J Agro Crop Sci (2013) ISSN 0931-2250

FLOODING STRESS

Different Physiological Reactions at Vegetative and

Generative Stage of Development of Field Bean Plants
Exposed to Flooding and Undergoing Recovery
E. Pociecha
Department of Plant Physiology, Faculty of Agriculture and Economics, University of Agriculture in Krakow Kraków, Poland

Keywords Abstract
enzymatic antioxidants; flooding stress;
growth; hydrogen peroxide Plants at various stages of growth are differentiated in their susceptibility to envi-
ronmental stresses and possibility of regeneration during recovery period after
Correspondence cessation of stress. The role of antioxidative enzymes in the stabilisation of the
E. Pociecha physiological state of field bean plants was studied. Catalase, peroxidises activity,
Department of Plant Physiology
and hydrogen peroxide accumulation were determined after 1 and 7 days of
Faculty of Agriculture and Economics
flooding and after 1 and 7 days of recovery from flooding. The influence of
University of Agriculture in Krakow
Podłużna 3 flooding on plants growth was analysed on the basis of leaf area index (LAI), rela-
30-239 Kraków tive growth rate (RGR) and net assimilation rate (NAR). Changes in antioxidative
Poland enzymes caused by flooding applied at vegetative stage were reversible. In genera-
Tel.: +48 12 425 3301 tive plants, antioxidative enzymes were activated to a greater extent during recov-
Fax: +48 12 425 3302 ery period and in contrast to vegetative plants did not attain the control level
Email: rrchilmo@cyf-kr.edu.pl
after that period. The reduction in growth was greater when flooding stress was
applied at vegetative stage. Vegetative plants regardless of the stabilisation of anti-
Accepted October 5, 2012
oxidant system were not able to restore the physiological function to attain the
doi:10.1111/jac.12009 optimal growth level after flooding. Probably other injuries caused by flooding
had a dominant effect on enzymatic antioxidant changes during stress period.

survive the flooding period by intensification of antioxida-

Introduction
Excess of water in soil is a common environmental abiotic
stress, which affects directly the plant roots and indirectly
the shoots (Henshaw et al. 2007a,b). Flooding may cause
severe injury and affects metabolism and functioning of
most crop plants (Jackson and Ricard 2003). The conse-
quences of flooding may be observed even after a long time
after the cessation of stress. According to Araki et al.
(2012), short-term flooding induced abnormally high res-
piration rates for nearly a month. As a result of stress, the
biomass and the final yield are restricted but the size of the
losses is dependent on physiological phase flooding stress
occurs at. In Poland, during vegetation period, many crop
plants are exposed to sporadic or permanent flooding at
various stages of growth. Because most of crop plants are
not adapted to the conditions of excess water, damages
caused by that stress are severe. However, some of the plant
possesses biochemical and morphological adaptation
mechanisms that allow them to succeed during temporary
flooding. One of the strategies of adaptation may be able to
tive system. Many authors have shown that flooding
strongly influences the whole metabolism. The submer-
gence led to significant decrease in photosynthesis, stomatal
conductance and chlorophyll content (Pociecha et al. 2008,
Else et al. 2009). This as well as the senescence of leaves
influences reactive oxygen species (ROS) production and
peroxidation of lipids. To protect against oxidative dam-
ages, plants are equipped with complex antioxidant systems
composed by low molecular mass antioxidants and
enzymes. Important for ROS detoxification are such
enzymes as catalase (CAT), superoxide dismutase (SOD),
ascorbic peroxidase (APX) and other peroxidases (PX;
mainly wall-bound ones), which decompose H2O2 to H2O
and O2 (Mittler 2002). The aim of the presented experi-
ment was to find differences in the physiological state
between field bean plants exposed to flooding stress at veg-
etative and generative stage of development. The aim was
also to provide information on plant abilities to follow
recovery. This process, if it proceeds successfully, is pivotal
for the maintenance of regular physiological functions in

© 2012 Blackwell Verlag GmbH, 199 (2013) 195–199 195

Pociecha
plants. The hypothesis assumed differences in enzymatic
antioxidants in the course of flooding and followed recov-
ery. Special emphasis was put on the changes of the mea-
sured parameters during recovery as the antioxidative
enzymes are involved in stabilisation of the physiological
state of plants after flooding stress. In the study, we also
attempt to characterise the influence of flooding on some
growth parameters.
Materials and Methods
Plants material
Seeds of the field bean cv. Nadwiślański were sown into
pots (5 dm3) filled with a mixture of peat substrate, brown
soil of loess origin and sand (2:2:1 v/v). Each experimental
treatment comprised 10 pots with three plants in each,
totalling 30 pots. Plants were grown in an air-conditioned
glasshouse at the temperature 23/17 °C (day/night),
under a 12-h photoperiod with a light intensity of
250 lmol m 2 s 1 PPFD (photosynthetic photon flux
density). The plants were fed every week with Hoagland
nutrient medium. At the vegetative stage (the 4th week) or
at the beginning of flowering (the 8th week), plants were
flooded by placing the pots in a reservoir of water up to
their rim for 7 days.
Catalase activity, non-specific peroxidase activity, ascor-
bate peroxidase activity and hydrogen peroxide accumula-
tion were determined after 1 and 7 days of flooding and
after 1 and 7 days of recovery from flooding (drainage of
the excess water from the treatment pots).
Biochemical assays
Catalase (EC 1.11.1.6) activity was estimated according to
Aebi (1984). Non-specific peroxidase activity was measured
spectrophotometrically according to the method described
by Bergmeyer (1965). Ascorbate peroxidase (EC 1.11.1.11.)
activity was estimated spectrophotometrically according to
Nakano and Asada (1981). Protein content was determined
according to the method described by Bradford (1976) with
bovine serum albumin as a calibration standard. Hydrogen
peroxide was detected in leaves with 3,3′-diaminobenzidine
(DAB) using a modification of the method described by
Thordal-Christensen et al. (1997). The determination of all
parameters was carried out in five replicates (in five leaves
from different plants of each treatment).
Growth parameters
The influence of flooding on plants growth was analysed
on the basis of estimation of the following parameters: leaf
area index (LAI), relative growth rate (RGR) and net
196
assimilation rate (NAR). All parameters were calculated
according to Shahidullah et al. (2010).
Statistics
Student’s t-test and standard error of means (SE) were used
when experimental treatments were compared. All calcula-
tions were carried out using Statistica 8.0 software package.
Graphs were constructed using the means and standard
errors for each data point.
Results
Enzymes activity and hydrogen peroxide accumulation
Statistical analysis showed that the CAT activity in leaves of
plants at vegetative stage rapidly increased after 7 days of
flooding in comparison with the control; however, activity
of that enzyme after cessation of flooding decreased to the
control values (Fig. 1a). Plants at generative stage demon-
strated the highest CAT activity after 1 day of flooding,
next decreased after 7 days and again increased after 7 days
of recovery (Fig. 1d).
Total peroxidase activity in vegetative plants gradually
increased during 7 days of flooding and after 7 days of
recovery came back to the control values (Fig. 1b). At gen-
erative stage, a dramatic increase in PX activity after 7 days
of flooding in comparison with the control was observed
(Fig. 1e). Next, a little decrease was noted during recovery
period, but PX activity was significantly higher in compari-
son with the control.
At vegetative stage, APX activity demonstrated a similar
pattern of changes as PX activity (Fig. 1c). A small increase
was noted after 7 days of flooding; next, a greater increase
was observed the 1st day of the recovery period. After
7 days of recovery, the APX activity in flooded plants was
equal to that in the control. At generative stage, flooding
also induced increase in APX activity but the highest values
were reached during recovery period (Fig. 1f). Flooded
plants were characterised by higher APX activity through-
out the time of experiment as compared with control.
One of the symptoms of the influence of flooding on
field bean plants was accumulation of hydrogen peroxide
indicated by brown DAB polymer formation. At vegetative
stage, the intensive DAB formation was observed on the 1st
day after flooding and during recovery (Fig. 2a). After
7 days of flooding, the strong reduction in DAB formation
was accompanied by an increase in CAT activity correlated
with an increase in CAT activity. At generative phase, the
strongest reduction in hydrogen peroxide concentration
was observed already on the 1st day after flooding
(Fig. 2b). These changes were also correlated with an
increase in CAT activity.
© 2012 Blackwell Verlag GmbH, 199 (2013) 195–199
 Antioxidants and Growth After Flooding Stress

(a) (d)

(e)
(b)

(f)
(c)

Fig. 1 Catalase, non-specific peroxidise and ascorbate peroxidase activity in leaves of field bean plants subjected to flooding at vegetative (a,b,c) or
generative (d,e,f) stage of development. Means ± S.E.

(a)
(b)
Fig. 2 The detection of hydrogen peroxide using 3′,3′-diaminbenzidine
(DAB) in leaves of field bean plants subjected to flooding at (a) vegeta-
tive or (b) generative stages of development.
Growth parameters
Flooding field bean plants resulted in reducing LAI and
RGR (Table 1). The decline was greater if the stress
occurred at vegetative stage (32%) than at generative
stage (7%). Such a strong factor as 1-week flooding
caused a further large decline in LAI during the follow-
ing recovery period. At vegetative stage the reduction as
compared to control was 80% while at generative stage
44%. Also decrease in RGR was observed during
recovery period, and similar to LAI, the decrease was
greater at vegetative stage (22%) than at generative
stage (8%).
If plants were subjected to flooding at vegetative stage,
during following recovery, a slight increase in LAI and
RGR was observed in contrast to plants flooded at genera-
tive stage where further decrease (LAI) or lack of changes
(RGR) were noted during recovery.

© 2012 Blackwell Verlag GmbH, 199 (2013) 195–199 197

Pociecha
RGR (mg g
LAI
Stage Period Control Flooded Control
Vegetative Flooding 0.315 0.215** 0.505
Recovery 1.161 0.238** 0.620
Generative Flooding 1.996 1.854* 0.642
Recovery 3.268 1.823** 0.693
*P < 0.05.
**P < 0.01.
NS, not significant (Student’s t-test).
NAR values increased during flooding period, and the
increase was greater in generative plants (33%) than in veg-
etative plants (22%). During recovery period, there was a
significant decrease in NAR at vegetative stage (89%) and
at generative stage (85%), similar to LAI and RGR.
Discussion
Plants at various stages of growth are differentiated in their
response and susceptibility to environmental stresses and
possibility of regeneration during recovery period after ces-
sation of stress. The analysis of growth at vegetative and
generative stages showed that field bean plants varied in all
parameters and that at vegetative stage field bean plants are
most sensitive to water excess in soil.
To assess whether the reason for the growth disturbances
is the ROS generation, the activity of selected antioxidative
enzymes and H2O2 accumulation were determined. The
involvement of the main antioxidant enzymes catalase and
peroxidases in the adaptation processes during the reaction
to root flooding and during recovery is known (Yordanova
et al. 2004). According to Sarkar et al. (2001), activity
antioxidative enzymes such as superoxide dismutase
(SOD), catalase and peroxidise were higher in flooding tol-
erant rice cultivar.
Within a few hours, the effect of disturbances in roots is
transmitted via the transpiration stream to the aerial parts
causing the earliest adaptation of shoots to the stress (Jack-
son et al. 2003). Root flooding causes a systemic reaction
aimed at releasing a defensive reaction in plants, including
the activation of antioxidative enzymes. ROS generation is
also accompanied by leaf senescence, which also occurs
during flooding (Hurng and Kao 1994). In turn, during
recovery period, when plants are re-exposed to the air and
oxygen, oxidative stress is induced again. The mechanism
of post-anoxic ROS generation includes lipid peroxidation
(Monk et al. 1994) and generation of hydrogen peroxide
by b-oxidation in peroxisomes and photorespiration
(Jackson and Ram 2003).
In plants characterised by resistance to different types of
environmental stress, including flooding, increased activity
198
1
Table 1 LAI, RGR and NAR after 7-day flood-
per ing applied at vegetative and generative stage
1 2
day) NAR (g g per m ) of development and after following 7 days of
Flooded Control Flooded recovery. Significant differences between con-
trol and flooded plants
0.504 NS 5.997 7.323*
0.485* 6.727 0.713**
0.635 NS 1.404 1.865**
0.637* 2.981 0.440**
of one or more of the antioxidant enzymes is usually
observed (Chirkova et al. 1998, Mittler 2002). In the study,
flooding induced generation of hydrogen peroxide and
activation of catalase and peroxidases. Brownish colour of
leaves indicates H2O2 accumulation, but the lack of brown
spots may be a consequence of enhanced activity of H2O2-
scavenging enzymes such as CAT and peroxidases. Hurng
and Kao (1994) stated that increased rates of hydrogen per-
oxide in leaves of flooded tobacco plants could lead to
increased capacities of the system scavenging the hydrogen
peroxide. They suppose that accumulation of H2O2 in
flooded tobacco leaves is regulated by catalase and glycolate
oxidase.
In the study, H2O2 accumulation was observed at both
vegetative and generative plants, but in the latter was
accompanied by higher activation of antioxidative enzymes
during the recovery period.
It seems that the most important role is played by CAT
because APX due to its low concentration in cells rather
has a regulatory function (Mittler 2002). Also, Miyagawa
et al. (2000) report that transgenic tobacco plants were able
to protect photosynthetic apparatus by neutralising H2O2
instead of chlorophyll APX.
It can be assumed that in generative plants, antioxidative
enzymes were more activated and did not attain the control
level in the course of recovery period. In vegetative plants,
changes in antioxidant enzymes caused by flooding applied
at vegetative stage were reversible. The 7-day period
allowed the recovery of antioxidant activity to the control
rates in vegetative plants (although high level of H2O2), but
not in generative ones. Generative plants possibly had lar-
ger reserves, which may have been used for the synthesis of
new proteins including the enzymatic antioxidants. In veg-
etative plants, probably most of energy was required for the
growth process.
Studies of growth patterns provide information about
how plants accumulate dry matter and how stress
events influence the plant. Optimum LAI and RGR at
flowering according Sun et al. (1999) is reflected in
yield. In the study, the investigated growth parameters
were reduced to a greater extent when plants were
© 2012 Blackwell Verlag GmbH, 199 (2013) 195–199

flooded at vegetative stage. As a result, vegetative plants
cannot synthesise organic compounds efficiently. This
problem does not affect so much of generative plants
thus have already reached the optimal size. Older plants
probably accumulate more carbohydrates reserves, which
allows them to survive the stress period and makes
them more resistant.
NAR after flooding period was higher than observed in
control plants. It may be explained by more intensive dry
mass accumulation, which compensates for the growth-
inhibiting effects of flooding (Norby and Kozlowski 1983).
After recovery period, the changes in NAR and RGR
were reduced in flooded plants, which indicates that stress
strongly affected field bean plants.
In conclusion, changes in antioxidative enzymes caused
by flooding applied at vegetative stage were reversible. In
generative plants, antioxidative enzymes were activated
more during recovery period and in contrast to vegetative
plants did not attain the control level after 7 days of that
period. The growth analysis showed that vegetative plants
were not able to restore the physiological function to opti-
mal level after flooding regardless of the stabilisation of
enzymatic antioxidant activity. On this basis, it can be sug-
gested that other injuries caused by flooding had a domi-
nant effect on enzymatic antioxidant changes in field bean
plants during stress period.
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