Botanical Studies (2008) 49: 9-18.
*
Corresponding author: E-mail: hflo@faculty.pccu.edu.tw;
Tel: +886-2-28610511 ext. 31101.
INTRODUCTION
Environmental stress severely affects plants because
it can throw the production and scavenging of reactive
oxygen species (ROS) in plants out of balance (Gratao
et al., 2005). One of the major biological consequences
of soil flooding is oxygen deficiency. Roots suffer from
periodic or prolonged deprivation of oxygen, which
interferes with respiration at the level of electron transport.
The lack of a suitable electron acceptor leads to saturated
redox chains, accumulation of the reduced form of nicotine
adenine dinucleotide phosphate (NADPH), and a decline
in the generation of adenosine triphosphate (ATP). In plant
cells, oxidative stress reactions are associated with toxic
free radicals from the reduction of molecular oxygen to
superoxide radicals (O
2
-
), singlet oxygen (¡PO
2
), hydroxyl
radicals (¡POH) and hydrogen peroxide (H
2
O
2
). These
free radicals can inactivate various Calvin-Benson cycle
enzymes and are involved in oxidative systems, marking
proteins for degradation (Kennedy et al., 1992; Chaudiere
and Ilious, 1999). The toxic radicals can be removed
both enzymatically and chemically to protect plant cells
against oxygen toxicity and counter the hazardous effects
of ROS under stress (Gratao et al., 2005). The complex
antioxidative defense system that has evolved in plants
is composed of antioxidative enzymes (i.e., ascorbate
peroxidase (APX), catalase (CAT), superoxide dismutase
(SOD), glutathione reductase (GR)) and metabolites
(i.e., ascorbic acid (ASA), as well as reduced glutathione
(GSH), oxidized glutathione (GSSG), and vitamin E)
(Gratao et al., 2005). High levels of some antioxidative
enzymes and antioxidants are found to be important in
tobacco (Hurng and Kao, 1994a; Hurng and Kao, 1994b),
corn (Yan and Dai, 1996), wheat (Biemelt et al., 1998),
soybean (VanToai and Bolles, 1991), rice (Ushimaro et al.,
1992), tomato, eggplant (Lin et al., 2004), and sweetpotato
(Lin et al., 2006) survival of oxidative stress after being
subjected to different levels of flooding. Some oxidative
enzymes or oxidants have been useful in screening for
flooding-tolerant plants (Lin et al., 2004).
PBZ (paclobutrazol; (2RS, 3RS)-1-4 (-chlorophenyl)-4,
4-dimethyl-2-1, 2, 4-triazol-1-yl-penten-3-ol) is a member
of the triazole family. Triazoles have both fungitoxic and
plant-growth regulatory effects. In addition, they can
protect plants against various stresses, including drought,
Paclobutrazol leads to enhanced antioxidative protection
of sweetpotato under flooding stress
Kuan-Hung LIN
2
, Chao-Chia TSOU
2
, Shih-Ying HWANG
4
, Long-Fang O. CHEN
3
, and Hsiao-Feng
LO
1,
*
1
Department of Horticulture and Biotechnology, Chinese Culture University, Taipei 110, Taiwan
2
Graduate Institute of Biotechnology, Chinese Culture University, Taipei 110, Taiwan
3
Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan
4
Department of Life Science, National Taiwan Normal University, Taipei 116, Taiwan
(Received May 16, 2007; Accepted September 28, 2007)
ABSTRACT.
The aim of this research was to study the effect of paclobutrazol pretreatment on the changes
of antioxidative enzymes and antioxidants in the flooding-stressed sweetpotato Ipomoea batatas (L.) Lam.
¡¥Tainung 57¡¦ was grown in plastic boxes in a screenhouse and maintained in optimal water conditions for
45 days followed by PBZ treatments (0 and 0.5 mg/plant) for 1 day. Then flooding was induced by raising
the water level to 5 cm above the soil medium surface for a 5-day period followed by drainage for 2 days.
A factorial experiment in randomized complete blocks with three replications was conducted. Young fully
expanded leaves from each plant were clipped to measure enzyme activities and antioxidant contents.
Increased ascorbate peroxidase activity, total glutathione, oxidized ascorbic acid, and total ascorbic acid
amounts on different days of flooding provided the sweetpotato with increased flooding tolerance. The levels
of glutathione reductase, ascorbate peroxidase, total glutathione, oxidized ascorbic acid, and malondialdehyde
were regulated and elevated by paclobutrazol pretreatment under non-flooded conditions. Paclobutrazol
pretreatment increased the levels of all antioxidative enzymes and antioxidants following different flooding
durations and drainage, and boosted the flooding tolerance of the sweetpotato.
Keywords: Antioxidant; Antioxidative enzyme; Flooding; Ipomoea batatas (L.) Lam.; Paclobutrazol.
BIOChemISTRy
pg_0002
10
Botanical Studies, Vol. 49, 2008
low and high temperatures, UV-B radiation, air pollutants,
fungal pathogens, and flooding. Therefore, the triazoles
have been characterized as plant multi-protectants (Kraus
and Fletcher, 1994; Voesenek et al., 2003). In plants,
chloroplasts are a major site of free radical production,
and PBZ protects plants by increasing antioxidant defense
systems. PBZ-treated plants have a more-efficient free
radical-scavenging system to detoxify active oxygen
(Kopyra and Gwozdz, 2003). Even though PBZ-induced
metabolic stress tolerance or protection is reportedly
due to some increased antioxidant enzymes (Pinhero et
al., 1997), less is known about the extent to which the
antioxidative response of PBZ application differs in the
flooding tolerance of sweetpotato.
The sweetpotato is the world¡¦s fifth most important crop
and is a major source of food and nutrition in developing
countries (Food and Agriculture Organization, 2002).
Heavy rain storms and floods can leave the soil saturated
for days before drainage, making flooding a problem in
many parts of the world. Attempts have been made to
breed for increased flooding tolerance and modify crop
cultivation or management practices and avoid flooding
injury. PBZ has been reported to confer protection on
plants experiencing stress by reducing oxidative damage
via elevation of antioxidants or reducing the activity of
oxidative enzymes. We hypothesized that pretreatment
with PBZ would increase the activities of antioxidative
enzymes or levels of antioxidants under flooding stress,
leading to higher flood tolerance in the sweetpotato. The
antioxidative system of the leaves of sweetpotato exposed
to waterlogged conditions was studied. The results provide
information that PBZ pretreatment increases sweetpotato
tolerance to waterlogging stress.
mATeRIALS AND meThODS
Plant materials, cultural practices, experimental
design, and treatments
The sweetpotato (Ipomoea batatas (L.) Lam.) cultivar,
Tainung 57, was used as the experimental material in this
study. Tainung 57 is a popular variety grown in Taiwan
for its storage roots. Cuttings about 30 cm in length
were planted in plastic boxes 60 cm long, 22 cm wide,
and 15 cm deep, containing medium consisting of sand,
vermiculite, and loamy soil in a volume ratio of 2:1:1.
Plants were planted in September 2001 in a greenhouse
of Chinese Culture University, Taipei. Plants were
evenly spaced every 50 cm to encourage similar growth
rates and sizes. Plants were watered with a half-strength
Hoagland solution (Lin et al., 2006) every other day to
maintain optimal irrigation and growth for 45 days before
imposition of flooding stress. The average day/night
temperatures were 33/23¢XC, and the average day length
was 13 h during the period of study.
A factorial experiment of three factors with different
levels was used in this investigation. Two concentrations
of PBZ (trade name, Bonsi; Zeneca Agrochemicals,
Fermhursk Haslemere Survey, UK), aqueous solutions
at 0 and 0.5 mg/plant level, were sprayed to study
the plant responses to flooding stress based upon our
previous experiments (Lin et al., 2006). Twenty-four
hours after PBZ treatment, plants were subjected to two
water conditions (non-flooded and flooded by tap water)
for 0, 1, 3, and 5 days followed by drainage for 2 days.
Three plants from each flooding period were harvested
at the same time of the day and used for the enzyme
measurements. All boxes of the same replication in each
flooding time treatment were placed in a 140 ¡Ñ 50 ¡Ñ
35-cm plastic bucket containing a water level 5 cm above
the soil medium surface. Plants without PBZ treatment
in a non-flooded condition were considered the control
to provide a basis to compare the effects of PBZ under
flooded and non-flooded conditions. The experiment was
performed twice independently for a randomized design
of growth environment, sampling day, and biochemical
analysis. Young, fully expanded leaves from each plant
were clipped for measurement of enzyme activities and
antioxidant contents.
enzyme extraction and activity determination
The cut leaves of each treatment were carried in
an icebox to the laboratory less than 5 min away and
immediately frozen in liquid nitrogen. They were then
stored in a -70¢XC freezer for later analysis. Samples were
prepared for SOD, CAT, APX, and GR activity analyses
by homogenizing 0.2 g of frozen leaf in 990 £gL of an ice-
cold 100 mM HEPES buffer (pH 7.0) containing 1 mM
PMSF (phenylmethysulfonyl fluoride) and 0.03 g PVP
(polyvinylpyrrolidone). The extracts were centrifuged at
13,000 g and 4¢XC for 15 min. The supernatants were then
collected in a fresh tube for the enzyme assays. Enzyme
activities were determined using a spectrophotometer.
CAT activity was assayed by measuring the initial rate
of the disappearance of H
2
O
2
according to the method of
Hwang and VanToai (1991). GR activity was performed
by oxidized GSH-dependent oxidation of NADPH using
the protocols described by Foyer et al. (1997). The assay
for APX activity was carried out as described by Nakano
and Asada (1981). SOD activity was determined using a
SOD Assay Kit-WST (Dojindo Molecular Technology,
Gaithersburg, MD). The specific activity of SOD was
calculated using the equation described in the protocol of
the kit.
Antioxidant extraction and content measu-
rement
The contents of total ASA and total glutathione were
determined by dissolving 0.2 g of homogenates in 1
mL of a 5% m-phosphoric acid solution. The extract
was then centrifuged at 13,000 g for 10 min and 4¢XC.
The supernatant was used for the total ASA and total
glutathione assays. Both total ASA and reduced ASA
contents were determined according to Cakmak and
Marschner (1992). The content of oxidized ASA (DHA,
pg_0003
LIN et al. ¡X Antioxidative system changes following PBZ pretreatment
11
dehydroascorbate or vitamin C) was calculated by
subtracting the reduced ASA content from the total ASA
content. Total glutathione content (GSH + GSSG) was
quantified as described by Anderson (1985). MDA is a
final decomposition product of lipid peroxidation and has
been used as an index for the status of lipid peroxidation.
MDA concentration was determined by the methods of
Kosugi and Kikugawa (1985).
The general chemicals used in the study were
purchased from Sigma (St. Louis, MO, USA). All
spectrophotometric analyses were conducted on a 530
UV/VIS spectrophotometer (Pharmacia Biotech, Uppsala,
Sweden). One unit of enzyme was defined as the amount
of enzyme required to decompose 1 £gmole of substrate
min
-1
g
-1
fresh weight (FW).
Determination of the leaf water potential (WP)
and total chlorophyll content (TCh)
The WP was measured on the third leaf from the top
of each plant using a pressure chamber (Plant Water
System, Skyeskpm 1400, Tokyo, Japan) (Sairam et al.,
1998). A leaf sample (0.2 g) was homogenized with 1.25
ml of 80% acetone and incubated for 10 min followed by
centrifugation at 13,000 g for 15 min at 25¢XC. Absorbances
of the supernatant were measured at 663.6 and 646.6
nm, the major absorption peaks of chlorophyll a and b,
respectively. The TCH was calculated as 8.02 A
663.6
+ 20.2
A
646.6
as described by Porra et al. (1989).
Statistical analysis
Measurements of enzymes were analyzed by a three-
factor completely randomized ANOVA that compared the
PBZ concentrations, flooding conditions, and duration of
treatment. For significant values, means were separated by
the least significant difference (LSD) test at p . 0.05, 0.01
or 0.001, using PC SAS 8.2 (SAS Institute, Cary, NC).
ReSULTS
The effects of PBZ pretreatment (P) on sweetpotato
subjected to flooding conditions (F) were monitored by
measuring changes in CAT, GR, APX, SOD, total ASA,
oxidized ASA, total glutathione, MDA, TCH, and WP
under various durations (D) of treatment. SOD, CAT, APX,
GR, MDA, TCH, and WP levels displayed significant
differences (p . 0.001 and 0.05) by the main effect of PBZ
(Table 1). For the main effects of flooding, there were
significant differences in the activities of SOD and APX,
and in the contents of total ASA, oxidized ASA, total
glutathione, TCH, and WP. Moreover, all the enzymes and
antioxidants, as well as TCH and WP, were significantly
affected by the duration of treatment. Table 1 also
illustrates that the SOD activity appeared to significantly
differ in terms of both the main effects (P, F, and D) and
the interaction effects (P¡ÑD, P¡ÑF, F¡ÑD, and P¡ÑF¡ÑD).
Figure 1a shows the SOD activities of plants with
and without PBZ pretreatment after 0, 1, 3, and 5 days
of flooding and the subsequent 2 days of drainage. SOD
activity with non-PBZ treatment under a non-flooded
condition (-P/-F) increased, reached its maximum value
(1.33 unit/g FW) after 3 days of treatment, and dropped
thereafter. In the case of PBZ-treated and non-flooded
conditions (+P/-F), the activity of SOD increased up to
day 5 of treatment followed by a decrease. The trend
of change in the SOD activity of untreated plants under
flooding conditions (-P/+F) was that it remained low
before drainage, then increased to its maximum (1.09
unit/g FW) after drainage. In addition, pretreatment with
PBZ followed by flooding stress (+P/+F) resulted in a
significant increase in SOD activity on days 3 and 5 of
flooding and drainage, compared to the -P/+F condition.
Table 1. ANOVA of paclobutrazol
concentration (P), flooding condition (F), duration of treatment (D), and their interactions (P ¡Ñ D,
P ¡Ñ F, F ¡Ñ D, P ¡Ñ F ¡Ñ D) for superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR),
total ascorbic acid (ASA), oxidized ASA, total glutathione, malondialdehyde (MDA), total chlorophyll (TCH), and water potential
(WP) contents in sweetpotato leaves.
Source of
variance
Degrees of
freedom
Significance
SOD C AT APX GR Total ASA Oxidized ASA Total glutathione MDA TCH WP
PBZ (P)
1 *** * * *** NS
NS
NS
*** *** ***
Flooding (F) 1 *** NS * NS ***
***
*
NS ** ***
Days (D)
4 *** *** * ** ***
***
***
* *** **
P ¡Ñ D
4 *** NS ** ** ***
*
**
*** *** **
P ¡Ñ F
1 *** * * * ***
***
NS
*** NS NS
P ¡Ñ F
4 *** NS NS NS ***
***
***
*** *** **
P ¡Ñ F ¡Ñ D
4 *** NS ** *** ***
***
***
*** *** NS
***: p . 0.001; **: p. 0.01; *: p . 0.05; NS: non-significant difference.
pg_0004
12
Botanical Studies, Vol. 49, 2008
Therefore, adding PBZ before flooding stress may promote
SOD activity in the sweetpotato.
As shown in Figure 1b, the -P/-F condition produced
significantly higher CAT activity than the -P/+F condition
after 3 and 5 days of flooding. This observation suggests
that flooding stress can result in reduced CAT levels and
the occurrence of oxidative damage. On the other hand,
CAT activity in the -P/-F condition was significantly higher
than that in the +P/-F condition after 1 day of flooding and
drainage. This implies that pretreatment with PBZ may
decrease CAT activity under non-stressful conditions.
Figure 1c presents the effects of PBZ pretreatment on
leaf APX activity during flooding and drainage. The pre-
addition of PBZ to plants in a non-flooded condition on
day 0 of flooding caused a significantly higher level of
APX. One day after the plants received PBZ in a non-
flooded condition, PBZ immediately induced an increase
in APX activity. Plants are highly regulated by PBZ,
which can drastically elevate APX activity. Compared to
plants without PBZ treatment and a non-flooded condition
(-P/-F), PBZ-treated plants under a non-flooded condition
(+P/-F) had higher APX activities on all days of treatment
except for day 1 of flooding. This observation reveals that
pretreatment with 0.5 mg PBZ per plant in a non-flooded
condition could lead to an increase of APX activity.
When across-day comparisons were made, the +P/+F
condition consistently displayed significantly higher
GR activity than the -P/+F condition after flooding was
imposed (Figure 1d). Higher GR activity was shown in
the +P/+F condition on days 1, 3, and 5 of flooding than
for the -P/+F condition. Pretreatment with 0.5 mg PBZ
per plant enhanced higher GR activity in the sweetpotato
during flooding stress. GR stimulated by pretreatment with
PBZ may play an important role in overcoming flooding
stress.
The total glutathione content in the -P/+F condition
was significantly higher than that in the -P/-F condition
on days 1 and 5 of flooding (Figure 2a). It is noteworthy
that the -P/+F condition (230.1 nmol/g FW) displayed
an increase threefold greater than did the -P/-F condition
(65.0 nmol/g FW) on day 5 of flooding. In the absence
of PBZ pretreatment, the total glutathione content in
the sweetpotato was induced by flooding stress, and
glutathione could be considered a flooding-tolerant
enzyme against flooding stress.
Figure 2b demonstrates comparisons of oxidized
ascorbate (or DHA) contents under PBZ/flooding
conditions throughout the period of treatment. The
oxidized ASA content in the +P/-F condition accumulated
at different rates from day 0 (0.2 £gmol/g FW) to day 7 (4.0
£gmol/g FW). Therefore, pretreatment with PBZ followed
by non-stressful conditions may increase the level of
oxidized ASA in a 7-day period.
All four PBZ/flooding treatments had the highest level
of total ASA on day 0 of treatment with no significant
difference (Figure 2c). The -P/+F condition (3.1 £gmol/g
Figure 1. Effects of pretreatment with paclobutrazol
on the (a) superoxide dismutase, (b) catalase, (c) ascorbate peroxidase, and (d)
glutathione reductase activity in leaves of sweetpotato subjected to flooding stress for different durations of treatment. Vertical bars
represent the mean ¡Ó standard error. +P, paclobutrazol pretreatment; -P, no paclobutrazol treatment; +F, flooding stress; -F, non-flooded
condition.
pg_0005
LIN et al. ¡X Antioxidative system changes following PBZ pretreatment
13
FW) had a significantly higher total ASA level than the
+P/+F condition (1.5 £gmol/g FW) on day 5 of flooding.
This implies that PBZ addition may reduce the total ASA
content under long-term flooding stress.
A significantly lower MDA content under the +P/-F and
+P/+F conditions compared to the -P/-F control condition
with 3-day flooding duration is shown in Figure 3a.
Therefore, PBZ pre-addition increased lipid peroxidation
under flooding stress, and might afford protection to the
plants by decreasing flood-induced oxidative stress.
As shown in Figure 3b, the level of the leaf WP in both
-P/+F and +P/+F plants decreased at different rates as the
flooding duration was extended followed by drainage. The
water status of PBZ-treated plants was better than that of
untreated plants under flooding stress. For example, the
WP of -P/+F plants was -6.7 bar, and it was -9.2 bar for
+P/+F plants on day 5 of flooding. Data for the WP in
both +P/+F and +P/-F plants showed significantly higher
negative values on days 3 and 5 of flooding compared
to the control -P/-F plants. Therefore, PBZ pretreatment
under flooding may induce an increase in the plant tissue
water level, subsequently affecting the leaf WP.
Figure 2. Effects of pretreatment with paclobutrazol
on the (a)
total glutathione, (b) oxidized ascorbate, and (c) total ascorbate
content in leaves of sweetpotato subjected to flooding stress
for different durations of treatment. Vertical bars represent the
mean ¡Ó standard error. +P, paclobutrazol pretreatment; -P, no
paclobutrazol treatment; +F, flooding stress ; -F, non-flooded
condition.
Figu re 3. Effects of pretreatment with paclobutrazol
on the
(a) malondialdehyde content, (b) water potential, and (c) total
chlorophyll conte nt in lea ves of s we etpotat o subjec ted to
flooding stress for different durations of treatment. Vertical
bars repres ent the m ean ¡Ó standard error. +P, paclobutrazol
pretreatment; -P, no paclobutrazol treatment; +F, flooding stress;
-F, non-flooded condition.
pg_0006
14
Botanical Studies, Vol. 49, 2008
TCH was used as a physiological parameter to monitor
the response of PBZ-pretreated sweetpotato under flooding
stress (Figure 3c). The TCH contents in -P/-F, -P/+F, and
+P/+F plants gradually decreased over the time course of
the experiment, with the exception of elevated values from
days 0 and 1 in -P/-F plants. During flooding stress, PBZ-
treated plants (+P/+F) maintained significantly higher
TCH amounts than did untreated plants (-P/+F). Plants
under a non-flooded condition (both -P/-F and +P/-F)
exhibited higher levels of TCH compared to those under
flooded conditions (both -P/+F and +P/+F) on days 1, 5,
and 7. Therefore, periods of flooding and drainage were
accompanied by lower TCH values.
DISCUSSION
Comparisons between untreated plants under
non-stressful conditions (-P/-F, as the control)
and untreated plants subjected to a flooded
condition (-P/+F)
Determination of the function of an observed
response is one of the most complex issues in plant
stress physiology. In this study, the involvement of the
antioxidative system in the regulation of free-radical
metabolism was followed by measuring changes in
enzyme activities and antioxidant contents for comparisons
between flooded and non-flooded conditions. On different
days of treatment, plants responded differently to flooding
stress according to the various components of their
antioxidative system. Flooding conditions produced
significantly higher APX, total glutathione, oxidized
ASA, and total ASA contents than non-flooded conditions
following no PBZ application on day 5 of flooding
(Figures 1c, 2a-2c). In addition, after drainage, plants in
the -P/+F condition exhibited significantly higher SOD
and GR activities compared to those in the -P/-F condition
(Figures 1a, 1d). In contrast, no signifcant differences
for most of the treatment due to flooding stress appeared
when CAT and MDA levels under -P/+F conditions were
compared to those under -P/-F conditions (Figures 1b, 3a).
Plants may prepare for oxidative damage by up-regulating
APX, total glutathione, oxidized ASA, and total ASA
under flooding conditions for 5 days. Therefore, increases
in these enzymes and metabolites were observed before
leaves became epinastic and senescent under flooding
stress, and these identified systems could be used for rapid
monitoring and early detection of flooding injury, i.e., in
the seedling stage. This means that hundreds of individual
plants might be screened per day, providing scope for the
discovery of individuals that exhibit tolerance to flooding
stress.
The effect of flooding on the plant¡¦s growth was also
observed in this study. The lower leaves of the plants
under flooding stress showed epinasty and senescence
after 3 days of flooding; however, under a non-flooded
condition, most leaves looked green and healthy (photos
not shown). Flooding stress had a harmful effect on the
sweetpotato, and changes in enzyme activities were related
to the degree of chlorosis and reduced TCH content of the
plant leaves during flooding. The leaves of flooded plants
maintained a certain level of antioxidants in their systems,
which scavenged at least part of the ROS. Antioxidative
enzyme activities and antioxidant contents play major
roles in maintaining the balance between free radical
production and elimination. Enhancement of APX activity
in a waterlogged environment may be an indicator of
superoxide production (Figure 1c). High levels of APX
should favor the scavenging of H
2
O
2
produced by SOD and
CAT. The APX found in organelles is believed to scavenge
H
2
O
2
produced from the organelles while the function of
cytosolic APX is probably to eliminate the H
2
O
2
that is
produced in the cytosol or apoplasts and has diffused from
organelles. In the study, we measured the variations of
total enzyme activity. In most of the higher plants, algae
and some bacteria, APX, SOD, POD, and CAT isozymes
were distributed in four distinct cellular compartments:
chloroplast (including stomata and thylakoid), microbody
(including glyoxysome and peroxisome), mitochondria,
and cytosome (Shigeoka et al., 2002; Jang et al., 2004;
Kim et al., 2004). In the chloroplast of sweetpotato, H
2
O
2
can be detoxified by the ASA-GSH-NADPH system
catalyzed by swAPX1, and thus help to overcome the
oxidative stress induced by abiotic and biotic stresses
(Park et al., 2004). High levels of glutathione are the
result of increased GSH biosynthesis. The level of MDA
is one of the measures of whether plant cells are damaged
by oxidative stress. Lower levels of MDA indicate better
oxidative stress tolerance. In this study, MDA content of
1-day flooded plant (-P/+F) was lower (or non-significant
different) than non-flooded (-P/-F) plant, indicating
low cell damage in the flooded TN57 (Figure 3a). The
increased level of total glutathione in 1-day flooded plant
might help to reduce the MDA level while the increased
APX activity might help 5-day flooded plant and drained
plant show the same level of MDA content as the non-
flooded plant, which would indicate that TN57 is tolerant
to flooding stress. Plants are more flooding-tolerant if
the increased ROS level under flooding can lead to an
enhanced ROS-scavenging system. ROS scavenging is
important in imparting tolerance against flooding stress
(Noctor and Foyer, 1998; Blokhina et al., 2003).
Previously, we reported that the GR activity in Taoyuan
2 sweetpotato, the leaves of which are consumed as a
popular vegetable, was significantly enhanced over 5 days
of continuous flooding, in comparison with non-flooded
conditions (Lin et al., 2006). The present study indicates
that APX, total glutathione, oxidized ASA, and total
ASA levels affected the defense mechanism of Tainung
57 plants under a flooded condition. Different varieties
displayed variations in their antioxidative systems,
and the differential expressions of each genotype were
associated with the flooding stress response. Specific
genotype responses to flooding stress were correlated with
resistance to oxidative stress. Gratao et al. (2005) reported
that responses to oxidative stress induced by biotic and
pg_0007
LIN et al. ¡X Antioxidative system changes following PBZ pretreatment
15
abiotic stress may vary depending on plant species, tissue,
and length of stress, apart from other specific aspects.
Almeselmani et al. (2006) studied the effect of high
temperature stress on the antioxidant enzyme activity in
five wheat genotypes. There was significant increase in the
activity of SOD, APX, and CAT in the late and very late
planting and at all stages of plant growth, i.e., vegetative,
anthesis, and 15 days after anthesis. However, GR activity
decreased under late and very late plantings compared
to normal planting. Lee and Lee (2000) reported that
chilling stress enhanced the activities of SOD, APX, and
GR in the leaves of cucumber while inducing a decrease
in CAT activity. Cho and Park (2000) demonstrated that
substantial increases in H
2
O
2
content and SOD and CAT
activities occurred under mercury-induced oxidative stress
in 30-day-old tomato plants in comparison with controls.
Enhancement of the aforementioned antioxidative
systems favors flooding resistance. Antioxidative enzymes
may augment antioxidants in the removal of ROS from
plant cells. These findings are important for farming in
frequently flooded areas, and also informative for further
genetic and physiological studies on sweetpotato flooding
tolerance.
Comparisons between untreated plants under
a non-flooded condition (-P/-F, control) and
pretreatment with PBZ followed by a non-
stressful condition (+P/-F)
Figures 1a, 1b, and 2c showed reduced SOD, CAT,
and total ASA contents and increased MDA content in
the interval of days 0 to 7 under a non-flooded condition
with 0.5 mg PBZ/plant pretreatment. However, Figures
1c, 1d, 2a, 2b, and 3a reveal that PBZ pretreatment
significantly increased the levels of APX, GR, total
glutathione, and oxidized ASA under 0 to 7 days of non-
stressful conditions. PBZ pretreatment might lead to
prodution of ROS. Although APX and GR activities were
enhanced to detoxify ROS, the reduced SOD, CAT, and
total ASA levels might be the reason behind the increased
MDA content. In our observations, compared to control
plants (-P/-F), PBZ-treated plants under a non-flooded
condition (+P/-F) appeared healthy and had greener leaves
throughout the duration of the experiment (photos not
shown). More work needs to be conducted to confirm the
effect of PBZ on the production of sweetpotato under non-
flooded condition.
Comparisons between untreated plants
subjected to flooding stress (-P/+F) and
pretreatment with PBZ followed by a flooded
condition (+P/+F)
Compared to the -P/+F condition, SOD (at 3 and 5
days), CAT, oxidized ASA, and total ASA (at 3 days),
APX (at 1 and 3 days), and GR (at 1, 3, and 5 days) in
PBZ-pretreated sweetpotato were significantly enhanced
under flooding stress (+P/+F) (Figures 1a-1d, 2b, 2c).
Furthermore, the +P/+F condition showed a significantly
higher MDA content than the -P/+F condition on days
1 and 5 of flooding and drainage (Figure 3a). PBZ
pretreatment caused changes in the levels of various
components of the antioxidative system under flooding
stress.
As waterlogging was prolonged, the leaf WP of plants
decreased more rapidly in untreated plants (-P/+F) than in
PBZ-treated plants (+P/+F) (Figure 3b), indicating that the
development of flooding stress in leaves was more gradual
or perhaps delayed by PBZ treatment. Chlorosis of most
waterlogged and untreated plants was visually higher than
in PBZ-treated plants subjected to flooding stress. These
observations imply that PBZ application might reduce
or delay flooding stress thereby allowing those plants to
survive and function during flooding. This ability can
perhaps be attributed to an avoidance of flooding stress, as
indicated by the higher WP in +P/+F plants than in -P/+F
plants during the flooding time course. It is not clear how
PBZ pretreatment improves the water status of leaves, but
it might cause more-efficient water uptake by plants, by
retarding water loss from plants during flooding stress, or
both.
Generally speaking, PBZ application before flooding
protects plants from the adverse effects of flooding.
PBZ application affected enzyme activities, which
had an impact on the flood tolerance and health of the
plant. An anoxic condition is one of the major problems
for plants under flooding stress. As a plant encounters
anoxic stress, a stronger antioxidative system illustrates
a superior flood-tolerant mechanism in terms of the
ability to scavenge H
2
O
2
, O
2
-
, ¡PO
2
, and ¡POH. By removal
of O
2
-
and increases in SOD and GR, the redox potential
and important components of the electron transport
system may be altered, hence facilitating improved stress
tolerance. Pinhero et al. (1997) showed that PBZ treatment
of maize induced several changes in the antioxidative
system profiles and especially enhanced the activities of
SOD, GR, and APX, along with the induction of chilling
tolerance. Kraus and Fletcher (1994) proposed that PBZ-
induced protection of wheat from damage caused by heat
stress was mediated by increased SOD, APX, and GR
activities. In our study, pretreatment with PBZ could be an
important strategy for altering the behavior and survival
of the sweetpotato under flooding stress. PBZ apparently
plays an important role in the antioxidative system under
flooding stress. Flooding stress protection conferred
by PBZ was mediated to some extent by an enhanced
antioxidative system. Our results suggest that flooding
stress effects on the sweetpotato can be mitigated by 0.5
mg PBZ/plant. Further characterization of the isoforms of
each antioxidative enzyme will be helpful in elucidating
whether or not there are distinct effects and responses
among isozymes to the PBZ pretreatment under flooding
stress. If the PBZ and flooding treatments have affected
one particular antioxidative isozyme, this may indicate
correlation to a cell physiological phenomenon due to the
specific organelle localization of this isozyme. To know
exactly which isoform is related to the stress response,
pg_0008
16
Botanical Studies, Vol. 49, 2008
isoenzyme assays, i.e., activity staining is needed.
Comparisons between untreated plants under a
non-flooded condition (-P/-F as the control) and
pretreatment with PBZ followed by a flooded
condition (+P/+F)
CAT and APX activities, and oxidized ASA and
total ASA contents of the sweetpotato pretreated with
PBZ, eventually showed the same levels after flooding
stress and drainage as did untreated plants under a non-
flooded condition (Figures 1b, 1c, 2b, and 2c). Moreover,
pretreatment with PBZ eventually enhanced SOD and GR
activities and total glutathione and MDA contents after
flooding stress and drainage compared to the untreated
and non-flooded condition (Figures 1a, 1d, 2a, and 3a).
Generally speaking, pretreatment with PBZ improved the
antioxidative system of the sweetpotato under flooding
followed by drainage for 2 days to a level at least similar
to that of the control. These results suggest that SOD,
GR, total glutathione, and MDA values are flooding-
specific and not expressed solely in response to PBZ
pretreatment. Plants with various antioxidative systems
respond differently to flooding stress according to the
durations of the flooding period and subsequent drainage
period. In ABA-treated turfgrass, SOD and CAT activities
also markedly increased after ABA treatment and were
maintained at higher levels during drought stress (Lu et
al., 2003). Agarwal et al. (2005) reported that 0.05 mM
of H
2
O
2
increased the activities of SOD, APX, CAT and
NADPH oxidase in wheat genotypes C306 and Hira. Liu
et al. (2007) mentioned that hematin promoted both SOD
and CAT activities of rice seeds under salt stress.
Flooding had effects on both the leaf WP and TCH.
WP can be used as a parameter of flooding tolerance. WP
decreased with progressive soil flooding, indicating that
water relations of sweetpotato suffered from flooding
injury. As PBZ application to flooding-stressed plants
can improve the water status of plants (Figure 3b), it
is reasonable to expect that this in turn may lead to a
favorable effect on the TCH content (Figure 3c). Flooding
may induce stomatal closure and consequently reduce
WP and TCH levels. TCH has been widely examined in
a number of plants to determine injury or tolerance to
various environmental stresses including drought, chilling,
heat, and radiation (Ahmed et al., 2002). Typically, the
TCH amount is reduced by stressful conditions. This
is consistent with our observations that flooding stress
aggravated senescence in the leaves of -P/+F plants
that was associated with a significant loss of TCH. The
lower leaves of the plants under flooding stress showed
senescence after 3 days of flooding while most leaves
looked green and healthy under non-flooded conditions.
Simultaneously, with the flooded plants (-P, +F), along
with the characteristic visual symptoms, a significantly
lower content of TCH was observed compared to controls
(-P, -F) (Figure 3C). In addition, the light induced
chlorophyll accumulation was gradually inhibited by
the increasing flooding time. Stomatal closure causes a
decrease in internal CO
2
concentrations. Subsequently,
a concomitant decline in photosynthesis results from
the diminished availability of CO
2
for carbon fixation
(Carvalho and Anancio, 2002). Reduction of the CO
2
concentration increases the amount of harmful ROS within
the leaf due to ongoing light reactions, which leads to
senescence of the plant. Along with the visible symptoms,
the reduced TCH could be used to monitor the flooding
induced damage in both green or senescent leaves.
The present work studied changes in the antioxidative
system involved in detoxification of ROS in sweetpotato
responding to flooding stress. Compared to -P/-F plants,
+P/+F plants had higher TCH contents (Figure 3c) in
coordination with higher functions of APX (Figure 1c, on
days 0, 1, and 3), GR (Figure 1d, on days 1, 3, 5, and 7),
total glutathione (Figure 2a, on days 1, 5, and 7), and total
ASA (Figure 2c, on days 3 and 7), and lower production of
MDA (Figure 3a, on days 0, 1, and 3), implying that PBZ-
treated plants had a better waterlogging tolerance capacity.
Oxygen is required for flooding-induced inactivation
of photosynthesis to take place, which suggests that the
formation of ROS seemed to be the cause of the damage
to chlorophyll during exposure to flooding. A decrease in
the TCH use of radiation absorbed by pigments can lead
of the production of potentially dangerous ROS (Ahmed
et al., 2002). The ROS were then enhanced in the leaves of
sweetpotato. High rates of electron transport together with
a flooding-induced decrease in carbon metabolism increase
the probability of electron transfer to oxygen in the Mehler
reaction, thus forming superoxides and finally increasing
oxidative stress. The ROS can potentially be detoxified
by an efficient chloroplastic oxidative system. In order
to keep the electron transport chain oxidized and prevent
ROS formation, electrons must be efficiently consumed
in the leaves of sweetpotato by the Calvin cycle or other
electron sinks.
CONCLUSIONS
The APX activity and contents of total glutathione,
oxidized ASA, and total ASA were characterized by
an increasing trend for different durations of flooding.
Leaves of the sweetpotato under flooding stress generate
ROS that may then be removed by the aforementioned
antioxidative enzymes and antioxidants. The presence
of these antioxidants may be a useful criterion in the
early screening of flooding-tolerant sweetpotato for
the ability overcome flooding stress. Furthermore, the
antioxidative system level under non-flooded conditions
was highly regulated by PBZ pretreatment. APX, GR,
total glutathione, oxidized ASA, and MDA levels in the
sweetpotato were significantly induced by 0.5 mg PBZ/
plant pretreatment for different durations of treatment.
Under non-stressful condition, PBZ pre-addition
increasedantioxidative enzyme and antioxidant levels
against insults from ROS and rendered it better able to
respond to flooding stress. Levels of all components of the
pg_0009
LIN et al. ¡X Antioxidative system changes following PBZ pretreatment
17
antioxidative system at different durations of treatment
were enhanced by adding PBZ before flooding, and
GR exhibited especially higher activity levels in plants
subjected to flooding treatments and drainage. PBZ
pretreatment showed significant effects on both leaf WP
and TCH content under flooding stress. PBZ may protect
the sweetpotato from flooding stress through its influence
on the oxygen-detoxifying system. Therefore, applying 0.5
mg PBZ/plant 24 h before flooding may mitigate flooding
stress.
Acknowledgements. This research was supported by
grants from the National Science Council, ROC. The
authors are grateful to Ms. Huei-Ting Huang and Shu-Yen
Pi for typing and editing this manuscript.
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