Impeded Carbohydrate Metabolism in Rice Plants under … · Malay Kumar ADAK, et al. Impeded Carbohydrate Metabolism in Rice Plants under Submergence Stress 117 Since the post-flowering

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


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.


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


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.


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.


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.


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


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


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.

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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

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