Ind J Plant Physiol.
(January–March 2013) 18(1):67–70
DOI 10.1007/s40502-013-0008-0
SHORT COMMUNICATION
Effect of waterlogging on physiological characteristics, yield
and quality of flue-cured tobacco
M. Anuradha • K. Sivaraju • V. Krishnamurthy
Received: 23 February 2012 / Accepted: 3 October 2012 / Published online: 16 April 2013
Ó Indian Society for Plant Physiology 2013
Abstract Flue-cured tobacco is an important commercial
crop, which is highly sensitive to the wet conditions. An
experiment was conducted with different periods of
waterlogging (0, 24, 48, 72 and 96 h) at flower bud initi-
ation stage to study its effect on gas exchange parameters,
activity of antioxidative enzymes, yield and quality of flue-
cured tobacco. The gas exchange parameters viz., photo-
synthetic rate, stomatal conductance and transpiration rate
and soluble proteins decreased with increase in waterlog-
ging period. The activities of peroxidase, polyphenol oxi-
dase and catalase as well as superoxide dismutase showed
variation under waterlogging condition which protects the
cells from reactive oxygen species generated under
waterlogging. With increase in period of waterlogging
yield was reduced by 23, 43, 76 and 82 % due to water-
logging for a period of 24, 48, 72 and 96 h, respectively.
Quality constituents viz., nicotine and reducing sugars
decreased with increase in the period of waterlogging at all
leaf positions on the plant. The results showed that tobacco
plants tried to counteract the adverse effects of waterlog-
ging by enhancing the activity of superoxide dismutase.
Keywords Waterlogging
Antioxidative enzymes

Yield
Quality
Flue-cured tobacco
Tobacco is an important commercial crop grown in India.
India occupies third position in the world with an annual
production of 750 million kg. As the country is endowed
with diverse agroclimatic zones, it grows all types of
M. Anuradha (&)
K. Sivaraju
V. Krishnamurthy
Central Tobacco Research Institute, Rajahmundry 533105, India
e-mail: radhama2k2@yahoo.co.in
tobacco, which are broadly classified as Flue-cured Vir-
ginia (FCV) and non-FCV types. Among the different
types of tobacco grown, flue-cured tobacco contributes to
huge amount of forex and excise earnings and grown under
varied agro climatic conditions. It is grown as a rain-fed
crop in light soils of Karnataka, with conserved moisture in
the heavy soils and under irrigated conditions in northern
light soils of Andhra Pradesh. In recent years due to
changing environment the crop is facing continuous heavy
rains during the crop growth period. The excess water in
the root zone stops gas exchange between the soil particles
and atmosphere and seriously impedes the flow of oxygen
to active root surfaces which in turn affects the growth and
development of the crop (Davis and Nielsen 1999). As
tobacco is very sensitive to waterlogging continuous rains
are causing heavy reduction in the yield and quality of flue-
cured tobacco. The objective of this study was to analyze
the effects of waterlogging on physiological and bio-
chemical changes, and yield and quality constituents (nic-
otine and reducing sugars) of flue-cured tobacco.
An experiment was conducted with popular flue-cured
tobacco variety Kanchan under different periods of
waterlogging (24, 48, 72 and 96 h along with control) in
five replicates. The cement pots were filled with 150 kg of
soil collected from CTRI Research Farm, Jeelugumilli,
West Godavari district, Andhra Pradesh. The soil is slightly
acidic in reaction with low soluble salts and chlorides.
Each pot received N, P2O5 and K2O @ 115, 60 and
120 kg ha-1, respectively. The plants were grown using
recommended practices. The tobacco plants were subjected
to waterlogging for 0, 24, 48, 72 and 96 h when the plants
attained the flower bud initiation stage. Later the plants
were allowed to grow. One week after recovery the gas
exchange parameters namely net photosynthetic rate, sto-
matal conductance and transpiration rate were recorded
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using the portable photosynthetic system (LICOR Model
6400). The fully formed leaf (5th leaf) from the top of the
plant was selected for all the observations. The leaf sam-
ples were homogenized in phosphate extraction buffer (pH
7, 0.1 M) and centrifuged at 18,000 rpm for 15 min at
4 °C. The supernatant was used for enzyme assay and
estimation of soluble proteins following the method of
Lowry et al. (1951). The activity of peroxidase was esti-
mated in a reaction mixture containing 2.5 ml of 0.1 M
phosphate buffer pH 7, 1 ml of 0.01 M pyrogallol, 1 ml of
0.005 M H2O2 and 0.1 ml of the enzyme and incubated for
5 min at 25 °C. The amount of pyrogallin formed was
estimated by measuring the absorbance at 420 nm and the
enzyme activity expressed as absorbance units mg-1 pro-
tein min-1 Polyphenol oxidase activity was estimated as
above without peroxide (Kar and Mishra 1976). Acid
phosphatase activity was estimated by the method of
Sadasivam and Manikam (1992). Enzyme was extracted
from leaf samples with 50 mM citrate buffer pH 5.3 for
acid phosphatase. Three ml of substrate solution (EDTA
40 mM, citric acid 40 mM and p-nitrophenyl phosphate
0.8 mM, pH 5.3) incubated at 37 °C for 5 min and 0.2 ml
of enzyme was added, mixed well and further incubated at
37 °C for 15 min. At the end of the incubation period
0.5 ml of this reaction mixture was added to 9.5 ml of
NaOH and the absorbance was measured at 405 nm.
Appropriate controls were maintained and liberated p-
nitrophenol was estimated using standard p-nitrophenol.
The enzyme activity was expressed as lg p-nitrophenol
released mg-1 protein min-1. The leaves were harvested as
and when matured and cured in an electric barn and
observations on yield characters were recorded. Cured leaf
samples collected from bottom, middle and top positions
were processed and analysed for quality constituents viz.,
nicotine and reducing sugars (Harvey et al. 1969). The
statistical analysis was done using the methods given by
Gomez and Gomez (1984).
The gas exchange parameters viz., photosynthetic rate,
stomatal conductance and transpiration rate decreased
gradually with increase in waterlogging duration from 0 to
96 h (Table 1). The reduction in the rate of net photosyn-
thetic rate was drastic beyond 48 h of waterlogging period.
Tobacco is very sensitive to waterlogging and plants grown
under wet soil conditions tend to become yellow, wilted
and show decrease in photosynthetic rate (Goodall 1958).
The reduction in net photosynthetic rate and transpiration
rate recorded due to waterlogging have also been reported
in many other crops (Bhagwat et al. 1986; Lee et al. 2004;
El-Shihaby et al. 2002).
Soluble protein content increased significantly in 24 h
waterlogged plants compared to control and decreased
significantly with increase in duration of waterlogging but
the protein content at 48 h waterlogged plants was signif-
icantly higher than control plants and at par with 72 h
treated plants (Table 2). Activities of peroxidase and pol-
yphenol oxidase increased significantly with increase in
duration of waterlogging up to 48 h and thereafter it was
decreased with increase in duration. Even under optimal
conditions many metabolic processes produce reactive
oxygen species (ROS). Among the four major active oxy-
gen species hydrogen peroxide and the hydroxyl radical are
most active, toxic and destructive. Antioxidative enzymes

Table 1 Effect of waterlogging

Period of Photosynthetic rate Stomatal conductance Transpiration rate
on gas exchange parameters in
waterlogging (h) (lmol m-2 s-1) (mol m-2 s-1) (mmol m-2 s-1)
tobacco
0 33.45 0.66 7.74
24 25.0 0.25 4.42
48 14.38 0.24 4.16
72 3.89 0.07 1.52
96 1.90 0.08 1.78
CD (0.05) 2.80 0.13 1.60

Table 2 Effect of waterlogging

Period of Soluble protein Peroxidase Polyphenol oxidase Acid phosphatase
on soluble protein content and
waterlogging (h) (mg g-1 FW) (DA420 mg-1 (DA420 mg-1 (lg of p-nitrophenol
activity of different enzymes in
protein min-1) protein min-1) released mg-1 protein min-1)
tobacco
0 59.5 0.206 0.084 2.673
24 76.72 0.257 0.101 3.558
48 66.89 0.325 0.135 3.141
72 56.72 0.209 0.082 2.585
96 54.22 0.200 0.070 2.185
CD (0.05) 5.30 0.05 0.008 0.072
FW Fresh weight

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Ind J Plant Physiol. (January–March 2013) 18(1):67–70 69

Table 3 Effect of waterlogging on yield characters the leaf positions on the plant. Nicotine content in the leaf
Period of Green leaf yield Cured leaf yield Grade index
increased with increase in the leaf position on the plant
waterlogging (g plant-1) (g plant-1) (g plant-1) from bottom to top. Reducing sugar content in the cured
(h) leaf increased due in the plants subjected to 24 h period of
waterlogging and thereafter it decreased with increase in
0 1108 159 125
duration of waterlogging (Table 4).
24 939 121 93
Under waterlogging condition, plants showed variation
48 421 91 69
isozymes of peroxidase, polyphenol oxidase and catalase
72 209 39 21
and increased the activity of superoxide dismutase into
96 148 28 14
protect the cells from reactive oxygen species generated
CD (0.05) 121 22 6
due to waterlogging. Induction of enzymes involved in the
antioxidative system has been shown for anaerobically
germinated rice seedlings after transfer to air (Ushimaru
Table 4 Effect of waterlogging on nicotine and reducing sugars at et al. 1997). Van Toai and Bolles (1991) suggested that
bottom, middle and top leaf positions of the plant
increase in superoxide dismutase activity during water-
Period of Nicotine (%) Reducing sugars (%) logging may probably has a critical role in the survival of
waterlogging the plant when oxygen levels increase as the flooding stress
(h) Bottom Middle Top Bottom Middle Top
abates. The results showed that tobacco plants showed
0 1.24 2.38 3.44 12.1 16.72 13.36 variation in the expression of some antioxidative enzymes
24 1.03 2.06 3.03 14.8 18.4 11.40 as well as enhanced the activity of superoxide dismutase to
48 0.96 1.90 2.5 6.74 11.93 11.79 counter the injurious effects of waterlogging for the sur-
72 0.82 0.96 1.02 6.29 7.73 3.73 vival of the plants but could not avoid the reduction in
96 0.82 0.76 0.86 3.74 4.74 3.70 yield and quality constituents of flue-cured tobacco.
CD (0.05) 0.20 0.13 0.37 1.41 0.82 1.18

play an important role in counteracting these ROS. Sub-
baiah and Sachs (2003) reported increase in hydrogen
peroxide and activity of peroxidase during waterlogging.
Acid phosphatase activity increased significantly with
increase in duration of waterlogging from 0 to 24 h and
decreased significantly with increase in duration but the
enzyme activity was significantly higher at 48 h water-
logged plants compared to control. Phosphatases are
reported to be induced under phosphorous (Pi) deficiency
during waterlogging in order to maintain certain level of Pi
inside the cells (Olmos and Hellin 1997). Sharma et al.
(2005) also reported significant linear increase in acid
phosphatase activity in sorghum during waterlogging
(Table 2).
With increase in duration of waterlogging yield and
quality were reduced significantly (Table 3). The reduction
in yield due to waterlogging for a period of 24, 48, 72 and
96 h were 23, 43, 76 and 82 % respectively. Nicotine and
reducing sugars were decreased with increase in the period
of waterlogging at all the leaf positions on the plant. Davis
and Nielsen (1999) reported that flooding of more than
48 h caused yield reduction of more than 60 % in flue-
cured tobacco compared to unflooded plants. Watson et al.
(1976) also reported reduction in yield due to waterlogging
in wheat, oat and barley. Nicotine content of cured leaf
decreased with increase in the period of waterlogging at all
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