Botanical Studies (2006) 47: 417-426.
*
Corresponding author:
Hsiao-Feng LO, e-mail: hflo@
faculty.pccu.edu.tw; Pi-Yu CHAO, e-mail
:
pychao@faculty.
pccu.edu.tw; Tel: +886-2- 28610511-31101.
INTRODUCTION
Drought and flooding are considered to be predominant
factors determining the global geographic distribution of
vegetation and restriction of crop yields in agriculture.
Environmental stress severely affects plants because the
production and scavenging of the reactive oxygen species
(ROS) in plants loses its equilibrium (Crawford and
Brandle, 1996). Symptoms of flooding or drought injury
include chlorophyll breakdown, protein degradation,
membrane permeability decrease, peroxidation, slower
leaf expansion, petiole epinasty, and stomatal closure
(Moran et al., 1994; Gogorcena et al., 1995). Stomatal
closure causes a decrease in internal CO
2
concentration.
Subsequently, a concomitant decline in photosynthesis
resulted from the diminished availability of CO
2
for
carbon fixation. Reduction of CO
2
concentration increases
the amount of harmful ROS within the leaf due to ongoing
light reaction, which leads to senescence and even death
of the plant (Schwanz et al., 1996; Carvalho and Amancio,
2002; Keles and Dunl, 2002; Sairam et al., 1997). Roots
suffer from periodic or prolonged deprivation of water
or 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
NAD(P)H, and a decline in the generation of ATP (Asada,
1992; Kennedy et al., 1992). In plant cells, the oxidative
stress reactions are associated with toxic free radicals
from the reduction of molecular oxygen to superoxide
radicals (O
2
-
£»), singlet oxygen (
1
O
2
), hydroxyl radicals
(¡POH), hydrogen peroxide (H
2
O
2
), and peroxyl radicals
(ROO¡P). These radicals can inactivate various Calvin-cycle
enzymes and are involved in oxidative systems (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 (Perata and Alpi, 1993).
In recent years, increasing attention has been paid
PHYSIOLOGY
The effects of flooding and drought stresses on the
antioxidant constituents in sweet potato leaves
Kuan-Hung LIN
1
, Pi-Yu CHAO
2,
*, Chi-Ming YANG
3
, Wen-Ching CHENG
4
, Hsiao-Feng LO
1,
*, and
Tsan-Ru CHANG
5
1
Department of Horticulture and Biotechnology, Chinese Culture University, Taipei 111, TAIWAN, ROC
2
Department of Food, Health and Nutrition Science, Chinese Culture University, Taipei 111, TAIWAN, ROC
3
Research Center for Biodiversity, Academia Sinica, Taipei 115, TAIWAN, ROC
4
Graduate Institute of Applied Science of Living, Chinese Culture University, Taipei 111, TAIWAN, ROC
5
Taipei Sub-station, Taoyuan District Agricultural Research and Extension Station, Council of Agriculture, Shulin 237,
TAIWAN, ROC
(Received January 12, 2006; Accepted April 14, 2006)
ABSTRACT.
Environmental stress results in generation of reactive oxygen species in plants and causes
oxidative stress. The aim of this work was to study the changes to the antioxidative system in the leaves of
three sweet potato varieties, Taoyuan 2, Simon 1, and Sushu 18 as affected by flooding and drought stresses.
The experimental design was completely randomized with a split plot arrangement of treatments. Young,
fully expended leaves from each plant were clipped for antioxidant activity measurement. We concluded that
genotypes exhibited their abilities and specificities on porphyrins, polyphenol, flavonoids, reduction power
and scavenging DPPH radical and superoxide anion. The polyphenol content and scavenging superoxide
anion percentage of the three sweet potato varieties under the stresses declined significantly. However, the
conjugated dienes inhibition percentage increased markedly under the stresses. Inhibiting the conjugated
dienes could mitigate flooding and drought stress effects and be useful as a flooding and drought-tolerant
index.
Keywords: Antioxidative activity; Drought stresses; Flooding stress; Sweet potato.
pg_0002
418
Botanical Studies, Vol. 47, 2006
by consumers to the health and nutritional benefits of
sweet potato leaves. A diet rich in leafy vegetables,
including sweet potato leaves, offers production against
some common diseases such as cardiovascular and
cerebrovascular events, cancer, and other age-related
degenerative diseases (Hayase and Kato, 1984; Huang
et al., 2004; Scalzo et al., 2005). These protective effects
are considered, in large part, to be related to the various
antioxidants contained in them. Evidence that free
radicals cause oxidative damage to lipids, proteins and
nucleic acids is overwhelming. Antioxidants, which can
inhibit or delay the oxidation of an oxidizer in a chain
reaction, would therefore seem to be very important in
the prevention of these diseases (Yen and Chung, 2000;
Leong and Shui, 2002). These benefits have stimulated
research to investigate the content, ability, capacity,
and function of antioxidative systems in leafy sweet
potato. Leafy vegetables contain antioxidant nutrients,
in addition to vitamin C or E and carotenoids, which
significantly contribute to their antioxidant capacity.
Phenolic substances such as flavnoids are the most
common compounds in leafy vegetables and have strong
antioxidant activity (Prior et al., 1998; Chu et al., 2000;
Hollman and Arts, 2000; Proteggente et al., 2002).
Sweet potato is the world¡¦s fifth most important crop
and is a major source of food and nutrition in developing
countries (The International Potato Center, Lima, Peru,
1998). It is a plant species resistant to drought stress
because its deep-root system accesses moisture. The
increasing demand for food with growth of the world
population has resulted in an increasing use of flooding-
sensitive or drought-sensitive crops in wetland, lowlands,
or drylands. In Taiwan, waterlogging is one of the primary
physiological constraints to sweet potato production in the
summer season every year due to short, intensive rainfall.
Moreover, the wet-dry tropical environment of Taiwan
is characterized by prolonged seasonal drought. During
the dry season (October to January), little or no rain falls,
and temperatures, solar radiation, and vapor pressure
deficits are high. Plants possess different antioxidant
properties, depending on their content of antioxidant
molecules, which is, in turn, strongly affected by the
specific plant genotype and environmental conditions
of the plant. The interaction of these different factors in
determining the antioxidant capacity and ability of a plant
should be established to better characterize agronomic
production. Little has been done to study antioxidative
activity in response to flooding or water-depletion stresses
in sweet potato. The long-term goal of our work is to
help breed flooding-tolerant and drought-tolerant sweet
potatoes to be grown in the summer season and winter
season, respectively. The present research project studied
the antioxidative systems of the leaves of sweet potato
exposed to flooding and drought conditions. The system
may be useful in screening for flooding-tolerant and
drought-tolerant plants. The results provide information
on the effects of various amounts of antioxidative capacity
and ability on the flooding and drought tolerances in the
sweet potato.
MATERIALS AND METHODS
Plant materials, cultural practice, experimental
design and treatments
Three sweet potato (Ipomoea batatas [L.] Lam)
cultivars, Taoyuan 2, Simon 1, and Sushu 18 were used.
¡¥Taoyuan 2¡¦ is a variety grown in Taiwan that is popular
for the consumption of its leaves. Sushu 18 is a drought-
tolerant variety from China (JAAS and SAAS, 1984). The
storage roots of Simon 1 contain high amounts of vitamin
K. The planting was conducted in the screen house of
Taoyuan District Agricultural Research and Extension
Station in Taiwan. Terminals about 30 cm in length were
taken from sturdy vines and cultivated with 25¡Ñ25 cm
density in June, 2001. The plants were watered three times
per week to the field capacity by drip irrigation system
and allowed to grow for 45 days before water stress and
flooding imposition. The soil was sandy loam with pH
6.8. Average day/night temperature was 34/26¢XC and the
average day length was 14 h during the study period.
The size of the experimental field was 450 m
2
. Water
treatments were carried out in a split-plot design with four
replications of completely randomized design. The three
varieties were arranged as the sub-plot with five plants
per replication. The main-plots included a 3-day flooding
(watering to the soil level), no water irrigation to the
field for a two-week period (drought treatment), and non-
flooding treatments (control). After each treatment, the
youngest fully expanded leaves of each plant were washed
and clipped for antioxidant activity measurement.
Sample preparation and extraction
The cut leaves of each treatment were lyophilized
and ground to powder. Five milligrams of powder were
extracted with a 5-fold volume of methanol at room
temperature, and then filtered through Whatman #1 filter
paper. The remaining residue was re-extracted thrice
until the residue was colorless. The three extracts were
combined, concentrated to a powder by freeze dryer
(LabConco, Japan), and then stored in a -20¢XC freezer for
later analysis.
Determination of polyphenol content
Polyphenol content was measured by the Folin-
Ciocalteu method (Taga et al., 1984; Singleton et al.,
1999). One hundred microliters of the methanolic extract
was added to 1 mL distilled water and 2 mL of Folin-
Ciocalteu reagent. The mixture was allowed to stand at
room temperature for 5 min, and 2 mL of 2% sodium
carbonate was added to the mixture followed by gentle
mixing. After standing at room temperature for 30 min,
the absorbance was read at 750 nm. Deionized water
was used as a blank. The standard calibration curve was
plotted using gallic acid. The content of polyphenol was
expressed as mg gallic acid equivalent / g extract.
pg_0003
LIN et al. ¡X Flooding and drought on sweet potato antioxidants
419
Carotenoid and porphyrin content measurement
Carotenoid and porphyrin concentrations were
determined as decribed Lichenthaler (1987) and Porra
et al. (1989) and modified by Yang et al. (1998). Five
milligrams of samples were homogenized with 5 mL of
80% acetone in a cooled mortar. Extract was centrifuged
for 5 min at 1,500 g, and the supernatant was stored. The
pellet was re-extracted with acetone and centrifuged again.
This process was continued until the supernatant was
colorless, and then the supernatant was pooled.
1. Absorbance was measured at 663.6, 646.6 and 440.5
nm, the major absorption peaks of chlorophyll a and b and
carotenoids, respectively. Carotenoids were calculated
using the following equation: (4.69 ¡Ñ A
440.5
- 1.96 ¡Ñ A
663.6
- 4.74 ¡Ñ A
646.6
) ¡Ñ volume of supernatant (mL) ¡Ñ dilution
factor / sample weight (g).
2. Absorbance was measured at 663.6, 646.6, 440.5,
575, 590 and 628 nm, the absorption peaks of chlorophyll
a, chlorophyll b, carotenoids, protoporphyrin, magnesium-
protoporphyrin and protochlorophyllide, respectively.
Porphyrin contents were summed (A+B+C) by the
following three equations:
A =
[(12.25 ¡Ñ A
663.6
- 2 .55 ¡Ñ A
646.6
) ¡Ñ volume of
supernatant (mL) ¡Ñ diluted factor / sample weight
(g)] / 892 ¡Ñ 1000
B =
[(20.31 ¡Ñ A
646.6
- 4 .91 ¡Ñ A
663.6
) ¡Ñ volume of
supernatant (mL) ¡Ñ diluted factor / sample weight
(g)] / 906 ¡Ñ 1000
C =
[(196.25 ¡Ñ A
575
- 4 6 .6 ¡Ñ A
590
- 58.68 ¡Ñ A
628
) +
(61.81 ¡Ñ A
590
- 23.77 ¡Ñ A
575
- 3.55 ¡Ñ A
628
) + (42.59
¡Ñ A
628
- 34.32 ¡Ñ A
575
- 7.25 ¡Ñ A
590
)] ¡Ñ volume of
supernatant (mL) ¡Ñ dilution factor / sample weight
(g).
Determination of flavonoid content
The flavonoids were determined according to the
method of Geissman (1995). 80% ethanol containing 1%
HCl of solvent was used to extract 0.05 g of the powder
samples. The mixture was vigorously shaken for 30 min,
followed by centrifuging at 4¢XC, 1,500 g for 15 min.
Sampling of the supernatants was taken to measure the
absorbance at 540 nm. Flavonoid contents were calculated
as A
540
¡Ñ volume of supernatant (mL)/sample weight (g).
DPPH free radical-scavenging assay
The free radical scavenging ability of the sweet potato
was measured using the protocols described by Shimada
et al. (1992) and Yoshiki et al. (2001). Briefly, an aliquot
of 4 mL of the methanolic extract
(4, 6 and 8 mg/mL)
was added to 1 mL of 10 mM DPPH (2, 2-diphenyl-1-
picrylhydrazyl) solution freshly prepared in methanol. The
mixture was left in the dark for 30 min, and decolorization
of DPPH donated H
+
was followed by measuring the
absorbance at 517 nm. DPPH radical-scavenging activity
was calculated from the absorption according to the
following equation: DPPH radical-scavenging activity %
= [(A
control
- A
sample
) / A
control
)] ¡Ñ 100.
Measurement of reducing power
The reducing property of the crude extract was
determined according to the method of Pulido et al. (2000).
Briefly, an aliquot of 0.5 mL of the methanolic extract (2,
4 and 8 mg/mL) was mixed with an equal volume of 0.2
M sodium phosphate buffer (pH 6.6) and 1% potassium
ferrocyanide. The mixture was incubated at 50¢XC for 20
min. Then an equal volume of 10% trichloroacetic acid
was added to the mixture, which was then centrifuged at
1,500 g for 10 min. One milliliter of the supernatant was
mixed with equal volume of distilled water and 0.2 mL
of 0.1% FeCl
3
¡P4H
2
O. After 10 min, the absorbance at 700
nm was measured. Increased absorbance of the reaction
mixture indicated elevated reducing power. Reduction
capacity = sample of A
700
- control of A
700.
Determination of scavenging activity of
superoxide radical
The superoxide anion scavenging activity of the
methanolic extracts was determined according to
the method of Robak and Gryglewski (1988) with
modifications. An aliquot of 1.0 mL of methanolic
extract (0.5 and 1
mg/mL) was added to an equal volume
of 120 £gM phenazine methosulphate (PMS), 936 £gM
dihydronicotinamide dinucleotide (NADH) and 300 £gM
nitro-blue tetrazolium (NBT) in 0.1 M phosphate buffer
(pH 7.4). The mixture was left at room temperature for
5 min, and the absorbance at 560 nm was measured. The
lower absorbance indicated higher scavenging activity.
Superoxide radical-scavenging activity % = [(A
control
-
A
sample
) / A
control
)] ¡Ñ 100.
The inhibition of conjugated dienes formation
in linoleic acid emulsion
The inhibition of conjugated dienes formation was
determined according to the method of Mitsuda et al.
(1966). An aliquot of 0.05 mL of each methanolic extract
(0.3125 mg/mL) was added to 1 mL of 10 mM linoleic
acid emulsion (pH 6.6). The mixture was shaken and
incubated at 37¢XC for 15 h. One hundred microliters
of the solution from a 0- and 15-h incubation period
was separately added to 3.5 mL of 80% methanol. The
absorbance at 234 nm was then measured. The percent
inhibition of linoleic acid peroxidation was calculated as:
Inhibition % = [(A
control
- A
sample
) / A
control
)] ¡Ñ 100
The general chemicals used in this study were
purchased from Sigma Co. (MO, USA) unless otherwise
noted. All spectrophotometer analyses were conducted on
a Hitachi U-2000 type spectrophotometer (Japan).
Statistical analysis
The measurements of antioxidants were analyzed by a
two-factor completely randomized ANOVA that compared
the water conditions and the varieties. For the significant
pg_0004
420
Botanical Studies, Vol. 47, 2006
values, means were separated by the least significant
difference (LSD) test at P¡Ø0.05, 0.01 or 0.001, using PC
SAS 8.2 (SAS Institute, Cary, NC). Among three varieties,
means with the same small letters were not significantly
different and presented in Tables 2 to 8. Among three
water treatments, means with the same capital letters were
not significantly different and shown in Tables 2 to 8. Each
value was the mean of four replicate analyses.
RESULTS
The effects of water treatments on the three sweet
potato varieties were monitored by measuring changes
of antioxidant contents and capacity. Except for the
contents of carotenoids and conjugated dienes inhibition,
main effects of variety (V) on the measured components
of the antioxidative system displayed significant
differences across varieties (P¡Ø0.01 and 0.05) (Table
1). Across different water treatment (W), there were
significant differences in the polyphenol, reduction power,
scavenging of superoxide anions, and conjugated dienes
inhibition. Additionally, the amounts in each component
of the antioxidative system exhibited a non-significant
difference in the interaction effect (V ¡Ñ W), except for
those of polyphenol and scavenging superoxide anion.
Table 2 presents the effect of water conditions on the
antioxidant content in the leaves of three sweet potatoes.
Porphyrin content did not show any significant difference
among water treatments. However, Sushu 18 showed
a significantly higher porphyrin content than Taoyuan
2 or Simon 1. Thus, different genotypes displayed
variations in their porphyrins. When polyphenol content
under three water treatments with different varieties was
compared, normal water treatment had significantly higher
polyphenol content than either flooding or drought stresses
(Table 2). Furthermore, in Sushu 18, polyphenol content
Table 1. ANOVA of main effects of variety (V), water treatment (W) and their interaction (V ¡Ñ W) for porphyrin, carotenoid,
polyphenol and flavonoid contents, and scavenging DPPH radical, reduction power, scavenging superoxide anion, and conjugated
dienes inhibition capacities.
Source of
variance
Degree of
freedom
Significance
Porphyrins
(£gg/g)
Carotenoids
(£gg/g)
Polyphenol
(£gg/g)
Flavonoids
(A540/g)
Scavenging
DPPH radical
(%)
Reduction
power
Scavenging
superoxide
anion (%)
Conjugated
dienes
inhibition (%)
Variety (V) 2
*
NS
**
**
*
*
*
NS
Water (W) 2
NS
NS
**
NS
NS
*
**
**
V ¡Ñ W
4
NS
NS
**
NS
NS
NS
NS
NS
**: P¡Ø 0.01; *: P¡Ø0.05; NS: non-significant difference.
Table 2. The effect of drought and flooding treatment on the porphyrin, carotenoid, polyphenol and flavonoid contents of sweet
potato leaves.
Variety
Water treatment Porphyrins (£gg/g) Carotenoids (£gg/g) Polyphenol (mg/g) Flavonoids (A540/g)
Taoyuan 2
Control
31540 b
1553 NS
0.35 aA
132.8 b
Drought
38570 b
1507 NS
0.20 aB
130.2 b
Flooding
32104 b
1624 NS
0.21 aB
146.7 b
Sushu 18
Control
51980 a
1657 NS
0.11 bA
157.7 a
Drought
43577 a
1714 NS
0.02 bB
176.0 a
Flooding
43746 a
1538 NS
0.01 bB
163.7 a
Simon 1
Control
36051 b
1576 NS
0.39 aA
138.8 b
Drought
28775 b
1480 NS
0.27 aB
132.7 b
Flooding
32854 b
1315 NS
0.24 aB
144.2 b
Among three varieties, means with the same small letters were not significantly different by the least significant difference (LSD) at
P¡Ø0.05 with completely randomized design.
Among three water treatm ents, means with the same capital letters were not significantly different by the least significant
difference (LSD) at P¡Ø0.05 with completely randomized design.
NS: non-significant difference; Each value was the mean of four replicate analyses.
pg_0005
LIN et al. ¡X Flooding and drought on sweet potato antioxidants
421
was significantly lower than Taoyuan 2 and Simon 1. The
pattern and trend of flavonoid content appeared similar
to those of porphyrin content in Table 2. A significantly
higher level of flavonoid was observed in Sushu 18 than
in Taoyuan 2 and Simon 1. Nevertheless, the flavonoid
content in response to various water treatments displayed
non-significant difference.
DPPH radical is scavenged by antioxidants through
the donation of hydrogen, forming the reduced DPPH-H.
The color changes from purple to yellow after reduction,
which can be quantified by its decrease of absorbance
at wavelength 517 nm. Table 3 indicates that Simon 1
exhibited a significantly higher scavenging DPPH radical
percentage than Sushu 18 and Taoyuan 2 at 4 mg/mL and
8 mg/mL of the extract, respectively. For Sushu 18, the
percentage of scavenging DPPH radicals was significantly
lower than either Taoyuan 2 or Simon 1 at 6 mg/mL of the
extract.
Table 4 illustrates that the reduction power percentage
was different among the varieties under water treatments.
Simon 1 demonstrated a significantly higher level of
reduction ability compared to Taoyuan 2 at 2 and 4 mg/
mL of the crude extract. Additionally, plants of 3 varities
under drought stress induced significantly higher reduction
ability than those under flooding treatment at 8 mg/mL of
the extract. Flooding might result in a decreased reduction
ability in 8 mg/mL of the extract following plant oxidative
damage.
In Taoyuan 2, the scavenging superoxide anion
percentage was significantly higher than Sushu 18 and
Table 3. The effect of drought and flooding treatment on scavenging DPPH radicals of sweet potato leaves.
Variety
Water
treatment
Scavenging DPPH radicals %
4 mg/mL
6 mg/mL
8 mg/mL
Taoyuan 2
Control
31.2 ab
46.2 a
43.2 b
Drought
27.8 ab
38.0 a
42.7 b
Flooding
20.0 ab
36.3 a
41.0 b
Sushu 18
Control
11.2 b
29.1 b
48.7 ab
Drought
19.3 b
25.9 b
51.9 ab
Flooding
15.5 b
24.1 b
60.2 ab
Simon 1
Control
30.3 a
37.4 a
59.1 a
Drought
41.1 a
40.3 a
65.0 a
Flooding
25.9 a
42.2 a
67.5 a
Among three varieties, means with the same small letters were not significantly different by the least significant difference (LSD) at
P¡Ø0.05 with completely randomized design.
NS: non-significant difference; Each value was the mean of four replicate analyses.
Table 4. The effect of drought and flooding treatment on the reduction power of sweet potato leaves.
Variety
Water
treatment
Reduction power
2 mg/mL
4 mg/mL
8 mg/mL
Taoyuan 2
Control
0.2 b
1.0 b
2.4 AB
Drought
0.6 b
0.7 b
2.6 A
Flooding
0.5 b
0.5 b
2.0 B
Sushu 18
Control
1.0 ab
1.7 a
2.4 AB
Drought
1.2 ab
2.0 a
2.9 A
Flooding
0.9 ab
1.6 a
2.3 B
Simon 1
Control
1.4 a
2.0 a
2.4 AB
Drought
1.0 a
1.7 a
2.8 A
Flooding
1.0 a
1.7 a
2.0 B
Among three varieties, means with the same small letters were not significantly different by the least significant difference (LSD)
at P¡Ø0.05 with completely randomized design.
Among three water conditions, means with the same capital letters were not significantly different by the least significant difference
(LSD) at P¡Ø0.05 with completely randomized design.
NS: non-significant difference; Each value was the mean of four replicate analyses.
pg_0006
422
Botanical Studies, Vol. 47, 2006
Simon 1 at 0.5 mg/mL of the extract (Table 5). Moreover,
significantly higher percentages of scavenging superoxide
anion were detected under normal water conditions
compared to flooding stress at both 0.5 and 1 mg/mL of
the extract. Additionally, significantly different results
among genotype were observed at 0.5 mg/mL of the
extract only. It is noteworthy that Taoyuan 2 under normal
water treatment (33.5%) displayed a threefold increase
over Simon 1 exposed to flooding stress (10.1%) at 0.5
mg/mL of the extract. Hence, the various water treatments
showed significantly different levels of scavenging
superoxide anions in the leaves of sweet potatoes. Either
flooding or water-deficit stress showed a significantly
higher conjugated dienes inhibition percentage than
normal water conditions (Table 5). The conjugated dienes
inhibition percentage appears to be involved in imparting
tolerance against both flooding and drought stresses.
DISCUSSION
The determination of the function of an observed
response is one of the most complex issues in plant
stress physiology. In trying to understand responses to
stresses involving drought and a waterlogged component,
many enzymes and metabolites induced by periods of
water depletion and flooding have been identified and
characterized (Price et al., 1991; Ushimaro et al., 1992).
Nevertheless, studies of the response of the antioxidative
system of sweet potato in regard to its ability and capacity
to survive water-deficit and flooding stresses are scarce.
Enhancement in the production, ability and capacity of
antioxidants may play an important role in metabolic
stress tolerance. The effects of drought and flooding
stresses on the antioxidant system of sweet potato leaf
were thus examined in this study. The involvement of
the antioxidative system in the regulation of free-radical
metabolism was examined by measuring changes in
antioxidant content, capacity, and ability under normal,
flooding, and drought conditions. Tables 2 to 5 illustrate
that the different varieties may prepare for oxidative
damage by up-regulating their antioxidant contents.
Porphyrins, polyphenol, flavonoids (Table 2), and
DPPH radical scavenging at 4, 6, and 8 mg/mL (Table
3), reduction power at 2 and 4 mg/mL (Table 4) and
superoxide anion scavenging at 0.5 mg/mL (Table 5) were
involved in this process. Among the three varieties, Sushu
18 exhibited significantly higher levels of porphyrins and
flavonoids (Table 2) than Simon 1 and Taoyuan 2 under
control, drought and flooding conditions. On the other
hands, polyphenol content for Taoyuan 2 and Simon 1
(Table 2), scavenging DPPH radicals percentage (4, 6
and 8 mg/mL) for Simon 1 (Table 3), reduction power (2
and 4 mg/mL) for Sushu 18 and Simon 1 (Table 4), and
scavenging superoxide anion percentage (0.5 mg/mL)
for Taoyuan 2 (Table 5) were significantly higher than
for other varieties. These results imply that genotypes
exhibited different abilities and specificities in their
antioxidative systems. Past studies (Li and Staden, 1998;
Sairam et al., 1998; Hwang et al., 1999; Herbinger et
al., 2002) have demonstrated that drought or flooding
sensitive species and cultivars have a lower antioxidant
capacity than do tolerant species and cultivars. In our
study, Chinese drought-tolerant entry ¡¥Sushu 18¡¦ exhibited
higher reduction power in 4 mg/mL extract probably due
to higher flavonoids content.
The higher extract concentration, the higher percentage
of scavenging DPPH radicals were found among the
varieties (Table 3). An opposite trend in scavenging
superoxide anion percentage can be observed in Table
5. Superoxide is a biologically important substance that
Table 5. The effect of drought and flooding treatment on the scavenging superoxide anions and conjugated dienes inhibition of
sweet potato leaves.
Variety
Water
treatment
Scavenging superoxide anions %
Conjugated dienes inhibition %
0.3125 mg/mL
0.5 g/mL
1 mg/mL
Taoyuan 2
Control
33.5 aA
20.8 A
54.1 B
Drought
30.3 aA
19.7 A
61.4 A
Flooding
23.8 aB
13.4 B
68.6 A
Sushu 18
Control
18.8 bA
23.4 A
56.9 B
Drought
13.6 bB
13.7 B
65.1 A
Flooding
11.0 bB
14.8 B
68.2 A
Simon 1
Control
16.2 bA
28.6 A
60.7 B
Drought
18.5 bA
16.0 B
64.8 A
Flooding
10.1 bB
17.2 B
68.4 A
Among three varieties, means with the same small letters were not significantly different by the least significant difference (LSD) at
P¡Ø0.05 with completely randomized design.
Among three water conditions, means with the same capital letters were not significantly different by the least significant difference
(LSD) at P¡Ø0.05 with completely randomized design.
NS: non-significant difference; Each value was the mean of four replicate analyses.
pg_0007
LIN et al. ¡X Flooding and drought on sweet potato antioxidants
423
can be decomposed and form stronger oxidative species
such as singlet oxygen and hydroxyl radicals (Korycka-
Dahl and Richardson, 1978). The highly reactive hydroxyl
radicals can cause oxidative damage to DNA, lipids, and
proteins (Grootvled and Jain, 1989). Too much superoxide
anion may also damage the cell membrane by the
adverse effect of stress, which leads to a decrease in the
scavenging superoxide anion percentage in plant, and fails
to bring about water-depletion or flooding tolerance. Thus,
different genotypes responded differently to scavenge
DPPH radicals or superoxide anions under various
water treatments, and the differential expression of each
genotype was associated with flooding or drought stress
response.
Tables 2, 4 and 5 demonstrate the impact of drought
and flooding stresses on the antioxidative system of
sweet potatoes. The polyphenol content and scavenging
superoxide anion percentage of all plants were
significantly reduced under flooding stress as compared
to normal water conditions (Tables 2 and 5). Reduction
power at the 8 mg/mL concentration of all plants under
drought stress was significantly higher compared to those
under flooding stress (Table 4). Under drought stress,
Sushu 18 and Simon 1 exhibited a significantly lower
percentage of scavenging superoxide anion at a 1 mg/mL
concentration than normal water treatment (Table 5). In
contrast, with all plants subjected to flooding or drought
stress, the conjugated dienes inhibition percentage was
significantly increased as compared to normal water
conditions (Table 5). Antioxidant activity plays a major
role in maintaining the balance between the production
and elimination of free radicals. Presumably, the
accumulation of a component amount of the antioxidative
system and ROS formation favored drought tolerance. An
increased level of ROS in the water-depleted plants could
lead to an increased capacity of the scavenging system of
ROS, particularly in reduction power ability (Table 4). The
impact of water deficit on plant growth and development
varies depending on the severity of the water limitation,
the duration of the stress, and the plants¡¦ developmental
stage.
Sweet potato production is limited to the hot and wet
summer season in Taiwan. Flooding has been an important
factor affecting summer sweet potato production. In the
flooded soil, oxygen limitation is one of the primary
threats to plants. Plants are able to maintain radical
damage through their natural defense mechanisms. Anoxic
stress is a major factor in flooding conditions. When roots
are submerged this condition inhibits aerobic respiration
and less energy is yielded. The roots translocate less
nutrients to the leaves. The solutes entering the leaves
via the transpiration stream may also decrease (Yan and
Dai, 1996). As the plants encounter anoxic stress, a higher
level oxidative system illustrates their superior tolerance
mechanisms in ROS scavenging over the plants. Our
results indicate that the percentage of conjugated dienes
inhibition increased under flooding and drought stresses,
and this can be considered as a mechanism for overcoming
these stresses. This antioxidative ability may be useful in
screening for flooding-tolerant and drought-tolerant plants.
Heavy rainfall in summer results in loss of fresh market
production of sweet potato. High light stress usually also
exerts its effect on sweet potato following heavy rainfall
during summer. Previously, we have found that reduced
susceptibility to waterlogging together with high-light
stress was related to increases of superoxide dismutase and
catalase activities in the leaves of sweet potatoes (Hwang
et al., 1999). Hence, different stress conditions might
generate different antioxidative mechanisms for tolerance.
From our observations, the lower leaves of each
variety looked epinastic and senescent after 3-days of
flooding and 14-days of drought. However, under control
conditions, most leaves appeared healthy and sported
green throughout the duration of the experiment (photos
not shown). Flooding and drought stresses had a harmful
effect on the leaves of sweet potatoes, and some of the
damage was irreversible once flooding or drought injury
was done. Antioxidative activities changed were related
to degree of reduced chlorosis of the plant leaves during
flooding and drought. When significant flooding-injury
or drought-injury in appearance occurred, oxy-radical
production increased. The polyphenol content, reduction
power, scavenging superoxide anion percentage, and
conjugated dienes inhibition percentage in the leaves
of sweet potatoes under flooding and drought stresses
were significantly affected. The leaves of sweet potatoes
became more tolerant to flooding or drought, and oxidative
processes may be associated with this tolerance. The
degree of flooding-injury or drought-injury seems to be a
result of enhancement of conjugated dienes inhibition, and
of decline in polyphenol and scavenging superoxide anion
levels in sweet potatoes. Enhancement of conjugated
dienes inhibition percentage under stresses may be an
indicator of prooxidant production. Many polyphenols can
exhibit antioxidant content as their extensive, conjugated
electron systems allow ready donation of electrons or
hydrogen atoms from the hydroxyl moieties to free
radicals. Most polyphenols are very effective scavengers
of hydroxyl and peroxyl radicals and can stabilize lipid
oxidation. They are chelators of metals and inhibit the
Fenton and Haber-Weiss reactions, which are important
sources of ROS (Yamasaki et al., 1997; Chang et al., 2002;
Debarry et al., 2005). In addition, polyphenols retain their
free radical scavenging capacity after forming complexes
with metal ions. Transition metal ions accelerate free-
radical damage. Antioxidant defenses protect the plant
against oxidative burst, but they are not 100% efficient,
and so free-radical damage must be constantly repaired.
ROS scavenging is important in imparting tolerance
to environmental stress (Dixon and Paiva, 1995). The
aforementioned antioxidative activity may be limiting
the defense mechanisms of susceptible plants under
waterlogged or drought conditions.
Our data show that the carotenoid content (Table 2)
pg_0008
424
Botanical Studies, Vol. 47, 2006
of all varieties was not significantly affected by water
conditions. Additionally, chelating Fe
+2
ion percent of the
three genotypes showed no significant difference among
the various water treatments with different concentrations
of the extract (data not shown). The Fe
2+
-binding
activity was measured by the decrease in the maximal
absorbance of iron (II)¡Vferrozine complex (Dinis et al.,
1994). Flooding and drought stresses could increase both
parameters in all plants to a level equivalent to those of
normal water conditions. On the other hand, it might be
that the increases of other antioxidant abilities compensate
for the need for carotenoids and chelating Fe
+2
ions
under stresses. Quercetin, kaempferol, and myricetin are
the three most common flavonols that are also the most
widely distributed flavonoids (Lee et al., 1995). The
cultivar difference in antioxidative activity may due to
pigments that possess effective antioxidative activity alone
or synergistically. These antioxidative systems might
influence a plant¡¦s ability to maintain a balance between
the formation and scavenging of ROS, making leaves
vulnerable to oxidative stress. Changes in intercellular
redox might be a consequence of flooding and drought
stresses. Some oxidative systems are stimulated by the
oxidative burst in the sweet potato cells, but some are not.
ROS are important modulators of the cellular signal of
transduction events following flooding-stress or drought-
stress injury. Plants are tuned to the absolute levels of
ROS because a small amount of change can result in
drastically different responses (Perata and Alpi, 1993).
Thus, carotenoids and chelating Fe
+2
ions are genotype-
and environment-independent antioxidative systems which
may stimulate the restoration of leaf oxidative damage.
CONCLUSION
In conclusion, different varieties of sweet potatoes
responded differently to oxidative injury flooding and
drought stresses according to various components of
their antioxidative systems. Among three varieties, Sushu
18 proved more resistant to drought and flooding than
Taoyuan 2 and Simon 1 under field conditions. Our results
indicate that the contents of all antioxidative systems
in sweet potatoes, except for carotenoids and chelating
Fe
2+
ions, were significantly affected by the stresses.
The polyphenol content and scavenging superoxide
anion percentage of sweet potato leaves decreased under
flooding and drought. On the other hand, the percentage of
conjugated dienes inhibition under flooding and drought
increased when plants under stress generated ROS that
may then have been removed by the above mentioned
antioxidative system. Inhibiting conjugated dienes was
related to antioxidative activity of leafy sweet potato under
the stresses and can be an index to screen stress-tolerance
plants. These findings may have greater significance for
farming in frequently flooded areas or dry-lands. Our
findings are also informative for further genetic and
physiological studies on sweet potato flooding or drought-
tolerance.
Acknowledgements. This research was supported by
grants from the Council of Agriculture, Taiwan. The
authors are grateful to Prof. Shin-Shinge Chang for
assistance and Mr. M.Y. Hwang for collecting papers and
rendering this manuscript.
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