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Rice Science, 2011, 18(2): 116−126 Copyright © 2011, China National Rice Research Institute Published by Elsevier BV. All rights reserved
Impeded Carbohydrate Metabolism in Rice Plants under Submergence Stress
Malay Kumar ADAK1, Nirmalya GHOSH
1, Dilip Kumar DASGUPTA2, Sudha GUPTA
1
(1Plant Physiology and Plant Molecular Biology Research Unit, Department of Botany, University of Kalyani, Kalyani 741235, India; 2Department of Crop Physiology, University of Calcutta, Calcutta 700009, India)
Abstract: The detrimental effects of submergence on physiological performances of some rice varieties with special
references to carbohydrate metabolisms and their allied enzymes during post-flowering stages have been documented
and clarified in the present investigation. It was found that photosynthetic rate and concomitant translocation of sugars into
the panicles were both related to the yield. The detrimental effects of the complete submergence were recorded in
generation of sucrose, starch, sucrose phosphate synthase and phosphorylase activity in the developing panicles of the
plants as compared to those under normal or control (i.e. non-submerged) condition. The accumulation of starch was
significantly lower in plants under submergence and that was correlated with ADP-glucose pyrophosphorylase activity.
Photosynthetic rate was most affected under submergence in varying days of post-flowering and was also related to the
down regulation of Ribulose bisphosphate carboxylase activity. However, under normal or control condition, there
recorded a steady maintenance of photosynthetic rate at the post-flowering stages and significantly higher values of
Ribulose bisphosphate carboxylase activity. Still, photosynthetic rate of the plants under both control and submerged
conditions had hardly any significant correlation with sugar accumulation and other enzymes of carbohydrate metabolism
like invertase with grain yield. Finally, plants under submergence suffered significant loss of yield by poor grain filling which
was related to impeded carbohydrate metabolism in the tissues. It is evident that loss of yield under submergence is
attributed both by lower sink size or sink capacity (number of panicles, in this case) as well as subdued carbohydrate
metabolism in plants and its subsequent partitioning into the grains.
Key words: photosynthesis; sucrose; starch; phosphorylase; grain yield; rice; submergence
Photosynthetic assimilation and its simultaneous
apportionment is the key path to realize the yield in
relation to physiological attributes in crop plants,
particularly those for cereals (rice is one of those).
Accordingly, photosynthesis in plants monitors a very
fine tuning in metabolic conversion of sucrose, the
immediate and most translocatable sugar moiety in
developing grains of panicles (Weber et al, 2000).
Moreover, sucrose becomes a readily abundant precursor
for inter-conversion of starch and other storage
carbohydrate or even other polysaccharides. Now, in
rice grains, starch is one of the predominant
accumulated compounds in the endosperm cells of
grains as amylose and amylopectin in precised ratio.
Starch in the rice grain contributes almost 90% of the
final dry weight and becomes a subject for
investigation on its biosynthesis and regulation (Duan
and Sun, 2005). Grain filling, a process of starch
accumulation, has been reported that there are 33
major enzymes in rice endosperm (Ehleringer, 2002).
The regulatory enzymes are sucrose synthase, invertase,
ADP-glucose pyrophosphorylase, starch synthase, and
starch de-branching enzyme (Yang et al, 2001). The
most important sucrose synthase and its activity are
reported to be positively correlated with the rate of
starch accumulation in cereal grains like rice (Jeng et al,
2007). Another enzyme, ADP-glucose pyrophosphorylase,
is a producer of ADP-glucose, the initial or primary
moiety of starch biosynthesis in the rice endosperm,
and regarded as a rate limiting enzyme (Nakamura
and Yuki, 1992). Invertase provides the continued
supply of the reducing sugars (as glucose) for one
substrate of ADP-glucose pyrophosphorylase and is
correlated with the rate of starch synthesis in the
grains of cereals (Hurkman et al, 2003). The steady
inter-conversion of sucrose to starch and its
consequent acquisition into grains (as a sink) becomes
a rate limiting step for the current photosynthetic rate,
particularly at the post-flowering stages when plants
undergo maturation phase after vegetative growth. Received: 9 September 2010; Accepted: 22 May 2011 Corresponding author: ADAK M. K. ([email protected])
Malay Kumar ADAK, et al. Impeded Carbohydrate Metabolism in Rice Plants under Submergence Stress
117
Since the post-flowering stages are predominantly
characterized by grain filling, the photosynthetic
efficiency and its adequate maintaining during the
flowering period and onwards become significantly
contributory for grain filling, and varieties are
discriminated according to their efficiencies. Again,
sustaining the photosynthetic rate particularly under
the exposure of abiotic stress is also another important
parameter for adequate grain filling on physiological
aspects (Kato et al, 2007).
In rice, stagnation of water within a limit of
depth is essential throughout its life cycle and it varies
as the plants proceed towards different growth stages.
In general, the submergence is an acute problem at the
early growth stages which causes serious damages of
plants by uprooting of the seedlings particularly at
coastal lowland where traditional semi-dwarf varieties
are cultivated (Ismail et al, 2009). However, occurrences
of submergence at the heading stage or all through the
post-flowering stages are also frequent by prolonged
duration of monsoon or elevated water level due to
flash flood. This often turns out into the situation of
partial or complete submergence of plants and is more
prone to coastal regions and in the catchments lowland
areas of river coupled with drainage congestion. This
happens to be a major concern to rice plants for poor
development of spikelets, infertile grain, reduced
grain weight and finally substantial curtail of yield
(Perata and Voesenek, 2007).
Physiologically, grain yield of rice is the function
of a steady photosynthetic rate of leaves (source
activity) and its sustenance throughout the post-
flowering stages and concomitant translocation of photo-
assimilates into developing grains (sink efficiency). At
the cellular level, this is characterized as the collection
of some metabolic pathways which facilitate the
conversion of soluble sugars into storage carbohydrates
(polysaccharides) in grains. It is very much prudent
that inter varietal discrimination of grain yield both at
quantitative and qualitative level is based on the
partitioning and acquisition of storage carbohydrates in
the developing grains. Moreover, if submergence
extends up to the grain filling stage, it will leave the
grain tissues either partially or non-filled condition,
thus resulting in yield loss. For those cultivars grown
in lowland, they frequently do not exhibit high yield
potential due to poor grain filling by less synthesis of
photosynthates and/or conversion and allocation of
storage carbohydrates i.e., starch into grains (Ao et al,
2008). The slow grain filling problem under this
adverse situation, specially for the cultivars under
submergence, is also attributed by inadequate
partitioning of storage carbohydrate into grains by
more carbohydrates towards the vegetative part than
developing spikelets or grains. It is also mentioned
that plant tissues under hypoxic or anoxic condition of
submergence often turns out to be an exposure of high
oxygen tension when the water level recedes,
particularly at the end of flowering period in rice. This
is another mode of oxidative shock to the plants that
also manifests into impaired cellular metabolism of
developing panicle (Damanik et al, 2010). Finally, a
significant perturbance of metabolic pathways is
ensured that collectively sets a slow or poor grain
filling. Therefore, admittedly the vulnerability of
submergence for growth and development of rice
plants, the physiological process of grain growth also
has to compromise from its normal pace of development.
The low sink capacity under submergence becomes a
bottleneck for realization of satisfactory yield and
plants have to suffer from less panicle and spikelet
numbers (Surendra et al, 2009).
Actually, submergence before heading becomes
seriously detrimental to the plant’s survival, growth
and development, and thus workers have paid their
attention to characterization of plants on the basis of
physiological responses.
In rice, extensive work has been done on the
effects of environmental factors, predominantly water
and heat stress, on the starch biosynthesis and
enzymes involved in sucrose-starch relationship.
Since water deficit in the soil sets a limitation for
synthesis of sucrose, a readily photosynthetic, more
likely, the down stream pathways for starch biosynthesis
are also affected. A significant correlation in reduction
of starch content in rice plants have been reported
with concomitant down regulation of sucrose synthase
and starch synthase activity under heat stress during
heading period (Hurkman et al, 2003). Ahmadi and
Baker (2001) also reported that in water stressed
plants, it is the subdued activity for ADP-glucose
pyrophosphorylase which is responsible for reduced
grain growth. However, an concerted action of all the
enzymes for starch biosynthesis and duly replenishment
Rice Science, Vol. 18, No. 2, 2011
118
by stable photosynthetic rate during grain filling are
the most contributory physiological traits which undergo
modulation under water stress condition. Still,
information is meager on changes in the activities of
key enzymes in sucrose-starch inter-conversion when
the plants are under excess water in the form of
submergence or water logging. However, under
submergence, rice plants have modulated pattern of
partitioning dry matter for grains by enhancing
remobilization of pre-stored carbohydrates from pre-
flowering photosynthesis (Tang et al, 2009). Therefore,
the slow and inadequate grain filling in submerged
rice varieties might be compounded with lesser
carbohydrates mobilization from pre-stored source (as
in culm and leaf sheath before flowering) and
impairment of readily supplied grain filling materials
through starch-sucrose metabolic pathway. Thus, a
clear understanding is still required for the actual fates
of rice plants for response to submergence, when
physiology of grain filling is concerned.
Therefore, it is evident that the physiological
constraints for grain filling under submergence, however,
is also attributed by inadequate photosynthesis coupled
with impaired carbohydrate metabolism, particularly
for inter-conversion of sucrose-starch pathways in the
developing grains. So, a study that has a scope for
unraveling the basic aspect of metabolism for
synthesis of grain filling compounds (like starch, in
the present case) and its concomitant transportation to
the sink would be an realistic approach to explain
subdued grain yield in rice. Thus, the objective of the
present investigation is to explain how submergence
influences the activities of some enzymes like sucrose
phosphate synthase (SPS), invertase, ADP-glucose
pyrophosphorylase (AGPase) and Ribulose bis-phosphate
carboxylase/oxygenase (Rubisco) during grain filling
stage in some indigenous rice varieties.
MATERIALS AND METHODS
Plant growth and treatments
Eight semi-dwarf indica rice varieties (120 days
duration), namely Nagra 14/41, Achra 108/1, FR-13A,
Badkalamkati, CR-683-140, CR-644, CR-1012 and
Kalamdani were employed in the study. The seeds of
those varieties were sown in separate seed beds in wet
season (at the middle of June) for seedling development.
The 15-day-old seedlings were transplanted in earthen
wire pots (7 seedlings of each variety per pot) filled
with alluvial soil (pH 6.8) containing basal doses of
nitrogen (80 kg/hm2 N, 40 kg/hm2 P2O5 and 40
kg/hm2 K2O). Each variety was replicated thrice
randomly in different pots with a spacing of 5 cm×5
cm for plant to plant and 15 cm×15 cm for pot to pot.
The plants were maintained under natural condition as
of wet or rainy season with the temperature averaged
from 35ºC to 28ºC, relative humidity of 80%±5% and
an average photoperiod of 11–12 h. When the plants
were at the booting stage (i.e. 75 days after
transplanting), another nitrogen dose was applied. At
50% flowering or anthesis (i.e. 95 days after
transplanting), all the pots were transferred to an open
top cemented tank maintaining a water depth of
roughly 100 cm for submergence. The submergence
period was continued for 28 d, however, it was split at
every alternate four days by successive two days of
de-submergence (i.e. taking out the plants from
submergence). It simply refers to the flash flood or
intermittent flooding as prevails in rice fallows in wet
season during submergence (June to August/September).
Another set comprising of all the same varieties in the
pots as mentioned was kept out of submergence and
regarded as control or normal condition. After 28 days
under submergence, the pots were transferred from
water and kept under the normal condition for another
five days to acclimatize the post submergence period.
Meanwhile, during submergence period, the plants
from both control and submergence conditions were
taken for observations on physiological and biochemical
parameters by 7 days interval throughout the
submergence period. For the biochemical parameters
studies, the 1st, 2nd and 3rd leaves from the main shoot
of the plants from each hill were sampled, and for
each at least three replications were taken. After
sampling, the materials were frozen in liquid nitrogen
and stored at -70ºC for further use.
Measurement of photosynthetic rate
The photosynthetic rate [Po, µmol/(m2·s)] of the
2nd leaf from the top of the main shoot was measured
as by an IRGA coupled with portable photosynthetic
system (LI-COR 6200, USA) at around 11:00 AM
[PAR=900–1000 E/(m2·s)]. The flow rate in the leaf
Malay Kumar ADAK, et al. Impeded Carbohydrate Metabolism in Rice Plants under Submergence Stress
119
chamber was maintained as 700–800 Mpa and relative
humidity was around 70%–80% with leaf temperature
at 35±2ºC.
Biochemical studies
For biochemical estimation, each variety was
sampled under each treatment and immediately frozen
in liquid nitrogen and stored at -70ºC for further use.
Estimation of starch and soluble sugar contents from
leaves and panicles was done from alcoholic extracts
following the standard method with acidified anthrone
reagent (Hodge and Hofreiter, 1962). The sugar
profile in developing panicles was determined by
separating those on thin layer chromatogram (E-Merk,
Germany) with butane, acetic acid and water as
solvent with different standards of sugars. Finally,
separated sugars were quantified by scraping those
spots, eluting in 80% alcoholic solvent followed by
analysis with the standard methods and expressed as
fresh weight (FW) basis according to Reddy and Mitra
(1984).
Enzyme assays
Starch synthase and starch branching enzymes
are the key enzymes for starch biosynthetic pathway,
and have also been reported in other studies for grain
filling in rice. Moreover, it has been observed that the
activities of these enzymes were subdued in inferior
spikelets under water stress than in normal spikelets
during grain filling stage (Yang et al, 2001). However,
during the submergence period, the activities of other
enzymes viz. SPS, invertase, AGPase and Rubisco
were not much referred. Therefore, in the present
investigation, the activities of SPS, invertase, AGPase
and Rubisco were assayed following the referred
methods with modification.
In vitro assay of Rubisco (E.C.4.1.1.39.) was
conducted by partial purification of leaf protein
extract (extraction buffer: 50 mmol/L Tris-HCl, pH
7.5, 1 mmol/L EDTA, 10 mmol/L MgCl2, 12.5%
glycerol, 1% PVP, 0.5 mmol/L DTT and 1% PVPP).
This was centrifuged at 10 000 r/min at 40ºC for 15
min. The supernatant was saved, followed by 80%
ammonium sulphate fractionation of protein and
subsequent dialysis for over night at 4ºC. The assay of
Rubisco was done with coupled enzyme assay as
referred by Jiang et al (1994). The reaction mixture
for assay was incubated at 37ºC in 1.5 mL volume of
50 mmol/L HEPES-KOH (pH 8.0), 1 mmol/L EDTA,
20 mmol/L MgCl2, 2.5 mmol/L DTT, 1 mmol/L
NaHCO3, 5 mmol/L ATP, 5 mmol/L PMSF, 0.15
mmol/L NADH, 10 U of phosphoglycerate kinase, 10
U of 3-phospho glycerate dehydrogenase, 10 U of
phosphocreatine kinase, 0.5 mmol/L RuBP (Ribulose
1,5-bisphosphate) and 50 µg of protein. The oxidation
of NADH was recorded by absorbance at 340 nm and
the activity was computed as referred by Jiang et al
(2006). The protein was estimated as suggested by
Bradford (1976). The assay of SPS (E.C 2.4.1.13) and
AGPase (E.C.2.7.7.27) were conducted from the
extracted protein of the frozen samples with respective
buffers followed by partial purification in ammonium
sulphate (80%) saturation and desalted by dialysis.
The faction of proteins recovered and lyophilized to
concentrate aliquot was assayed for different enzymes.
For assay of AGPase, 50 µg extract of protein was
incubated at 37ºC with 1 mL of reaction mixture
containing 75 mmol/L HEPES (pH 7.9), 5 mmol/L MgCl2,
1 mmol/L ADP-glucose, 1 mmol/L Na-pyrophosphate,
2 mmol/L NAD, 4 U/mL phosphoglucomutase, 10
U/mL glucose-6-P dehydrogenase. The OD value was
read at 340 nm at 24ºC and the enzyme activity was
computed (Weber et al, 2000). SPS activity was
assayed according to Saman et al (1995) in a 2 mL
reaction mixture of 10 mmol/L UDP-glucose, 10
mmol/L fructose-6-P, 40 mmol/L glucose-6-P, 50
mmol/L MOPS (pH 7.9), 15 mmol/L MgCl2, 2.5
mmol/L DTT and 50 µL of diluted enzyme extract.
After incubation at 25ºC for 10 min, the reaction was
terminated by 70 µL of 30% KOH for 10 min in
boiling water bath. Treat the mixture with 15 µL ice
cold anthrone solution for 30 min followed by boiling
at 40ºC for 1 min, and finally read the absorbance at
620 nm. The activity of enzyme was computed as
µmol sucrose produced per gram fresh tissue per min.
Invertase (E.C.3.2.1.26) activity was recorded
from the enzyme extract with 1.5 mL assay mixture
containing citrate buffer (pH 3.8), sucrose (200
mmol/L) and properly diluted enzyme extract (50 µg
protein) under incubation of 37ºC for 90 min
(Hubbard et al, 1989). The reaction was terminated
with 1.5 mol/L NaOH (pH 6.5) at 100ºC for 30 min.
Rice Science, Vol. 18, No. 2, 2011
120
Glucose and fructose products were measured according
to Jones and Outlaw (1981). Total protein was estimated
with Bradford reagent with Bovine serum albumin
(Bradford, 1976).
At harvest, the plants were evaluated for grain
yield and yield components (panicle number, spikelet
number, 1000-grain weight) for each variety under
each treatment.
Statistical analysis
All the observations were recorded with three
replications and the data were expressed as mean±SE.
The statistical analysis was performed by one-way
ANOVA analysis, taking P≤0.05 as significant.
RESULTS
Periodical observations of physiological and
biochemical aspects of plants were made at specific
days after flowering (DAF) and significant variations
were recorded under normal and submerged conditions.
Likewise, the changes of the photosynthetic rate of the
rice varieties throughout the post-flowering stages
under the submerged condition remained significantly
subdued as compared to those of the varieties under
the normal condition (Fig. 1). It is interesting to note
that the varieties under the normal condition
maintained a steady photosynthetic rate through the
post-flowering stages (up to 21 DAF). However,
under submergence, plants showed a steady decline in
photosynthetic rate much earlier and even from 7
DAF. On an average during the post-flowering stages,
plants under submergence had curtailed their
photosynthetic rate significantly (P≤0.05) by almost
38.9% than those under the normal condition (Table
1).
It is well known that the most unique and notable
enzyme for CO2 fixation in C3 plants such as rice is
Rubisco. Thus, an assay of this enzyme recorded some
interesting results for the plants under the normal and
submerged conditions. The enzyme activity was
maintained a steady rate up to 7 DAF in the plants
Table 1. Photosynthetic rate, RuBP carboxylase/orygenase (Rubisco) activity, Km for Rubisco activity, sucrose content, sucrose phosphate synthase (SPS) activity, Km for SPS activity of rice varieties during post-flowering stages under normal and submerged conditions.
Photosynthetic rate [µmol/(m2·s)] Rubisco activity [μmol/(g·min)] Km for Rubisco activity (μmol/L) Variety
Normal Submergence Normal Submergence Normal Submergence
Nagra 14/41 36.4±0.3 20.9±0.2 465.7±4.1 301.1±2.9 12.4±0.2 85.5±0.8 Achra 108/1 39.3±0.4 25.9±0.3 396.9±3.2 300.2±2.6 13.9±0.1 90.1±0.9 Badkalamkati 41.3±0.4 31.8±0.2 440.1±4.2 265.8±2.5 17.6±0.5 95.6±0.9 FR-13A 55.1±0.6 32.4±0.3 369.3±3.6 275.8±2.7 11.1±0.1 78.3±0.7 CR-683-10 32.4±0.2 31.8±0.3 475.2±4.5 310.1±2.9 14.2±0.3 87.7±0.8 CR-644 41.2±0.4 17.2±0.1 353.2±3.3 214.1±1.8 15.6±0.6 89.9±0.8 CR-1012 34.0±0.3 16.7±0.1 388.5±3.6 114.1±1.1 10.3±0.1 90.1±0.9 Kalamdani 28.6±0.2 17.9±0.2 224.1±2.1 70.2±0.7 13.3±0.2 93.3±0.9 Mean 38.5±0.3 23.5±0.2 388.5±3.7 206.5±2.1 13.6±0.4 88.8±0.8
Sucrose content (μg/g) SPS activity [μmol/(g·min)] Km for SPS activity (μmol/L) Variety
Normal Submergence Normal Submergence Normal Submergence
Nagra 14/41 160.3±1.6 61.3±0.6 87.2±0.7 30.3±0.2 142.7±1.1 310.3±2.2 Achra 108/1 127.5±1.2 40.1±0.3 93.1±0.8 35.5±0.3 150.9±1.2 360.6±2.9 Badkalamkati 166.0±1.6 56.1±0.5 87.2±0.5 33.6±0.1 140.5±1.3 315.1±2.1 FR-13A 151.3±1.5 50.2±0.4 110.0±0.9 36.7±0.2 148.8±1.2 317.3±1.9 CR-683-10 153.0±1.1 40.1±0.2 79.5±0.5 27.3±0.3 140.3±1.3 305.5±2.7 CR-644 129.8±1.4 50.2±0.1 87.5±0.8 31.3±0.1 165.5±1.5 320.5±2.3 CR-1012 146.2±1.3 51.3±0.5 101.3±0.9 30.7±0.3 156.8±1.4 306.8±3.1 Kalamdani 132.7±1.2 40.3±0.3 76.5±0.6 24.3±0.2 171.1±1.6 338.1±3.2 Mean 145.8±1.6 48.7±0.4 90.2±0.8 31.2±0.3 152.1±1.4 321.7±3.1
Values are means ±SE (n=3).
Fig. 1. Photosynthetic rate of rice varieties under normal and submerged conditions at different days after flowering.
Malay Kumar ADAK, et al. Impeded Carbohydrate Metabolism in Rice Plants under Submergence Stress
121
under submergence, but thereafter it fell more rapidly
as compared to the plants under the normal or control
condition (Fig. 2). The average activity of Rubisco
was significantly higher (1.88-fold) in the plants under
normal than those under the submerged condition
(Table 1). The loss of enzyme specific activity at the
post-flowering period was also significant for the
plants under submergence. This depletion of specific
activity could also be supported by the loss of protein
and changes in the affinity for substrate as also
recorded from its values of Km (Table 1).
Simultaneously, a significant (P≤0.05) depletion
of the sucrose content in the leaves of the plants under
submergence (66.6%) was recorded as compared to
those of the plants under the normal condition (Fig. 3
and Table 1). However, the photosynthetic rates of
those varieties had hardly any significant correlations
with sucrose synthesis through the post-flowering
stages, but the enzyme for sucrose synthesis i.e. SPS
from leaves was recorded a higher value under the
normal condition and it was almost 2.89-fold higher at
the post-flowering stages than that in the submerged
plants (Fig. 4 and Table 1). Maintenance of activity
was recorded throughout the post-flowering stages
under the normal condition, but a gradual decline in
the activity was recorded from 7 DAF and onward
under submergence. A significant deviation of specific
activity and Km had also proved the detrimental effects
of submergence on this enzyme in the plants under the
submerged condition (Table 1). Moreover, a significant
correlation was recorded with accumulating sucrose
content in the leaves with the enzyme activity under
the normal condition.
One of the sucrose hydrolyzing enzymes, the
invertase, was taken into consideration in the
developing panicles in the present experiment.
Interestingly, the higher activity of invertase (1.98
fold) of the plants under the submerged condition than
that under normal conditon was consistent through the
post-flowering stages at least up to 21 DAF and
thereafter sharply declined (Fig. 5 and Table 2). When
the products of invertase activity were taken into
account, plants under the normal condition recorded
with more accumulated reducing sugars in developing
panicles throughout the post-flowering stages. But the
panicles of submerged varieties had significantly
decreased the amount of reducing sugars over control
by 52.2% throughout the post-flowering stages (Table
2). The reducing sugar produced by invertase would
be a limiting factor as a precursor for synthesis of
storage compounds like starch. Presumably, maintaining
the respiration of developing panicle tissues that
consume more reducing sugars as respiratory substrate
would have supported it, particularly when the plants
were under submergence, a form of hypoxic condition.
By chromatographic separation of different sugar
profiles in panicles and their estimation revealed that
Fig. 2. RuBP-carboxylase/oxygenase (Rubisco) activity in leaves of rice varieties under normal and submerged conditions at different days after flowering.
Fig. 3. Sucrose content in the leaves of rice varieties under normal and submerged conditions at different days after flowering.
Fig. 4. Activity of sucrose phosphate synthase (SPS) in leaves of rice varieties under normal and submerged conditions at different days after flowering.
Rice Science, Vol. 18, No. 2, 2011
122
glucose, fructose and galactose were variably
accumulated irrespective of plants under the normal
and submerged conditions. However, sugar contents
were insignificant (P≤0.05) in both the cases of normal
and submerged conditions (Data for each type of
sugars are not presented here).
Moreover, the AGPase activity, a starch
synthesizing enzyme in plants, was recorded
significantly subdued under the submerged condition
(86%) to that of the normal condition (Fig. 6 and
Table 2). Also, the accumulation of starch was
significantly higher in the panicles of the plants under
normal than those under submergence and it was 2.78-
fold higher in case of former (Fig. 7 and Table 2). The
activity of AGPase in the panicles as expected was
significantly down regulated under submergence to
that under the normal condition and it was well
correlated with starch accumulation. It is interesting to
note that the plants under submergence was able to
keep the rate stable at least up to 14 DAF, still not
compatible significantly to those under normal, but
thereafter the photosynthetic rate declined steadily.
The exposure to submergence had also left
detrimental effects on yield and yield components in
the varieties with significant variation in comparison
to those under the normal condition. Likewise,
number of panicles and 1000-grain weight of the rice
varieties subdued by 12.0% and 19.3% respectively
under submergence (Table 3). Spikelet number as
recorded was also detrimentally affected under
submergence, irrespective of varieties, by 13.3% when
compared to the normal condition (Table 3).
Supposedly, inadequate grain filling under submergence
as revealed from the low grain weight (recorded as
Table 2. Invertase activity, ADP-glucose pyrophosphorylase (AGPase) activity, starch content, reducing sugar content of rice varieties during post-flowering stages under normal and submerged conditions.
Invertase activity [U/(μg·min)]
AGPase activity [U/(μg·min)]
Starch content (mg/g)
Reducing sugar content (mg/g) Variety
Normal Submergence
Normal Submergence Normal Submergence
Normal SubmergenceNagra14/41 31.77±0.2 64.27±0.6 11.35±0.08 1.31±0.008 163.32±1.6 63.94±0.5 25.2±0.2 12.5±0.1 Achra 108/1 30.57±0.3 56.72±0.5 10.66±0.07 0.76±0.001 179.78±1.7 63.9±0.7 26.6±0.1 13.6±0.2 Badkalamkati 23.84±0.1 47.74±0.3 27.62±0.10 2.35±0.004 171.00±1.5 59.72±0.6 24.8±0.3 11.9±0.1 FR-13A 36.87±0.3 63.99±0.5 14.52±0.08 1.06±0.002 147.00±1.6 56.20±0.4 23.4±0.3 10.2±0.2 CR-683-10 24.36±0.1 58.98±0.4 7.35±0.05 1.66±0.003 142.96±1.3 50.10±0.5 27.8±0.2 13.8±0.1 CR-644 29.02±0.2 53.06±0.4 14.32±0.06 0.31±0.002 206.24±1.9 77.52±0.7 28.9±0.4 14.1±0.1 CR1012 37.32±0.4 70.94±0.6 8.23±0.07 0.31±0.001 171.40±1.5 59.84±0.8 23.6±0.5 9.2±0.2 Kalamdani 26.89±0.2 60.75±0.5 12.51±0.09 0.65±0.001 148.58±1.3 46.30±0.5 25.9±0.2 13.1±0.2 Mean 30.08±0.3 59.67±0.5 13.33±0.08 1.86±0.005 166.28±1.5 59.69±0.6 25.8±0.3 12.3±0.1
Values are means±SE (n=3).
Fig. 5. Invertase activity in the panicles of rice varieties under normaland submerged conditions at different days after flowering.
Fig. 6. ADP-glucose pyrophosphorilase (AGPase) activity in the panicles of rice varieties under normal and submerged conditions at different days after flowering.
Fig. 7. Starch content of the panicles of rice varieties under normal and submerged conditions at different days after flowering.
Malay Kumar ADAK, et al. Impeded Carbohydrate Metabolism in Rice Plants under Submergence Stress
123
1000-grain weight) of the varieties could be caused by
the depletion of starch accumulation under inundated
condition of submergence. Finally, when grain yield
was taken to justify the impact of all those parameters
mentioned above, it was found that plants had suffered
a significant loss of grain yield by 13.7% under
submergence as compared to normal condition (Table 3).
DISCUSSION
Submergence, a form of inundation is quite
abundant in occurrence in rice fallows that sets a
bottleneck for proper functioning of cellular status by
its various facets. However, submergence predominantly
exposes plants to some sort of hypoxia and/or anoxia
as well as a condition prevailing with a cellular
environment that is characterized by an elevation of
steady-state concentration reactive oxygen species
(Setter et al, 1997). The depletion of photosynthetic
rate [µmol/(m2·s)] under submergence has been
documented previously and primarily it was based on
the loss of chlorophyll fluorescence, lowering of
stomatal conductance, intercellular CO2 concentration
as well as denaturing of the photosynthetic
machineries. Moreover, inundation owing to the
submergence also limits the carboxylation by low/
intermediate intercellular CO2 concentration that may
also subside the RuBP-carboxylase activity, rather
more favoring the oxygenation (Buchanan et al, 2004).
This deviating ratio of carboxylation to oxygenation
under submergence is more serious for switching over
the tissues to make it more prone to photorespiration
and thus plants become devoid of acquisition of sugar,
particularly at the post-flowering stages (Adak and
Das Gupta, 2002). The loss of CO2 so fixed,
characterizes the rice plants more photorespirer out of
high O2 tension, particularly at the de-submergence
period. In addition, the post-flowering period of rice
happens to be the most contributory for grain filling
which was limited under photorespiratory condition
and finally manifested into substantial loss of yield
(Kumar et al, 2006). Therefore, adequate current
photosynthetic rate of those varieties and their
reliability to sustain throughout the post-flowering
stages could be imperative for varietal performances
under the submerged condition for rice. Disintegration
of cellular membrane covering photosynthetic pigments
and binding proteins on chloroplast membrane become
significantly functional to curtail the photosynthesis
under submerged rice plants (Mommer and Visser,
2005). Admittedly, the synthesis of sucrose, the
predominant translocating products of current photo-
synthesis is stringent under the control of CO2 flux
into the leaves which was recorded as depleted under
submergence in the present experiment.
Now, total amount of fixed carbon available for
translocation depends upon subsequent metabolic
fates of the readily fixed photosynthetic products like
sucrose. Likewise, sucrose synthesis in leaves and its
following translocation determines the partitioning of
photosynthates into various parts of plants including
panicles and consequently determines the yield in case
of cereals like rice. It is well recorded that sucrose
accumulation in the submerged leaves were impeded
by the down regulation of SPS activities. In fact, the
increase in the rate of CO2 fixation in leaves generally
results in an increase in sucrose synthesis and its
transport if other factors remain non-limiting.
Moreover, in the present experiment, the photosynthetic
rate of submerged rice leaves was significantly
curtailed as compared to non-submerged ones and
thus impairing sucrose synthesis and its transport to
grains or other storage tissues as well. There is a limit
to the fixed carbon normally allocated into starch, the
predominant grain filling compound in the panicles of
rice (Moradi et al, 2007). It also leaves a clue that
under submergence, plants which fail to accumulate
substantial amount of starch in the panicles could
primarily be accounted by the down regulation of
starch biosynthetic pathways. AGPase, the primary
enzyme for starch biosynthesis, is one such enzyme
(Yang et al, 2001). A significant and rapid loss in the
activity of AGPase in the panicles became the
criterion for less developed grains in the submerged
plants, which could be expected at least in the present
experiment. A steady state of starch-sucrose interr-
elationship is the key determinant of allocation of
fixed carbon in photo assimilation in plants. Therefore,
impeded activity of AGPase in the panicles might be
clarified in the support of less developed grains in rice
under water deficit and other abiotic stress also
(Visser et al, 2003). The allocation of storage
Rice Science, Vol. 18, No. 2, 2011
124
carbohydrate/starch in culm and leaf sheath instead of
panicles also deprives the plants from proper grain
filling under submergence as also reported previously.
Moreover, to make provision for ample supply of
respiratory substrate by the hydrolysis of stored starch
or even from sucrose also restricts the mobilization of
those towards the grains. The fine-tuning of sucrose-
starch inter-conversions by metabolic control is the
actual key to allocate the fixed carbon and its
diversion into different compounds. Reports suggested
that under waterlogged condition, rice plants could
alternatively replenish sugars, mostly reducing types by
hydrolyzing the sucrose and other storage
carbohydrates on demand of respiratory substrate
(Sujatha et al, 2008). Since current photosynthesis is
readily depleted under inundated condition of
submergence, plants use its alternative source of
respiratory substrate from storage carbohydrates, and
this also sets a bottleneck for adequate accumulation
of starch in the panicles for grain filling (Kawano et al,
2009). Additionally, higher rate of sucrose hydrolysis,
i.e. invertase activity provides ample reducing sugars
as a substrate for sustaining minimum respiratory rate
of the vegetative tissues under anoxic/hypoxic
condition of submergence. Thus, understanding the
depletion of starch accumulation in developing grains,
especially in relation to sucrose metabolism, the
coordination of sink capacity also becomes imperative
to justify. The sink capacity otherwise, the panicle
number in the present experiment was significantly
reduced under submergence compared to normal as
recorded. In general, if submergence prevails
vegetative growth during early phases and extends
thereafter, plants in general fail to develop adequate
number of panicles as compared to those under non-
submerged condition. Under this circumstance, plants
also suffer from less panicle number, which sets a
bottleneck for reduction in yield potential as well. In
the present experiment, though plants were kept under
submergence during heading, still we notice that some
panicles started to degenerate and later on spikelets
under this condition sheds off. Finally, we observed
reduction in panicles and spikelets numbers as
compared to the control under submergence at the
harvest stage. Moreover, under submergence, the
changes in sink activity in terms of both SPS and
invertase function in the leaves and panicles
respectively had made it more complex with reduced
numbers of panicles and spikelets. The activity of
invertase in the sink is thought to be involved in early
sink growth and sucrose synthesis, and is associated
with polysaccharide synthesis during sink growth
(Quick and Shaffer, 1996). Under submergence, the
plants were recoded in relation to higher activity of
invertase, which was attributed to the maintenance of
low sucrose content in the panicles. Actually, invertase
activity offers a regulatory point for diversion of fixed
carbon into various metabolic pathways (Fridman and
Zamir, 2003). The hydrolyzed products of the sucrose
by invertase are compensated as respiratory substrate
for the sink tissues (panicles) rather than conversion
of grain filling materials like starch. Thus, starch
synthesis might have been suffered from limitation of
substrates for AGPase under the submerged condition.
In general, utilization of imported sugars by the sink is
maximized on high abundance of sugar produced from
current photosynthesis in leaves, since it ensures a
gradient of sucrose for uptake by the sink for further
conversions. However, on condition of diminished
sugar supply from leaves as found under submergence,
utilization is increasingly restricted in the sink tissues
but more towards the sites of vegetative parts.
Therefore, the greater the ability of the sink tissues to
store or metabolize imported sugars, the larger
potential to compete for assimilates exported by the
sources (Chen and Wang, 2008). In rice, the number
of panicles and the size of panicles are consistently
regarded as selection criteria for realizing adequate
productivity or grain yield. Supposedly, under the
condition of submergence, rice varieties exert a less
demand for partitioning of assimilates towards sink
(developing grains in panicles) from source (leaves),
thus setting a bottleneck for adequate assimilate
partitioning in favor of developing grains, which
consequently manifested into subdued yield under
submergence.
However, the limitation of carbohydrate metabolism
is not governed by a single factor like photosynthesis,
rather it is associated with the conversion of readily
photo assimilates into sucrose and starch moieties.
Still, development of less number of panicles in the
varieties under submergence could possibly be
Malay Kumar ADAK, et al. Impeded Carbohydrate Metabolism in Rice Plants under Submergence Stress
125
another constraint for adequate sink capacity that
might restrict the sufficient translocation of sugars
into the grains. Thus, it is clear that poor grain filling
of the rice varieties under submergence could pose a
serious problem and frequently limits the yield
potential. The physiological performance of the rice
varieties in the present experiment was evaluated only
on the basis of pot experiment, and the behaviour of
plants would be much more compounded under field
conditions, because there is ample chances for the
plants to be shocked by oxidative stress, which
happens when the water level recedes intermittently.
This immediate aerobic exposure/high oxygen tension
to plants after prolonged submergence is critical for
metabolic aspect of grain filling as reported previously
(Damanik et al, 2010), thus it is evident that plants are
more vulnerable to detrimental effects out of oxidative
stress under submergence during grain filling under
field condition than pot experiment. This could lead to
the possible discrimination between evaluation of rice
varieties under simulated condition of pot experiment
and natural field condition. However, submergence
during this period is unavoidable and by default the
countermeasures could only be the selection of those
rice varieties superior in grain filling even under the
submerged condition. The rice varieties with prolonged
sustenance of photosynthetic potential, proper
functioning of starch synthesis machineries and higher
translocation efficiency to developing spikelets might
be most contributory for this situation. Moreover, for
those varieties, transport of preserved carbohydrates
from vegetative parts to grains would be preferred to
realize adequate productivity. Modification over the
assimilate translocation of rice varieties under this
condition could be materialized by the use of proper
field practices, long/medium duration varieties and
chemical or hormonal exercise for delayed senescence
or ripening of panicles and finally, molecular
approaches for modulation of gene expression for
metabolism of grain filling. Besides the panicles,
however, other factors within the plants like root
activity, root generated osmotic or chemical signal
and their transportation, source-sink synchronization
for assimilate partitioning would be other selective
criteria for further investigation in regulation of grain
filling under such constraints of submergence.
REFERENCES
Adak M K, Das Gupta D K. 2002. Metabolic activities in some rice
varieties under submergence stress. Ind J Plant Physiol, 6: 312–
316.
Ahmadi A, Baker D A. 2001. The effects of water stress on
activities of key regulatory enzymes of sucrose to starch
pathways in wheat. Plant Growth Regul, 35: 81–91.
Ao H, Wang S, Zou Y, Peng S, Tang Q, Chen Y, Xiong C, Xiao A.
2008. Study of yield stability and dry matter characteristics of
super hybrid rice. Sci Agric Sin, 41: 1927–1936. (in Chinese
with English abstract)
Bradford M M. 1976. Methods for quantification of microgram
quantities of protein utilizing the principle of protein dye binding.
Ann Biochem, 72: 248–254.
Buchanan B B, Gruissem W, Jones R L. 2004. Flooding and
oxygen deficit. In: Biochemistry and Molecular Biology of
Plants. New Delhi: International Pvt. Ltd.: 1177–1179.
Chen H J, Wang S J. 2008. Molecular regulation of sink source
transition in rice leaf sheath during heading period. Acta Physiol
Plant, 30: 639–649.
Duan M J, Sun S S. 2005. Profiling the expression of genes
controlling rice grain quality. Plant Mol Biol, 59: 165–178.
Ehleringer J. 2006. Photosynthesis: Physiological and ecological
considerations. In: Taize L, Zeiger E. Plant Physiology. 4th Edn.
Sunderland, MA: Sinauer Associates, Inc.: 222–223.
Table 3. Number of panicles, number of spikelets, 1000-grain weight and grain yield of rice at harvest under normal and submerged conditions.
No. of panicles per m2 No. of spikelets per m2 (×102) 1000-grain weight (g) Grain yield (g/m2) Variety
Normal Submergence Normal Submergence Normal Submergence
Normal Submergence
Nagra14/41 161.3±1.7 142.1±1.4 297.3±3.1 242.5±2.4 27.6±0.2 19.4±0.1 373.1±3.8 310.7±3.1 Achra 108/1 127.0±1.2 101.1±0.9 187.5±1.9 164.7±1.5 22.1±0.1 18.4±0.2 313.2±3.5 285.1±2.7 Badkalamkati 121.6±1.2 110.3±1.1 183.6±1.8 155.7±1.4 21.3±0.2 15.6±0.1 293.5±2.9 241.2±2.3 FR-13A 135.2±1.4 114.5±1.1 241.3±2.6 217.4±2.0 24.1±0.3 20.8±0.3 326.4±3.2 301.8±2.9 CR-683-10 130.3±1.3 121.1±1.2 172.6±1.9 152.3±1.4 19.2±0.2 15.6±0.2 201.1±2.4 174.2±1.7 CR-644 131.7±1.3 125.2±1.1 157.8±1.6 131.3±1.2 20.1±0.3 16.8±0.1 251.8±2.1 201.5±2.0 CR-1012 104.3±1.0 84.8±0.8 157.1±1.4 141.5±1.4 21.2±0.2 18.7±0.2 223.4±2.3 182.5±1.8 Kalamdani 135.2±1.3 121.3±1.1 249.0±2.7 220.6±2.1 23.2±0.3 19.0±0.3 342.9±3.8 310.3±3.1 Mean 130.8±1.3 115.0±1.1 205.8±2.0 178.3±1.8 22.4±0.2 18.0±0.2 290.7±3.1 250.9±2.4
Values are means ±SE (n=3).
Rice Science, Vol. 18, No. 2, 2011
126
Fridman E, Zamir D. 2003. Functional divergence of a synthetic
invertase gene family in tomato, potato and Arabidopsis. Plant
Physiol, 131: 603–609.
Hodges J E, Hofreiter B T. 1962. Methods in Carbohydrate
Chemistry. New York: Academic Press: 67–69.
Hubbard N L, Huber S C, Mason Pharr D. 1989. Sucrose phosphate
synthase and acid invertase as determinants of sucrose
concentration in developing muskmelon (Cucumis melo L.)
fruits. Plant Physiol, 91: 1527–1534.
Hurkman W J, McCue K F, Altenbach S B, Korn A, Tanaka C K,
Kothari K M, Jonhson E L, Bechtel D B, Wilson J D, Anderson
O D. 2003. Effects of temperature on expression of genes
encoding enzymes for starch biosynthesis in developing wheat
endosperm. Plant Sci, 164: 173–181.
Ismail A M, Ella E S, Vergara G S, Mackill D J. 2009. Mechanism
associated tolerance in rain-fed lowland rice (Oryza sativa L.)
during germination and early seedling growth. Ann Bot, 103:
197–203.
Jeng T L, Wang C S, Chen C L, Sung J M. 2007. Expression of
granule bound starch synthase in developing rice grain. J Sci
Food & Agric, 87: 2456–2463.
Jiang D A, Hirasawa T, Ishihara K. 1994. Depression of photosynthesis
in rice plant with low root activity following soluble starch
application to the soil. Jpn J Crop Sci, 63: 531–538.
Jiang W, Zang C J, Chen G X, Wang P, Shi D W, Lu C G. 2006.
Response of photosynthetic fluctuation to less temperature in
flag leaves of rice genotypes at milky stage. Rice Sci, 12: 112–
118.
Jones J, Outlaw W. 1981. Enzymatic assay for sucrose. In: Konberg
H L. Techniques in Carbohydrate Metabolism. Amsterdam:
Elsevier: 1–8.
Kato T, Shinmura D, Taniguchi A. 2007. Activities of enzymes for
sucrose-starch conversion in developing endosperm of rice and
their associoation with grain filling in extra heavy panicle types.
Plant Prod Sci, 10: 442–450.
Kawano N, Ito O, Sakagami J I. 2009. Morphological and physiological
responses of rice seedlings to complete submergence (flash
flood). Ann Bot, 103: 161–169.
Kumar R, Sarawagi A K, Ramos C, Amarante S T, Ismail A M.
2006. Partitioning of dry matter during drought stress in rainfed
lowland rice. Field Crop Res, 8: 1–11.
Mommer L, Visser E J W. 2005. Under water photosynthesis in
flooded terestial plants: A matter of leaf plasticity. Ann Bot, 96:
581–589.
Moradi F, Ismail A M. 2007. Response of photosynthesis,
chlorophyll fluorescence and ROS-scavenging system to salt
stress during seedling and reproductive stages of rice. Ann Bot,
103: 197–209.
Nakamura Y, Yuki K. 1992. Changes in enzyme activities associated
with carbohydrate metabolism during development of rice
endosperm, Plant Sci, 82: 15–20.
Perata P, Vosenek A C J. 2007. Submergence tolerance in rice
requires Sub1A, ethylene response factor like gene. Trend Plant
Sci, 12: 43–46.
Quick W P, Schaffer A A. 1996. Sucrose metabolism in source and
sink. In: Zamarki E, Schaffer A A. Photoassimilate Distribution
in Plants and Crops: Sucrose-Sink Relationship. New York:
Marcel Dekker: 115–158.
Reddy M D, Mitra B N. 1984. Effects of complete submergence
on vegetative growth, grain yield and some biochemical changes
in rice plants. Plant & Soil, 87: 365–374.
Damanik R I, Mahmood M, Ismail M R, Ahmad S, Zain A M.
2010. Responses of antioxidative enzymes in Malaysian rice
(Oryza sativa L.) cultivars under submergence condition. Acta
Physiol Plant, 32: 739–747.
Saman P, Seneweera A, Basra S, Edward W, Barlow J, Conroy P.
1995. Diurnal regulation of leaf blade elongation in rice by CO2.
Plant Physiol, 108: 1471–1477.
Setter T L, Ellis M, Laureles E V, Senadhira D, Mishra S B,
Sarkarung S, Datta S. 1997. Physiology and genetics of submergence
tolerance in rice. Ann Bot, 79: 67–77.
Sujatha K B, Uprety D C, Nageswara Rao D, Raghuveer Rao P,
Dwivedi N. 2008. Up-regulation of photosynthesis and sucrose-
P synthase in rice under elevated carbon dioxide and temperature
conditions. Plant Soil Environ, 54: 155–162.
Surendra P, Shivanna H, Naik S T. 2009. Performances of rice
genotypes under semi-deepwater condition during kharif at Sirsi.
IRRN (0117–4185): 1–4.
Tang T, Xie H, Wang Y X, Lu B, Liang J S. 2009. The effects of
sucrose and absisic acid interaction on sucrose synthase and its
relationship to grain filling of rice (Oryza sativa L.). J Exp Bot,
60: 2641–2652.
Visser E J W, Voesenek L A C J, Vertapetian B B, Jackson M B.
2003. Flooding and plant growth. Ann Bot, 9: 107–109.
Weber H, Rolletschek H, Heim U, Golobek S, Gubataz S, Wobus S.
2000. Antisense inhibition of ADP-glucose pyrophosphorylase
in developing seeds of vicia narbonensis moderately decrease in
starch but increase in protein and affects seed maturation. Plant
J, 24: 33–43.
Yang J, Zhang J, Wang Z, Zhu Q, Wang W. 2001. Hormonal
changes in the grains of rice subjected to water stress during
grain filling. Plant Physiol, 127: 315–323.
Yang J C, Peng S B, Gu S L, Visperas R M, Zhu Q S. 2001.
Changes in activities of three enzymes associated with starch
synthesis in rice during grain filling. Acta Agron Sin, 27: 157–
164. (in Chinese with English abstract)