in leaf relative water content.
Exogenous ABA also caused reduction in shoot growth, similar to the effects
of drought stress but to the lesser extends (Fig. 1c). In
addition, application of exogenous ABA also showed similar effects in the shoot
tissue as seen in the droughted plants. On the other hand, root lengths were
not significantly affected by drought stress. Both root fresh and dry weights,
however, were significantly reduced under drought stress. Root fresh and dry
weights were reduced from 0.09 and 11.52 mg plant-1 in the control
plants to 0.02 and 6.46 mg plant-1 in the drought plants (Fig.
1d, f).
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Fig. 1(a-f): |
Effects of drought and exogenous ABA on shoot length (a),
root length, (b) shoot fresh weight, (c) root fresh weight and (d) shoot
dry weight (e) and root dry weight (f) in mature leaves. Data represent
the Means±SE of five measurements per data point. Values showing
the same letter are not significantly different |
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Fig. 2 (a-d): |
Effects of drought and exogenous ABA on (a) relative water
content, (b) chlorophyll a, (c) chlorophyll b, (d) and total chlorophyll
in mature leaves. Data represent the Means±SE of five measurements
per data point. Values showing the same letter are not significantly different |
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Fig. 3 (a-d): |
Effects of drought and exogenous ABA on (a) total soluble
sugar, (b) sucrose, (c) fructose, (d) and starch (d) in mature leaves. Data
represent the Means±SE of five measurements per data point. Values
showing the same letter are not significantly different |
In contrast to the shoot tissue, application of exogenous ABA to the unstressed
plant caused no reduction in the root biomass.
Leaf relative water content was significantly reduced by drought stress. Leaf
relative water content was reduced from 91.89% in the control plants to 65.73%
in the drought plant (Fig. 2a). Exogenous ABA had no effects
in leaf relative water content in the unstressed plants. On the other hand,
exogenous ABA increased relative water content in the droughted plants to 75.93%
compared to 65.73% in the droughted plants (Fig. 2a). In addition,
drought stress also caused reduction in chlorophyll a, b and total chlorophyll
contents. Chlorophyll a, b and total chlorophyll were reduced from 0.97, 0.34
and 1.31 mg g-1 FW, respectively, in the control plants to 0.37,
0.13 and 0.51 mg g-1 FW, respectively, in the droughted plants (Fig.
2b, d). Although application of exogenous ABA showed slight
increases in chlorophyll contents compared to the droughted plants, the increases
were not significantly different.
Carbohydrate contents: Water deficit stress caused a significant increase
in leaf total soluble sugars. Total soluble sugar was increased from 232.40
μmol g-1 FW in the control plants to 623.98 μmol g-1
FW in the droughted plants (Fig. 3a). Sucrose and fructose
were also increased similarly (Fig. 3b, c).
Application of exogenous ABA also resulted in a higher increase in total soluble
sugars compared to the droughted plants. Exogenous ABA caused increase in leaf
total soluble sugar, sucrose and fructose to 672.89, 309.65 and 203.18 μmol
g-1 FW, respectively. Application of exogenous ABA to the unstressed
plants, however, showed no difference in total soluble sugar compared to the
control plants. Water restriction also resulted in an increase in leaf starch
content (Fig. 3d). On the contrary, exogenous ABA showed no
increase in starch accumulation compared to the control plants (Fig.
3d).
Enzyme activities: Accumulation of sugars in the stressed plants was
also reflected in an increase in SPS activities. SPS activities increased from
0.66 μmol product mg-1 protein h-1 in the control
plants to 1.11 μmol product mg-1 protein h-1 in the
droughted plants (Fig. 4a). Exogenous ABA also caused increases
in SPS activities to 0.96 μmol product mg-1 protein h-1.
In addition, application of exogenous ABA to the unstressed plants had no effect
on SPS activities.
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Fig. 4 (a-c): |
Effects of drought and exogenous ABA on activities of (a)
sucrose phosphate synthase, (b) acid invertase, (c) and alkaline invertase,
(d) in mature leaves. Data represent the Means±SE of five measurements
per data point. Values showing the same letter are not significantly different |
On the other hand, water deficit stress and ABA had no significant effect on
both acidic and alkaline invertase activities (Fig. 4b, c).
DISCUSSION
Upon water deficit stress, the seedling growth was largely decreased. This
may be partly due to lower turgor pressure and decreased photosynthetic rate
in the cells (Cha-Um et al., 2007; Regier
et al., 2009). Exogenous ABA also caused reduction in shoot growth,
similar to drought condition. The hormone has been known to trigger stomatal
closure in order to limit water loss under water stress, thus limiting photosynthetic
CO2 assimilate (Liu et al., 2005).
Exogenous ABA was also reported to reduce photosynthetic rate, stomatal conductance
and transpiration rate in cotton (Pandey et al.,
2003).
The reduction in leaf relative water content was provoked by the water deficiency
in soil (Hassanzadeh et al., 2009). Exogenous
ABA helps the plants to better maintain cellular water level which were also
reported in chickpea, Ilex paraguariensis and Polypodium vulgarei (Sansberro
et al., 2004; Bagniewska-Zadworna et al.,
2007; Kumar et al., 2008). In this study,
application of exogenous ABA resulted in increased sugar accumulation which
may partly responsible for improving leaf relative water content. In addition,
application of ABA are known to affect plant growth and development, mimicking
the effects of water stress, thereby helping plants to better survive stress
conditions (Farooq et al., 2009a).
Under drought condition, sugar accumulation was observed. The increase of sucrose
was accompanied by significance increase in SPS activities. Stressed-induced
overproduction of sugars has been reported in numerous species and is believed
to play an important role in drought tolerance. In soybean, total soluble carbohydrates,
sucrose and reducing carbohydrates were increased under water deficit (Adejare
and Umebese, 2008; Lobato et al., 2008). In
robusta coffee (Coffea canephora Pierre var. kouillou) leaves,
drought tolerance clones showed increases in hexoses and sucrose in response
to drought whereas their levels in drought sensitive clones remained unchanged
(Praxedes et al., 2006). Accumulation of sugars
can help to maintain osmotic balance as well as protect enzymes and membrane
against deleterious effects of destabilizing ions (Farooq
et al., 2009a). Furthermore, application of exogenous ABA even triggers
more sugar accumulation, but decreases starch content. ABA also causes increases
in sugar accumulation as well as decreased starch content in Polypodium vulgare
(Bagniewska-Zadworna et al., 2007). It has been
suggested that under drought stress, the products from starch hydrolysis could
be the substrate for sucrose synthesis (Lee et al.,
2008). ABA-induced sugar accumulation may partly increase osmotic adjustment
in which helps the plants to better survive under drought condition.
In conclusion, this study demonstrates that accumulation of sugars was associated with drought stress. Accumulation of sugar is considered to play an important role in drought tolerance. Exogenous ABA was also shown to help the plants to maintain their relative water content and enhanced sugar accumulation under drought stress. ACKNOWLEDGMENTS This research is supported in part by Khon Kaen University Research Fund (No. 523701).
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