Mar Biol (2006) 150:1–15
DOI 10.1007/s00227-006-0323-3
R E S E A R C H A RT I C L E
Salinity stress and hydrogen peroxide regulation of antioxidant
defense system in Ulva fasciata
I-Fan Lu Æ Ming-Shiuan Sung Æ Tse-Min Lee
Received: 10 June 2005 / Accepted: 27 March 2006 / Published online: 6 May 2006
 Springer-Verlag 2006
Abstract The regulation of antioxidant defense sys-
tem in macroalgae exposed to salinity stress was
examined in Ulva fasciata Delile. As compared to the
30& control, a long-term (4 days) exposure to hypos-
aline (5, 15&) and hypersaline (60, 90, 120, 150&)
conditions inhibited growth rate and TTC reduction
ability. A decrease in maximum quantum efficiency
(Fv/Fm ratio) and the maintenance of superoxide
dismutase activity under salinity stress indicate the
potential generation of reactive oxygen species in
chloroplasts. An exposure to 15, 60, and 90& de-
creased seawater H2O2 contents but increased thallus
H2O2 contents that are positively correlated with
TBARS and peroxide contents. Alleviation of oxida-
tive damage and H2O2 accumulation at 15 and 90& by
a H2O2 scavenger, dimethylthiourea, suggests that
oxidative damage occurring under moderate hyposa-
line and hypersaline conditions is ascribed to accumu-
lated H2O2. Increased glutathione reductase activity
and glutathione content and decreased ascorbate con-
tent are responsible for accumulated H2O2 at 15, 60,
and 90&, while ascorbate peroxidase activity increased
only at salinity ‡ 90&. Catalase and peroxidase activ-
ities also increased at 60 and 90& for H2O2 removal,
but only catalase showed activity increase at 15&. For
the regeneration of ascorbate, the activities of both
dehydroascorbate reductase and monodehydroascor-
bate reductase were increased at 5 and 15& while only
Communicated by M. Kühl, Helsingør
I-Fan Lu Æ M.-S. Sung Æ T.-M. Lee (&)
Institute of Marine Biology,
National Sun Yat-sen University, Kaohsiung 80424
Taiwan, Republic of China
e-mail: tmlee@mail.nsysu.edu.tw
monodehydroascorbate reductase activity increased at
60 and 90&. It is hypothesized that the availability of
antioxidants and the activities of antioxidant enzymes
are increased in U. fasciata to cope with the oxidative
stress occurring in hyposaline and hypersaline condi-
tions.
Introduction
Salinity fluctuation has been considered to be the pri-
mary factor limiting the growth of macroalgae from
rocky intertidal regions and estuaries (Lobban and
Harrison 1997). It is becoming important to understand
how algae respond and adapt to salinity stress. The
physiological and biochemical responses of algae to
salinity stress, including osmotic adjustment, ion
homeostasis and compartmentation, metabolite accu-
mulation, growth and development, photosynthesis,
and respiration, have been extensively studied (Kirst
1990; Parida and Das 2005). It is known in higher
plants that the generation of reactive oxygen species
(ROS) can be enhanced by salinity, thus leading
to oxidative stress (Singha and Choudhuri 1990;
Hernández et al. 1993; Fadzilla et al. 1997; Wang et al.
1998; Rios-Gonzalez et al. 2002). ROS are produced by
chloroplast and mitochondrion electron transport flow
and also from peroxisome and membrane-bound
NADPH oxidase (Doke and Miura 1995). It is known
that oxidative stress results from the disruption of
cellular homeostasis of ROS production driving from
the excitation of O2 to form singlet oxygen (O21) and
the transfer of 1, 2 or 3 electrons to O2 to form
superoxide (O2)), hydrogen peroxide (H2O2) and hy-
123
2 Mar Biol (2006) 150:1–15
droxyl radical (HOÆ), respectively (Halliwell and Gut-
teridge 1989); the generating ROS causes oxidative
destruction of the cell components through oxidative
damage of membrane lipids, nucleic acid and protein
(Davis 1987; Wise and Naylor 1987; McKersie and
Leshem 1994; Imlay and Linn 1998). To counteract the
toxicity of ROS, the defense systems in scavenging of
cellular ROS have been developed in plants to cope
with oxidative stress via the non-enzymatic and enzy-
matic systems (Noctor and Foyer 1998; Asada 1999).
Small antioxidant molecules such as water-soluble
ascorbate (AsA) and glutathione (GSH) and water-
insoluble a-tocopherol and carotenoids are the main
non-enzymatic agents for ROS scavenging (Noctor and
Foyer 1998; Smirnoff and Wheeler 2000; Munné-Bosch
and Alegre 2002). Several enzymes are involved in the
detoxification of ROS; superoxide dimutase (SOD;
EC 1.15.1.1) can convert O2) to H2O2 and then H2O2
is removed by ascorbate peroxidase (APX; EC
1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2)
in the ascorbate–glutathione cycle (AGC) (Asada
1999). APX utilizes AsA to reduce H2O2 by the oxi-
dation of AsA to the monodehydroascorbate (MDHA)
radical, which is reduced to AsA either by photo-
synthetic electron flow through ferredoxin or by
NAD(P)H-dependent monodehydroascorbate reduc-
tase (MDHAR; EC 1.6.5.4). If NAD(P)H is limited,
MDHA would be spontaneously disproportionated to
AsA and dehydroascorbate (DHA) (Mittler 2002) and
then DHA will be reduced to generate AsA by dehy-
droascorbate reductase (DHAR; EC 1.8.5.1) using
GSH as an electron donor; the GSSG formed after
GSH oxidization is reduced to GSH by GR utilizing
reducing equivalents from NAD(P)H. Catalase (CAT;
EC 1.11.1.6) (Willekens et al. 1997) and several kinds
of peroxidase (POX; EC 1.11.1.7) (Asada and Takah-
ashi 1987) are also involved in H2O2 removal. Several
reports have shown there is a close association of
antioxidant capacity and salinity tolerance in higher
plants (Dionisio-Sese and Tobita 1998; Hernández
et al. 1999; Amor et al. 2005; Kim et al. 2005). It sug-
gests that the ability in scavenging ROS and preventing
and repairing their damaging effects on macromole-
cules is critical for salt tolerance plants.
Studies on the salinity effects on the production of
ROS and the regulation of antioxidant defense sys-
tems are still limited in algae. A recent investigation
of long-term hyposaline and hypersaline treatments
on a halotolerant eukaryotic alga Dunaliella tertio-
lecta showed that activities of MDHAR and APX
increased upon exposure to extreme high salinity
while an exposure to extreme low salinity increased
GSH and a-tocopherol contents but did not affect
123
antioxidant enzyme activities; its responses are dif-
ferent between hyposaline and hypersaline stresses
(Jahnke and White 2003). Ulva, a macroscopic mar-
ine alga belonging to Chlorophyta, frequently appears
in the intertidal regions of rocky shores and estuaries,
in which it suffers fluctuations of salinity due to
interaction tides, rainfall, and evaporation. Our pre-
vious investigation on the effects of salinity stress on
U. fasciata has shown there is a different response of
this intertidal chlorophyte to hyposaline and hypers-
aline conditions with polyamine accumulation under
hyposaline conditions (Lee 1998; Lee and Chen 1998)
and proline accumulation under hypersaline condi-
tions (Lee and Liu 1999). It is documented that
macroalgae from intertidal waters have developed the
ability to resist salinity changes (Lobban and Harri-
son 1997; Kirst 1990). It is known that the antioxi-
dant defense mechanisms against ROS are pivotal for
algal survival under stressful conditions; higher anti-
oxidant contents and antioxidant enzyme activities
are associated with higher stress tolerance in algae
(Butow et al.1994; Collén and Davison 1999a, b). It is
interesting for us to know whether antioxidant de-
fense is operated in U. fasciata against oxidative
stress occurring in salinity stress. To our knowledge,
the oxidative responses of U. fasciata to hyposalinity
and hypersalinity have not been examined. Attempts
to elucidate whether oxidative stress is induced by
salinity changes in U. fasciata and whether antioxi-
dant resistance mechanism is a strategy for this
chlorophyte to counteract salinity changes are made
by the determination of the levels of lipid peroxida-
tion estimated by thiobarbituric acid reacting sub-
stance (TBARS) contents, the contents of thallus and
seawater H2O2 and also the contents of water-soluble
antioxidants (AsA and GSH), and the activities of
SOD, CAT, POX, APX, GR, MDHAR, and DHAR
after 4 days of exposure to varying salinities (5, 15,
30, 60, 90, 120, and 150&). TTC reduction ability as
viability parameter and the fluorescence parameter,
Fv/Fm, as photosynthetic electron transport perfor-
mance of photosystem II were also determined to
evaluate the general responses of U. fasciata to
varying salinities. Further, the attempt was also made
to investigate whether H2O2 is involved as elicitors
causing oxidative damage and as a potential signal
inducing antioxidant defense system in U. fasciata by
examining the responses of TTC reduction ability,
thallus H2O2 production, the levels of lipid peroxi-
dation (TBARS) and the activities of H2O2 scav-
enging enzymes to a H 2O 2 scavenger,
dimethylthiourea (DMTU) (Levine et al. 1994), in 15
or 90& condition.
Mar Biol (2006) 150:1–15
Materials and methods
Algal materials, treatments, and seawater samples
Ulva fasciata of 15–20 cm in height were collected on
July–September 2004 from the intertidal regions of
Hsitzu Bay at Kaohsiung in southern Taiwan. After
harvesting, whole plants were washed with natural
seawater to remove the attached sands and then the
rhizoidal portions were removed to avoid microbial
contamination in the following culture. Thalli were
pre-incubated at 25C for 14 days in 30& nutrient-
enriched artificial seawater (ASW) containing
403.5 mM NaCl, 10 mM KCl, 10 mM CaCl2, 30 mM
MgSO4, 10 mM Tris–HCl (pH 8.0), N- (NH4+ and
NO3)) and P- (PO43)) free Provasoli nutrient solutions
(Provasoli 1968), 3 mM NaHCO3, 400 lM NaNO3,
and 20 lM Na2HPO4. The light time was 12 h and
the photosynthetically active radiation (PAR,
400–700 nm) was 100–120 lmol photons m)2 s)1 in the
absence of algae, achieved by 20 W cool-fluorescent
lamps (FL20D, China Electric Apparatus Ltd., Taiwan,
ROC). Before salinity treatment, 3-cm-long thallus
segments were excised and 30 g wet weight (WW) of
thallus segments were incubated in a Petri dish
(internal diameter 30 cm) containing 2 l of 30&
nutrient-enriched ASW for a 24-h incubation at
25 ± 1C under light condition (150 lmol photons
m)2 s)1 PAR provided by 40 W cool-fluorescent
lamps). After incubation, healthy thallus segments of
0.7 g WW were transferred to a polycarbonate vessel
(Magenta GA-7 vessel, Sigma, St. Louis, MO, USA)
containing 300 ml of 30& nutrient-enriched ASW for
treating with varying salinity. The nutrient-enriched
ASW media of 5 and 15& were prepared by the
combination of N- (NH4+ and NO3)) and P- (PO43))
free Provasoli nutrient solutions (Provasoli 1968),
3 mM NaHCO3, 400 lM NaNO3, 20 lM Na2HPO4 in
a ASW diluted from 30& ASW and 60–150& nutrient-
enriched ASW media were achieved by increasing
NaCl contents in 30& nutrient-enriched ASW. The
photoperiod was 12 h and irradiance was set at
150 lmol photons m)2 s)1 PAR provided by 8 cool-
fluorescent lamps (40 W). The incubation temperature
was 25 ± 1C. ASW media were changed every day.
To explore the involvement of H2O2 on hyposalin-
ity- and hypersalinity-induced oxidative damage and
antioxidant defense system, thallus segments (0.75 g
WW) were first cultured in a vessel containing 300 ml
of 30& nutrient-enriched ASW containing DMTU of
0, 25 or 100 lM for 12 h under darkness and then
transferred to 15 or 90& ASW for 1 day; then thallus
segments were sampled.
3
After treatments, thallus segments were sampled
and divided into three parts: the first part was imme-
diately used for TTC reduction ability assay, the sec-
ond part for the determination of lipid peroxidation
and the contents of peroxide, H2O2 and water-soluble
antioxidants was fixed in liquid nitrogen and kept in a
)20C freezer till analysis, and the third part was fixed
in liquid nitrogen and lyophilized under )40C for
enzyme assay.
For the determination of seawater H2O2 and per-
oxide contents, seawater samples were collected
everyday and assayed immediately. In this study, each
incubation vessel was a replicate in all statistical anal-
ysis; there were three replicates for each treatment.
Determination of TTC reduction ability and PSII
electron transport activity
To determine the cellular activity, freshly sampled
thallus segments of approximately 0.05 g WW were
incubated at 25C in 1.5 ml of 50 mM potassium
phosphate buffer (pH 7.4) containing 0.8% (w/v) 2,3,5-
triphenyltetrazolium chloride (TTC) and 35& ASW
under darkness for 16 h (Chang et al. 1999). After
washing with 10 ml of 35& ASW for three times,
intracellular insoluble formazan was extracted twice
with 5 ml of 95% ethanol at 80C for 20 min and
ethanol extracts were combined. After having made up
to 10 ml, the absorbance of combined ethanol extract
was determined at A530. The A530 values of salinity-
treated thallus segments were calculated as the per-
centage of the control, that is, the 30& ASW
treatment.
Before the determination of the maximum photo-
synthetic quantum efficiency, Fv/Fm, three thallus seg-
ments randomly selected from a vessel were
transferred to a Petri dish (15 cm diameter) with
250 ml ASW media of the same salinity and incubated
in the darkness for 15 min. The Fv/Fm ratio of each
thallus segment was determined with the underwater
pulse amplitude modulated (PAM) fluorometer
(DIVING-PAM, Walz, Effeltrich, Germany). For each
salinity treatment, three vessels with totally nine thal-
lus segments were determined.
Determination of lipid peroxidation, peroxide,
and H2O2 contents
Thallus segments of approximately 0.1 g WW were
ground in liquid nitrogen and then 1 ml of 5% (w/v)
trichloroacetic acid (TCA) was added. After centrifu-
gation at 12,000g for 10 min at 4C, the supernatant
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4 Mar Biol (2006) 150:1–15
was collected as TCA extract for the determination of
lipid peroxidation, peroxide, and H2O2.
The levels of lipid peroxidation were determined
from the TBARS contents resulting from the thiobar-
bituric acid (TBA) reaction as described by Health and
Tallman (1968). The supernatant of 0.25 ml was mixed
with 1 ml of 0.5% (w/v) TBA in 20% (w/v) TCA and
the mixture was incubated in a water bath at 95C for
30 min. After cooling and centrifugation at 12,000g for
10 min at 4C, the absorbance of the reaction solution
was measured at 532 and 600 nm. After subtracting the
non-specific absorbance at 600 nm, the TBARS con-
tents were determined by its extinction coefficient of
155 mM)1 cm)1.
Thallus and seawater peroxide contents were
determined by the absorbance of A480 and extinction
coefficient of 13.93 lM)1 cm)1 according to Sagisaka
(1976). Because seawater was changed everyday, the
contents of peroxide in seawater were determined
everyday and they were combined as cumulative sea-
water peroxide contents over 4 days.
Thallus H2O2 contents were determined based on
the decomposition of H2O2 by peroxidase as de-
scribed by Okuda et al. (1991). KOH (4 M) of
11.51 ll was added to 0.2 ml supernatant to adjust
pH to 7.5 and the mixture was centrifuged at 12,000g
for 1 min under 4C. The supernatant was collected
and applied to a 1-ml column of Amberlite IRA-410,
and residual H2O2 was washed out by 800 ll of dis-
tilled water. After elution, the contents of H2O2 in
the elute were determined within 10 min. For the
determination of H2O2 in the elute, 0.4 ml of
12.5 mM 3-dimethylaminobenzoic acid (DMAB),
0.4 ml of 10 mM 3-methyl-2-benzothiazoline hydraz-
one (MBTH) and finally 0.02 ml of 0.25 U ml)1
horseradish peroxidase (Sigma, MO, USA) were ad-
ded and then the reaction was measured at 590 nm
for 3 min. The amount of thallus H2O2 was estimated
from the H2O2 standard curve that was determined
as described above.
Seawater H2O2 contents were also determined
following the same method for thallus H2O2 deter-
mination. Sampled seawater of 1 ml was immedi-
ately passed through a 1-ml column of Amberlite
IRA-410 and then the residual H2O2 in the column
was eluted by applying Milli-Q water of 800 ll.
Elutes were combined and adjusted to pH 7.0 with
4 M KOH. Seawater H2O2 contents were calculated
based on the calculation from a series of H2O2
concentrations prepared in nutrient-enriched ASW.
H2O2 contents in seawater were determined daily
and they were combined as cumulative seawater
H2O2 over 4 days.
123
Determination of ascorbate and glutathione
contents
Thallus segments of approximately 0.1 g dry weight
(DW) were ground in liquid nitrogen and then 1 ml of
5% (w/v) TCA was added. After centrifugation at
12,000g for 10 min at 4C, the supernatant was col-
lected as TCA extract for the determination of water-
soluble antioxidant contents.
The measurement of total ascorbate and reduced
AsA contents were modified from the method of Hod-
ges et al. (1996). Total ascorbate contents were deter-
mined in 1-ml mixture containing 200 ll TCA extract,
50 mM potassium phosphate buffer (pH 7.4), 3 mM
EDTA and 1 mM dithiothreitol (DTT). The mixture
was incubated at 25C for 10 min, 100 ll of N-ethyl-
maleimide was added and then 400 ll of 0.61 M TCA,
400 ll of 0.8 M orthophosphoric acid and 400 ll of a,a¢-
bipyridyl were added. Finally, 200 ll of FeCl3 was added
and the mixture was incubated at a 55C water bath for
10 min and the absorbance was detected at A525. Re-
duced AsA contents were determined by adding dis-
tilled water instead of DTT and N-ethylmaleimide and
then followed the same method as above. Total AsA and
reduced AsA contents were estimated from the stan-
dard curve of 0–40 nmol L-AsA determined by the
above methods. Oxidized AsA contents were calculated
by the subtraction of reduced AsA from total AsA.
Total glutathione contents were determined by the
method according to Griffiths (1980). The contents of
glutathione (reduced form) were estimated from the
standard curve of 0–20 nmol GSH. After the removal
of GSH by 2-vinylpyridine derivatization, GSSG con-
tents were determined and GSH contents were calcu-
lated by the subtraction of GSSG contents from total
glutathione contents.
Determination of antioxidant enzyme activity
Lyophilized thallus discs of approximately 0.08 g DW
were first homogenized in liquid nitrogen and 0.8 ml of
0.1 M sodium phosphate buffer (pH 6.8) containing 1%
(w/v) polyvinylpolypyrrolidone (PVPP) and 0.25%
(v/v) Triton X-100 was then added. After centrifugation
at 12,000g for 10 min at 4C, the supernatant was used
for enzyme activity assay of SOD, CAT, POX, GR,
MDHAR and DHAR. For the determination of APX,
0.1 g DW of lyophilized thallus discs was homogenized
in liquid nitrogen and 1 ml of extraction buffer [0.1 M
sodium phosphate buffer (pH 6.8) containing 1% (w/v)
PVPP, 0.25% (v/v) Triton X-100 and 0.5 mM L-ascor-
bate] was added. After centrifugation at 12,000 g for
10 min at 4C, the supernatant was used for APX assay.
Mar Biol (2006) 150:1–15
The soluble protein contents were determined by coo-
massie blue dye binding method (Bradford 1976) with
bovine serum albumin as standard curve.
The CAT activity was measured at A420 for H2O2
decomposition rate using the extinction coefficient of
40 mM)1 cm)1 according to Kato and Shimizu (1987).
Guaiacol POX activity was determined by the formation
rate of tetraguaiacol detected at A470 with the extinction
coefficient of 26.6 mM)1 cm)1 according to Kato and
Shimizu (1987). SOD activity was determined by the
inhibition of photochemical inhibition of nitro blue
tetrazolium according to Giannopolitis and Ries (1977).
APX activity was determined at A290 for oxidized AsA
according to the extinction coefficient of 2.8 mM)1 cm)1
(Nakano and Asada 1981). GR activity was monitored
by A340 for b-NADPH oxidization as GSSG reduction
according to Schaedle and Bassham (1977).
Both MDHAR and DHAR activities were deter-
mined according to Hou and Lin (1997). AR activity
was measured by monitoring the absorbance at 265 nm
for 3 min in the reaction mixture that consisted of
enzyme extract, 50 mM Na-phosphate buffer (pH 7.0),
0.3 mM GSH, 0.06 mM Na2EDTA and 0.2 mM DHA.
MDHAR activity was measured by monitoring the
absorbance at 340 nm for 3 min in 50 mM Na-phos-
phate buffer (pH 7.6), 0.1 mM b-NADPH and 0.1 U
AsA oxidase (Sigma, MO, USA) and 2.5 mM AsA.
Non-enzymatic reduction of DHA or MDHA in
phosphate buffer was measured in a separate cuvette at
the same time.
Chemicals and statistical analysis
Chemicals were purchased from Merck (Germany) or
Sigma (USA). Statistics were analyzed by SAS (SAS v
8.1, NC, USA). The present results were the mean of
three replicates with a vessel as a replicate. The effects
of salinity on TTC reduction ability, TBARS contents,
peroxide contents, H2O2 contents, water-soluble anti-
oxidant contents, and enzyme activities were analyzed
by one-way analysis of variance (ANOVA). The dif-
ference among means was analyzed by Duncan’s new
multiple range test following by significant ANOVA at
P < 0.05.
Results
Water contents, growth rate, photosynthesis, TTC
reduction ability and the contents of TBARS,
peroxide and H2O2
The changes in salinity lead to a change of water
contents of U. fasciata and in turn also of the cellular
5
ion contents (Na+, K+, and Cl)) (Lee and Liu 1999).
Here, the data from Fig. 1a show that as compared to
the 30& ASW-cultured thalli, the relative water con-
tents after 4 days of hyposaline treatment increased as
salinity decreased from 30 to 5& and on exposure to
hypersalinity, the relative water contents decreased
fast and approached a steady level when salinity
was ‡ 60&, (ANOVA, P < 0.05 and Duncan’s new
multiple range test, P < 0.05).
After 4 days of exposure to different salinities, the
growth rate, photosynthetic Fv/Fm ratios, and TTC
reduction ability were affected by salinity (one-way
ANOVA, P < 0.05). As compared to the 30& control,
growth rate (Fig. 1b), Fv/Fm ratio (Fig. 1c), and TTC
reduction ability (Fig. 1d) were decreased by decreas-
ing and increasing salinities.
Lipid peroxidation (TBARS contents) (one-way
ANOVA, P < 0.0001) and peroxide contents
(P = 0.0006 for thallus peroxide and P = 0.0065 for
seawater peroxide) as oxidative stress parameters were
significantly affected by salinity (Fig. 2). As compared
to the 30& control, the contents of both TBARS
(Fig. 2a) and thallus peroxide (Fig. 2b) increased at 60
and 90& while seawater peroxide contents were in-
creased by decreasing and increasing salinity except
150& (Fig. 2c).
Next we investigated the salinity effect on H2O2
production by thallus segments. A large quantity of
H2O2 detected in seawater after incubation of thallus
segments seems to suggest that thalli of U. fasciata
could secrete H2O2 into seawater. Therefore, in addi-
tion to thallus H2O2 contents, the H2O2 contents in
seawater were also quantified in this study. The H2O2
contents of thalli and seawater were only determined at
day 4; seawater medium at day 1, day 2, and day 3 were
not collected and thus seawater H2O2 contents were
not determined at day 1, day 2, and day 3. As shown in
Fig. 3, salinity affected thallus (one-way ANOVA,
P = 0.0032) and seawater (P < 0.0001) H2O2 contents.
The contents of thallus H2O2 increased at 5, 15, 60 and
90& as compared to the 30& control but those at 120
and 150& were similar to the 30& control (Fig. 3a). At
day 4, seawater H2O2 contents were decreased by
decreasing and increasing salinities (Fig. 3b).
Ascorbate and glutathione contents
Ascorbate and glutathione are not only important an-
tioxidants against oxidative stress but also the sub-
strates for APX and GR in the AGC (Noctor and
Foyer 1998; Smirnoff and Wheeler 2000). Therefore,
the contents of ascorbate and glutathione were deter-
mined to test whether water-soluble antioxidants are
123
6 Mar Biol (2006) 150:1–15

Fig. 1 Changes in relative 18

water contents (a), specific
growth rate (b), Fv/Fm ratios a A. Relative water content

(g H2O.g-1 DW)
(c) and TTC reduction ability
(d) in Ulva fasciata in 12
response to different
salinities. Data are presented b
as means ± SD (n = 3). 6 bc
Different letters above or c c c c
below symbols indicate
significant level at P < 0.05
by Duncan’s new multiple
a
range test 30 B. Specific growth rate
(% DW)
b
20
bc
cd
10 cde
e de
0

0.9 a C. Fv/Fm
Fv/Fm ratio

b b
0.6 bc
c

0.3 e
ef

0.0

120 a
D. TTC reduction activity
ab
(% of control)

90 b
b
60

30 c
c c
0

0 30 60 90 120 150

Salinity (o/oo)

involved as the defense system against salinity-induced
oxidative stress in U. fasciata. The contents of total
AsA (ANOVA, P < 0.0001), AsA (P < 0.0001) and
DHA (P = 0.0010) and the ratios of AsA/DHA
(P = 0.0080) were significantly influenced by salinity
(Fig. 4). Total AsA contents were increased by 15&
but decreased by 120 and 150& (Fig. 4a). The contents
of AsA were also increased by 15& and gradually
decreased by increasing salinity (Fig. 4b) while those of
DHA were increased by low salinity (5 and 15&) and
also by 90& (Fig. 4c). To explore the regeneration of
AsA, the ratio of AsA/DHA was calculated. The data
in Fig. 4d showed that the ratio of AsA/DHA de-
creased with decreasing and increasing salinity as
compared to the 30& control.
Total glutathione (ANOVA, P < 0.0001), GSH
(P = 0.0020) and GSSG (P < 0.0001) contents and
GSH/GSSG ratios (P = 0.0039) were affected by
salinity (Fig. 5). As compared to 30& control, the
contents of total glutathione (Fig. 5a) and GSSG
(Fig. 5c) were increased by hyposalinity (5 and 15&)
and also by 60 and 90&. GSH contents and GSH/
GSSG ratios significantly increased at 15& and de-
creased at 120 and 150& (Fig. 5b).
Enzyme activities
Salinity significantly affected the activity of SOD based
on mg protein (ANOVA, P < 0.0001) that increased
when salinity was ‡ 120& (Fig. 6a). SOD activity

123
Mar Biol (2006) 150:1–15 7

Fig. 2 Changes in the

0.6
contents of TBARS (a) and A. MDA b
peroxide in thallus (b) and a

(µmol.g DW)
seawater (c) in Ulva fasciata 0.4
in response to different

-1
salinities. Data are presented
as means ± SD (n = 3). 0.2 c c
Different letters above or c
c c
below symbols indicate
significant level at P < 0.05 0.0
by Duncan’s new multiple
range test 15
B. Thallus total peroxide a
(µmol.g DW) 12

9
b
-1

6 b
bc bc
3 c c

0
40
C. Seawater peroxide ab a
(µmol.g DW)

30
ab
ab bc
-1

20

10 c
c
0

0 30 60 90 120 150

Salinity (o/oo)

based on g dry weight was also influenced by salinity
(ANOVA, P < 0.0001); it only increased at 5&. SOD
activity based on g dry weight showed a different pat-
tern as compared to the activity based on U h)1 mg)1
protein; the increase in the activity of SOD based on
mg protein at 120 and 150& did not appear for SOD
activity based on g dry weight (Fig. 6a). This difference
is mainly due to a marked decline of soluble protein
contents at 120 and 150&.
The activities of CAT and POX on the basis of mg
protein or g dry weight were all affected by salinity
(ANOVA, P < 0.0001), but two enzymes showed a
different pattern at different salinities (Fig. 6b, c).
CAT activity based on mg protein was increased not
only by decreasing salinities but also increased by
increasing salinities with the maximum at 120&. Based
on g dry weight, the responses of CAT activity to hy-
posalinity showed a similar trend to SOD activity
based on mg protein. Under hypersaline conditions,
SOD activity increased fast to a maximum at 60–90&
and then decreased (Fig. 6b). POX activity based on
mg protein and g dry weight showed the same pattern,
both increased only at 60 and 90& (Fig. 6c).
Activities of APX (one-way ANOVA, P < 0.0001)
and GR (P = 0.0031) based on mg protein and g dry
weight were affected by salinity (Fig. 6d, e). GR
activity based on mg protein increased linearly as
decreasing salinities while that based on g dry weight
peaked at 15& and then showed a drop at 5&
(Fig. 6d). Upon exposure to hypersalinity, GR activity
based on mg protein and g dry weight peaked at 60&
and then declined to a plateau only slightly higher than
the 30& control (Fig. 6d). APX activity based on mg
protein and g dry weight was linearly increased by
decreasing salinities (Fig. 6e). APX activity was also
increased by hypersalinity, but its pattern in response
to hypersaline conditions was different between mg
protein and g dry weight basis (Fig. 6e). APX activity
based on mg protein increased to a plateau when
salinity was ‡ 90& whereas that based on g dry weight
increased to a peak at 90& and then dropped fast at
120 and 150& (Fig. 6e).
Based on mg protein and g dry weight, the activities
of both DHAR (one-way ANOVA, P < 0.0001) and
MDHAR (P = 0.0001), the enzymes involving in the
regeneration of AsA, were affected by salinity (Fig. 7).

123
8 Mar Biol (2006) 150:1–15
Fig. 3 Changes in the
500
contents of H2O2 in thallus
(a) and seawater (b) in Ulva 400
(nmol.g DW)
fasciata in response to
different salinities. Data are 300
-1
presented as means ± SD
200
(n = 3). Different letters
above or below symbols 100
indicate significant level at
P < 0.05 by Duncan’s new 0
multiple range test
80
(µmol.g DW)
60
-1
40
20
0
0 30
DHAR activity based on mg protein and g dry weight
did not change in response to hyposalinity while that
based on mg protein increased only at 150& with a
2-fold increase as compared to the 30& control, and
that based on g dry weight showed a significant decline
at 120 and 150& (Fig. 7a). MDHAR activity on the
basis of mg protein and g dry weight was significantly
increased by extreme hyposalinity (5&) and showed a
linear increase as salinity increased from 60 to 90&,
then decreased to around zero at both 120 and 150&
(Fig. 7b).
Effects of DMTU on oxidative responses
To elucidate the roles of O2) and H2O2 as elicitors for
induction of oxidative damage in U. fasciata exposed to
hyposaline and hypersaline conditions, thallus seg-
ments were first pre-incubated in 30& ASW medium
containing varying DMTU (25 and 100 lM) concen-
trations under darkness for 12 h and then transferred
to 15 and 90&, respectively, for 1 day. TTC reduction
ability, the contents of thallus H2O2 and TBARS, and
the activities of SOD, CAT, POX, APX, GR, DHAR
and MDHAR were determined (Table 1). DMTU
treatment recovered the TTC reduction ability of 15&-
and 90&-treated thallus segments, and reduced the
increase of thallus H2O2 and TBARS contents. The
increase in the activities of CAT, POX, APX, GR, and
DHAR at 15 and 90& was inhibited by DMTU
(P < 0.05), while SOD and MDHAR activities were
not affected by DMTU (P > 0.05).
123
a
A. Thallus H2O2
ab a
bc c
c
c
a B. Seawater H2O2
b
c c c c c
60 90 120 150
o
Silinity ( /oo)
Discussion
An exposure to hyposaline and hypersaline conditions
not only influenced the water status of U. fasciata but
also resulted in growth inhibition, in which the extent
of growth inhibition increases with decreasing and
increasing salinities (30& as the control). Both the
respiratory dehydrogenase-dependent TTC reduction
ability and the photosystem II activity (Fv/Fm ratio)
were also decreased by decreasing and increasing
salinities; the inhibition is more significant under hy-
persaline condition. In comparison to a 10% reduction
of Fv/Fm ratio for hyposaline conditions, there was a
30–40% reduction for moderate hypersaline conditions
and an 80% reduction for extreme hypersaline condi-
tions, suggesting that the activity of photosystem II is
more sensitive to hypersaline stress. However, the re-
sponses of photosystem II activity to salinity altera-
tions depend on algal species. In the marine microalga
D. tertiolecta, the Fv/Fm ratio was not affected by hy-
persaline conditions but showed a 20% reduction in
response to hyposaline conditions (Jahnke and White
2003).
The blockage of photosynthetic electron transport
would lead to over production of ROS (Asada 1994).
Thus, the decline in photosystem II activity in U. fas-
ciata following salinity stress implies enhanced ROS
generations. The data show that one of ROS species
generated by U. fasciata following salinity stress is
H2O2 that appears to be intimately related to oxidative
damage as indicated by the concomitant increase of
Mar Biol (2006) 150:1–15 9

Fig. 4 The contents of total 12

AsA (a), reduced AsA (b), a
A. Total AsA
oxidized AsA (c), and

(nmol.g-1 DW)
9 b
reduced AsA/oxidized AsA b
b
ratio (d) in Ulva fasciata in b
response to different 6
salinities. Data are presented
as means ± SD (n = 3). 3
Different letters above or c c
below symbols indicate 0
significant level at P < 0.05
by Duncan’s new multiple a
12
range test (nmol.g-1 DW) B. AsA
9

b b
6
c c
3
d d
0

4 ab a C. DHA
(nmol.g DW)

3 ab
-1

2 cd bc

1 cd
d
0

12
a D. AsA/DHA
9
ab
6

bc bc
3 c c
c
0

0 30 60 90 120 150

Salinity (o/oo)

thallus H2O2 and peroxide contents and the extent of
lipid peroxidation at 60–90&. The prevention of lipid
peroxidation at 90& by DMTU treatment indicates the
involvement of H2O2 in oxidative damage in U. fasci-
ata exposed to moderate hypersaline conditions.
Moreover, the extent in the decline of Fv/Fm ratio
following salinity stress was increased with decreasing
or increasing salinities. In this way, it can be expected
that the generation of ROS is enhanced in salinity-
stressed U. fasciata with a progressive accumulation of
H2O2 and other ROS as decreasing and increasing
salinities. However, marked H2O2 accumulation was
detected only in moderate hyposaline (15&) and hy-
persaline (60–90&) conditions. H2O2 did not accumu-
late under extreme hypersaline conditions (120 and
150&). It is known that H2O2 is generated photosyn-
thetically and non-photosynthetically; the photosyn-
thetic production of O2) at photosystem I could be
spontaneously disproportionated to H2O2 or converted
to H2O2 by SOD. It is postulated that the severe
inhibition of photosystem II activity in U. fasciata
grown under extreme salinities (120 and 150&) re-
flected by marked decline of Fv/Fm ratios to approxi-
mately 0.15–0.23 decreases electron transport to
photosystem I and thus reduces the Mehler reaction-
mediated O2) production. As a consequence, the for-
mation of H2O2 decreases. Because SOD activity
based on mg protein or g dry weight was not sup-

123
10 Mar Biol (2006) 150:1–15

Fig. 5 The contents of total a

glutathione (a), GSH (b), 1200
A. Total GSH
GSSG (c), and GSH/GSSG ab

(nmol.g DW)
ratio (d) in Ulva fasciata in 900 bc
response to different c

-1
600
salinities. Data are presented d
as means ± SD (n = 3).
300
Different letters above or e e
below symbols indicate 0
significant level at P < 0.05
by Duncan’s new multiple
range test a
800 B. GSH
(nmol.g DW)
600
-1

400
bc b
b
200 bc
c c
0

800 C. GSSG
a
(nmol.g DW)

600 a
-1

b bc
400 c
200
d d
0

a
2.0 D. GSH/GSSG

1.5

1.0
b b b
0.5 b

0.0
0 30 60 90 120 150
o
Salinity ( /oo)

pressed at 120& and at 150&, the dismutation of O2)
to H2O2 mediated by SOD, which constitutes the first
line of defense components against ROS in plants
(Alscher et al. 2002), is not an essential element
involving the regulation of antioxidant defense system
in U. fasciata against salinity-induced oxidative stress.
A large quantity of H2O2 in seawater after incu-
bation of U. fasciata thallus segments in 30& medium
reflects that H2O2 generated by thalli can be secreted
extracellularly. The pattern that the lower seawater
H2O2 contents, the higher thallus H2O2 contents at
15, 60, and 90& implies that the secretion of H2O2
might be inhibited under moderate hyposaline and
hypersaline conditions. Macroalgae such as the chlo-
rophyte Ulva rigida C. Ag. (Collén et al. 1995) and
the brown macroalga Fucus sp. (Collén and Davison
1999a, b) are able to secrete cellular H2O2 into the
surrounding medium to avoid H2O2 accumulation.
This secretion of H2O2 into the surrounding water has
been considered a mechanism for algae to avoid
harmful accumulation of cellular H2O2 (Collén et al.
1995; Barros et al. 2003). We thus hypothesize that
the inhibition of H2O2 secretion by U. fasciata in
moderate hyposaline and hypersaline conditions re-
sults in harmful accumulation of cellular H2O2 under
15, 60, and 90& conditions.
It is known that increasing antioxidant pools and its
scavenging capacity are essential for plant defense
against oxidative stress (Noctor and Foyer 1998). In
dinoflagellates (Butow et al. 1994) and in a brown
macroalga Fucus (Collén and Davison 1999a, b),
higher antioxidant contents are correlated with higher

123
Mar Biol (2006) 150:1–15 11

Fig. 6 The activities of SOD 4000 10000

(a), CAT (b), POX (c), APX A. SOD (b) a
(d) and GR (e) in Ulva 3000 (b) 8000

(U.h .mg protein)

fasciata in response to (b) (b) (b)

(U.h .g DW)
(a) 6000
different salinities. Data are 2000 (b)

-1
-1
presented as means ± SD 4000

-1
(n = 3). Different letters

-1
1000
above or below symbols b
c c 2000
indicate significant level at c c c
0
P < 0.05 by Duncan’s new 0
(a) (a) a 4
multiple range test. Letters in B. CAT

(µmol.min-1.mg-1 protein)
1.8
parenthesis represent (ab) b

(µmol.min-1.g-1 DW)
significant difference for 3
(b)
activity based on g)1 DW 1.2 c c
2
(bc)
0.6 c (c)
d (e) 1

e
0.0 0

0.09 C. POX a 0.4

(µmol.min-1.mg-1 protein)

(µmol.min-1.g-1 DW)
0.3
0.06

(a) (a) 0.2

0.03
b b b
b b 0.1
0.00 (c) (b) (c) (c)
(b) 0.0
D. GR 25
5 a
(µmol.min-1.mg-1 protein)

4 20

(µmol.min-1.g-1 DW)
3 15
(b) (a)
b 10
2
c c c
1 5
(c) b (e)
d (d) (d) (de)
0 0
E. APX (a) a
1.5
(µmol.min-1.mg-1 protein)

b 9

(µmol.min-1.g-1 DW)
1.0 b
6
(b)
0.5 c (b)
d d c
3
0.0
(d) (d) (c) (c)
0
0 30 60 90 120 150

o
Salinity ( /oo)

tolerance to stress. Here, we have found that the radicals, superoxide, singlet oxygen and H2O2 and it is
antioxidant defense systems were induced by salinity also used for regeneration of the lipophilic antioxi-
stress in U. fasciata; however, not only the ROS dant a-tocopherol (Smirnoff 1996; Noctor and Foyer
generation but also the antioxidant metabolism and 1998). AsA also involves in photoprotection, in the
antioxidant enzymes are differently regulated under regulation of photosynthesis and in preserving the
different salinity conditions. In the moderate hypos- activities of enzymes that contain prosthetic transition
aline condition (15&), increasing AsA and GSH metal ions (Smirnoff 1996). Our observation that the
pools and their regeneration rate together with high amounts of total AsA as well as reduced AsA were
CAT and DHAR activities are responsible for the increased by 15& indicates that sufficient AsA
detoxification of accumulated H2O2. It is known that availability is provided by enlarging the AsA pool.
AsA has multiple functions in cellular metabolism; for Although AsA/DHA ratios decreased, DHAR activ-
example, AsA is the substrate of APX for enzymatic ity increased. This may imply that the regeneration of
detoxification of H2O2. It reacts directly with hydroxyl AsA is stimulated by exposure to 15&. In addition to

123
12 Mar Biol (2006) 150:1–15

Fig. 7 The activities of 0.5 2.0

DHAR (a) and MDHAR (b) A. DHAR (a) a
in Ulva fasciata in response to 0.4

(µmol.min-1.mg-1 protein)
different salinities. Data are (ab) 1.5

(µmol.min-1.g-1 DW)
presented as means ± SD 0.3 (b) (b)
(b)
(n = 3). Different letters 1.0
above or below symbols 0.2
b
b b
indicate significant level at b b
b
P < 0.05 by Duncan’s new 0.5
0.1
multiple range test. Letters in (c) (c)
parenthesis represent
0.0 0.0
significant difference for
activity based on g)1 DW 3 B. MDHAR
(a) 12
(µmol.min-1.mg-1 protein) (ab)
(ab)

(µmol.min-1.g-1 DW)
2
8
(c)
(cd)
1 ab 4
a abc
d f f
de (e) (e)
0 0
0 30 60 90 120 150
Salinity (o/oo)

AsA, GSH was also involved in the defense system by
increasing the glutathione pool for scavenging H2O2
in 15&-treated U. fasciata. An increase in GR activity
at 15& also reflects that the ability to regenerate
glutathione was enhanced, but the GSH/GSSG ratios
did not increase. Because it has been documented
that GSH functions as an antioxidant to react non-
enzymatically with singlet oxygen, superoxide and
hydroxyl radical and also protects proteins against
denaturation caused by the oxidation of protein thiol
groups (Noctor et al. 2002), GSH can be non-enzy-
matically used to remove ROS. Further, GSH is also
utilized by glutathione peroxidase in the removal of
lipid and alkyl peroxide (Eshdat et al. 1997) or by
glutathione-S-transferase to reduce peroxide (Cum-
mins 1999). Possibly, GSH is consumed at a faster
rate than the regeneration rate, thus leading to
notable reduction of GSH/GSSG ratios in 15&-trea-
ted U. fasciata. In response to the moderate hyposa-
linity, the mechanisms for the amelioration of
oxidative stress in U. fasciata is different in compari-
son to a halotolerant microalga D. tertiolecta which
increases GSH and thylakoid a-tocopherol contents
instead of antioxidant enzymes as an antioxidant de-
fense system in hyposaline conditions (Jahnke and
White 2003). In contrast to our study, there was a
slight decrease of AsA contents in this salt tolerant
D. tertiolecta exposed to hyposaline media (Jahnke
and White 2003).
In moderate hypersaline conditions (60–90&), the
SOD activity remained unchanged for O2) dismuta-
tion. A similar result has been observed in a green
microalga D. tertiolecta in response to high salt stress
(Jahnke and White 2003). It is also found in pea
(Hernández et al. 2000) and cucumber (Lechno et al.
1997). But SOD activities in tomato (RodriguezRo-
sales et al. 1999), pea (Hernández et al. 1999), cotton
(Gossett et al. 1994), rice (Lee et al. 2001), and barley
(Liang 1999) show an increase upon exposure to high
salt conditions. Present evidence shows that generated
H2O2 in moderate hypersaline conditions is degraded
by CAT and POX together with AGC. There was an
increase in GR, APX and MDHAR activities at 60 and
90&. APX has been suggested to be involved in the
degradation of H2O2 generated under high light con-
dition (Collén and Pedersen 1996). The efficient
regeneration of AsA and GSH mediated by MDHAR
and GR makes AGC working. A similar result has
been observed in a green microalga D. tertiolecta in
response to high salt stress that APX and MDHAR
activities increased as defensive responses to remove
ROS and keep cellular AsA level constant (Jahnke and
White 2003). But they did not find increased CAT
activity under high salt conditions. It is in contrast to
our observation that CAT as well as POX had signifi-
cant increase on exposure to high salinity. Evidently,
the antioxidant defense system is differently regulated
between algal species. It is likely that SOD activity in
U. fasciata is kept constant for O2) dismutation and the
generated H2O2 is subsequently degraded by CAT and
POX together with AGC in moderate hypersaline
conditions.

123
Mar Biol (2006) 150:1–15 13

A different antioxidant defense system is activated

0.013c
Table 1 Effects of 0, 25 and 100 lM DMTU (dimethylthiourea) on TTC reduction ability (% of initial), the contents of H2O2 (nmol g)1 DW) and TBARS (lmol g)1 DW), and
the activities of SOD (U · 103 h)1 g)1 DW), CAT, POX, APX, GR, DHAR, and MDHAR (lmol min)1 g)1 DW) of 15&-, 30&- and 90&-treated U. fasciata thallus segments

TBARS thiobarbituric acid reacting substance, TTC 2,3,5-triphenyltetrazolium chloride, APX ascorbate peroxidase, CAT catalase, DHAR dehydroascorbate reductase, GR
1.03a

0.001
0.97c

0.02c

0.08c
0.05c
0.07c
0.56c
5.9a
by extreme salinities. The increase of SOD and CAT

100 lM
activities at 5& indicates that SOD and CAT are uti-

±
±
±
±
±
±
±
±
±
±
0.109

0.029
74.3
3.01

6.52
0.12

0.42
0.31
0.35
2.91
lized for enzymatically scavenging of ROS at 5&.
Besides, antioxidant availability is elevated at 5& by
increasing AsA and GSH contents and enhancing

0.011b
1.08ab

0.02ab
4.09b

0.22b

0.07b
0.08b

1.02b
0.011
6.6a
regeneration of these antioxidants as indicated by in-

Data are presented as means ± SD (n = 3) with different symbols indicating difference among different DMTU concentrations for each salinity treatment
creased GSH/GSSG ratios and increased GR, DHAR,
±
±
±
±
±
±
±
±
±
±
25 lM

23.54
0.189

0.093
52.5

5.61
0.98

0.87
0.98
0.72
6.39
and MDHAR activities. However, the defense system
working at 120 and 150& is different to that at 5&.
Among antioxidant enzymes, only APX and CAT,
29.05a
0.021a
1.83a
0.27a
0.013
1.01a
0.62a
0.23a
0.92a
4.8b

especially based on mg protein, showed an increase

upon exposure to 120 or 150&. The amount of an-
±
±
±
±
±
±
±
±
±
±
354.96
24.3

7.21
3.98

4.09
1.09
0.397

0.452
11.31

12.12 tioxidants (AsA and GSH) was also increased to

0 lM
90&

scavenge ROS, but they accumulated to a less extent in

comparison with 60 and 90&. This may be due to the
0.013a
0.01b

0.06b
0.01a
0.78a
1.13c

0.02c

0.03c
0.43c

fact that AsA as well as GSH were utilized for direct

4.9a

scavenging H2O2 or as the substrate for APX to scav-

100 lM

±
±
±
±
±
±
±
±
±
±

enge H2O2 generated under these severe hypersaline

0.87

5.98
0.11

0.35
0.32
0.21
1.16
102.3

0.063

0.041

conditions. Moreover, the suppression of activities of

GR, DHAR, and MDHAR at 120 and 150& demon-
0.007a

0.008a
5.27b

0.02b

0.07b
0.06b
0.09b
0.53b
1.11a

strates that AsA and GSH are consumed without

2.4a

glutathione reductase, MDHAR monodehydroascorbate, SOD superoxide dismutase, POX peroxidase

regeneration. It has been reported in several higher

±
±
±
±
±
±
±
±
±
±
25 lM

plants that total AsA and GSH contents decreased

99.5

7.01
0.09

0.98
0.35
0.52
3.71
19.56
0.059

0.042

under high salt stress (Gossett et al. 1994; Hernandez

et al. 1999; Hernandez et al. 2002), but in some cases,
0.008a

0.001a
8.78a

0.92a
0.01a

0.19a
0.13a
0.16a
0.36a

they are increased by high salt conditions (Lechno

6.7a

et al. 1997). The present study proved that the anti-

±
±
±
±
±
±
±
±
±
±

oxidant defense systems at 5& are different to those at

0 lM

105.8
90.17
0.052

0.031
6.78
0.23

1.78
0.92
1.17
5.21
30&

both 120 and 150&.

H2O2 has been known as the signal for the induction
0.11ab
0.005a

0.005a
0.32a
1.21c

0.03c

0.02c
0.01c
0.48c

of antioxidant defense system in plants in response to

5.3a

biotic and abiotic stresses (Neil et al. 2002). The inhi-

100 lM

±
±
±
±
±
±
±
±
±
±

bition of salinity-induced increase in antioxidant en-

0.063

0.049
95.9
2.49

6.05
0.15

0.39
0.29
0.31
1.18

zyme activities by DMTU, the scavenger of H2O2,

supports the idea that the induction of antioxidant
0.003a

0.011a
5.09b

0.77b
0.14b

0.05b
0.11b
0.57b
0.12a

defense system by moderate hyposalinity and hy-

3.6a

persalinity in U. fasciata is mediated by H2O2. The

±
±
±
±
±
±
±
±
±
±
25 lM

induction of APX gene expression by H2O2 has also

90.8

6.13
1.87

0.67
0.78
0.96
2.99
21.33
0.058

0.056

been observed in germinating rice embryos (Morita

et al. 1999) and in Arabidopsis leaves (Karpinski et al.
0.011a

0.006a
0.86b
4.51a

0.98a

0.09a
0.27a
0.03a
0.93a

1999). Evidently, H2O2 not only acted for triggering

6.7b

oxidative stress but also served as a signal for the

±
±
±
±
±
±
±
±
±
±

induction of antioxidant defense system in U. fasciata.

68.5

6.36
3.05

0.56
5.62
1.98
3.85
0.073

0.041
125.78
0 lM
15&

The results of present investigation indicate that

U. fasciata growing in intertidal areas and experiencing
salinity fluctuations has developed a defense system to
TTC reduction ability

cope with the oxidative stress induced by salinity

TBARS contents

changes. The balance between the production and the

H2O2 contents

removal of H2O2 in U. fasciata is disrupted by adverse

MDHAR

salinities, and H2O2 is one of ROS that is known to

DHAR

easily diffuse from the sites of production to other cel-

POX
APX
SOD
CAT

GR

lular compartments due to no charge like O2), and its

123
14
flux and accumulation are monitored by ascorbate and
glutathione, together with the antioxidant enzymes. The
availability of antioxidants and the activation of CAT,
guaiacol POX, and enzymes in the AGC served as the
defense system against oxidative stress occurring in
U. fasciata exposed to hyposaline and hypersaline con-
ditions. Antioxidant amounts and its regeneration and
ROS detoxifying enzyme activity are differently regu-
lated between moderate and extreme salinities, and also
between hyposaline and hypersaline stress.
Acknowledgements The scholarship from the National Science
Council (NSC93-2815-C-110-033-B), Executive Yuan, Taiwan,
Republic of China, to I-Fan Lu is acknowledged.
References
Alscher RG, Erturk N, Heath LS (2002) Role of superoxide
dismutases (SODs) in controlling oxidative stress in plants. J
Exp Bot 53:1331–1341
Amor NB, Hamed KB, Debez A, Grignon C, Abdelly C (2005)
Physiological and antioxidant responses of the perennial
halophyte Crithmum maritimum to salinity. Plant Sci
168:889–899
Asada K (1994) Production and action of active oxygen species
in photosynthetic tissues. In: Foyer CH, Mullineauxs RH
(eds) Causes of photooxidative stress and amelioration of
defense system in plants, CRC Press, Boca Raton pp 77–103
Asada K (1999) The water–water cycle in chloroplasts: scav-
enging of active oxygen and dissipation of excess photons.
Ann Rev Plant Physiol Plant Mol Biol 50:601–639
Asada K, Takahashi M (1987) Production and scavenging of
active oxygen radicals in photosynthesis. In: Kyle DJ, Os-
mond CB, Arntzen CJ (eds) Photoinhibition, vol 9. Elsevier,
Amsterdam, pp 227–288
Barros MP, Pedersen M, Colepicolo P, Snoeijs P (2003) Self-
shading protects phytoplankton communities against H2O2-
induced oxidative damage. Aquat Microbiol Ecol 30:275–
282
Bradford MM (1976) A rapid and sensitive method for the
quantification of microgram quantities of protein utilizing
the principal of protein-dye binding. Anal Biochem 72:248–
254
Butow B, Wynne D, Tel-Or E (1994) Response of catalase
activity to environmental stress in the freshwater dinofla-
gellate Peridinium gatunense. J Phycol 30:17–22
Chang WC, Chen MH, Lee TM (1999) 2,3,5-Triphenyltetrazoli-
um reduction in the viability assay of Ulva fasciata (Chlo-
rophyta) in response to salinity stress. Bot Bul Acad Sin
40:207–212
Collén J, Davison IR (1999a). Reactive oxygen production and
damage in intertidal Fucus spp. (Phaeophyceae). J Phycol
32:54–61
Collén J, Davison IR (1999b) Reactive oxygen metabolism in
intertidal Fucus spp. (Phaeophyceae). J Phycol 35:62–69
Collén J, Pedersen M (1996) Production, scavenging and toxicity
of hydrogen peroxide in the green seaweed Ulva rigida. Eur
J Phycol 31:265–271
Collén J, Del Rio MJ, Garcia-Reina G, Pedersen M (1995)
Photosynthetic production of hydrogen peroxide by Ulva
rigida C. Ag. (Chlorophyta). Planta 196:225–230
123
Mar Biol (2006) 150:1–15
Cummins I (1999) A role for glutathione transferases functioning
as glutathione peroxidases in resistance to multiple herbi-
cides in black-grass. Plant J 18:285–292
Davis KJA (1987) Protein damage and degradation by oxygen
radicals. I. General aspects. J Biol Chem 262:9895–9901
Dionisio-Sese ML, Tobita S (1998) Antioxidant responses of rice
seedlings to salinity stress. Plant Sci 135:1–9
Doke N, Miura Y (1995) In vitro Activation of NADPH-
dependent superoxide generating system in a plasma
membrane-rich fraction of potato tuber tissues by treatment
with an elicitor from Phytophtora infestans or with digitonin.
Physiol Mol Plant Physiol 46:17–28
Eshdat Y, Holland D, Faltin Z, Ben-Hayyim G (1997) Plant
glutathione peroxidases. Physiol Plant 100:234–240
Fadzilla NM, Finch RP, Burdon RH (1997) Salinity, oxidative
stress and antioxidant responses in shoot cultures of rice. J
Exp Bot 48:325–331
Giannopolitis CN, Ries SK (1977) Superoxide dismutase. I.
Occurrence in higher plants. Plant Physiol 59:309–314
Gossett DR, Millhollon EP, Lucas C (1994) Antioxidant re-
sponse to NaCl stress in salt-tolerant and salt-sensitive cul-
tivars of cotton. Crop Sci 34:706–714
Griffiths OW (1980) Determination of glutathione and gluta-
thione disulphide using glutathione reductase and 2-vinyl-
pyridine. Anal Biochem 106:207–212
Halliwell B, Gutteridge JMC (1989) Free radicals in biology and
medicine. Clarendon, Oxford
Health RL, Tallman G (1968) Photoperoxidation in isolated
chloroplasts. I. Kinetics and stoichemtry of fatty acid per-
oxidation. Arch Biochem Biophys 125:189–198
Hernández JA, Corpas FJ, Gomez M, del Rı́o LA, Sevilla F
(1993) Salt induced oxidative stress mediated by activated
oxygen species in pea leaf mitochondria. Physiol Plant
89:103–110
Hernández JA, Campillo A, Jimenez A, Alarcon JJ, Sevilla F
(1999) Response of antioxidant systems and leaf water
relations to NaCl stress in pea plants. New Phytol 141:241–
251
Hernández JA, Jimenez A, Mullineaux P, Sevilla F (2000) Tol-
erance of pea plants (Pisum sativum) to long-term salt stress
is associated with induction of antioxidant defenses. Plant
Cell Environ 23:853–862
Hernández JA, Nistal B, Dopico B, Labrador E (2002) Cold
and salt stress regulates the expression and activity of a
chickpea cytosolic Cu/Zn superoxide dimutase. Plant Sci
163:507–514
Hodges DM, Andrews CJ, Johnson DA, Hamilton RI (1996)
Antioxidant compound responses to chilling stress in dif-
ferentially sensitive inbred maize lines. Physiol Plant
98:685–692
Hou WC, Lin YH (1997) Dehydroascorbate reductase and
monodehydroascorbate reductase activities of trypsin
inhibitors, the major sweet potato (Ipomoea batatas [L.]
Lam) root storage protein. Plant Sci 128:151–158
Imlay JA, Linn S (1998) DNA damage and oxygen radical tox-
icity. Science 240: 1302–1309
Jahnke LS, White A (2003) Long-term hyposaline and hypers-
aline stresses produce distinct antioxidant responses in the
marine alga Dunaliella tertiolecta. J Plant Physiol 160:1193–
1202
Karpinski S, Reynolds H, Karpinska B, Wingsle G, Creissen G,
Mullineaux P (1999) Systemic signaling and acclimation in
response to excess excitation energy in Arabidopsis. Science
284:654–657
Mar Biol (2006) 150:1–15
Kato M, Shimizu S (1987) Chlorophyll metabolism in higher
plants. VII. Chlorophyll degradation in senescing tobacco
leaves: phenolic-dependent peroxidative degradation. Can J
Bot 65:729–735
Kim SY, Lim JH, Park MR, Kim YJ, Park TI, Se YW, Choi KG,
Yun SJ (2005) Enhanced antioxidant enzymes are associ-
ated with reduced hydrogen peroxide in barley roots under
saline stress. J Biochem Mol Biol 38:218–224
Kirst GO (1990) Salinity tolerance of eukaryotic marine algae.
Ann Rev Plant Physiol Plant Mol Biol 40:21–53
Lechno S, Zamski E, Tel-Or E (1997) Salt stress-induced re-
sponses in cucumber plants. J Plant Physiol 150:206–211
Lee TM (1998) Investigations of some intertidal green macro-
algae to hyposaline stress: Detrimental role of putrescine
under extreme hyposaline conditions. Plant Sci 138:1–8
Lee TM, Chen MH (1998) Hyposaline effect on polyamine
accumulation in Ulva fasciata (Ulvales, Chlorophyta). Bot
Bull Acad Sin 39:167–174
Lee TM, Liu CH (1999) Correlation of decreased calcium con-
tents with proline accumulation in the marine green mac-
roalga Ulva fasciata exposed to elevated NaCl contents in
seawater. J Exp Bot 50:1855–1862
Lee DH, Kim YS, Lee CB (2001) The inductive responses of the
antioxidant enzymes by salt stress in the rice (Oryza sativa
L.). J Plant Physiol 158:737–745
Levine A, Tenhaken R, Dixon R, Lamb C (1994) H2O2 from the
oxidative burst orchestrates the plant hypersensitive disease
resistance response. Cell 79:583–593
Liang YC (1999) Effects of silicon on enzyme activity and so-
dium, potassium and calcium concentration in barley under
salt stress. Plant Soil 209:217–224
Lobban CS, Harrison PJ (1997) Seaweed ecology and physiol-
ogy. Cambridge University Press, New York
McKersie BD, Leshem Y (1994) Stress and stress coping in
cultivated plants. Kluwer Academic Publishers, New York
Mittler R (2002) Oxidative stress, antioxidatants and stress tol-
erance. Trend Plant Sci 7:405–410
Morita S, Kaminaka H, Masumura T, Tanaka K (1999) Induction
of rice cytosolic ascorbate peroxidase mRNA by oxidative
stress; the involvement of hydrogen peroxide in oxidative
stress signalling. Plant Cell Physiol 40:417–422
Munné-Bosch S, Alegre L (2002) The function of tocopherols
and tocotrienols in plants. Crit Rev Plant Sci 21:31–57
Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by
ascorbate-specific peroxidase in spinach chloroplast. Plant
Cell Physiol 22:867–880
Neil S, Desikan R, Clarke A, Hurst RD, Hancock JT (2002)
Hydrogen peroxide and nitric oxide as signaling molecules
in plants. J Exp Bot 53:1237–1247
15
Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping
active oxygen under control. Ann Rev Plant Physiol Plant
Mol Biol 49:249–279
Noctor G, Gomez L, Vanacker H, Foyer CH (2002) Interactions
between biosynthesis, compartmentation and transport in
the control of glutathione homeostasis and signalling. J Exp
Bot 53:1283–1304
Okuda T, Matsuda Y, Yamanaka A, Sagisaka S (1991) Abrupt
increase in the level of hydrogen peroxide in leaves of
winter wheat is caused by cold treatment. Plant Physiol
97:1265–1267
Parida AK, Das AB (2005) Salt tolerance and salinity effects on
plants: a review. Ecotoxicol Environ saf 60:324–349
Provasoli L (1968) Media and prospects for the cultivation of
marine algae. In: Watanabe A, Hattori A (eds) Cultures and
collections of algae. In: Proceeding of the U.S., Japan
Conference, Hakone, Japanese Society of Plant Physiology
1968, pp 63–75
Rios-Gonzalez K, Erdei L, Lips SH (2002) The activity of anti-
oxidant enzymes in maize and sunflower seedlings as af-
fected by salinity and different nitrogen sources. Plant Sci
162:923–930
RodriguezRosales MP, Kereb L, Bueno P, Donaire JP (1999)
Changes induced by NaCl in lipid content and composition,
lipoxygenase, plasma membrane H+ ATPase and antioxi-
dant enzyme activities of tomato (Lycopersicon esculantum,
Mill) calli. Plant Sci 143:143–150
Sagisaka S (1976) The occurrence of peroxide in a perennial
plant, Populas gelrica. Plant Physiol 94:308–309
Schaedle M, Bassham JA (1977) Chloroplast glutathione
reductase. Plant Physiol 59:1011–1012
Singha S, Choudhuri MA (1990) Effect of salinity (NaCl) stress
on H2O2 metabolism in Vigna and Oryza seedlings. Bio-
chem Physiol Pflanzen 186:69–74
Smirnoff N (1996) The function and metabolism of ascorbic acid
in plants. Ann Bot 78:661–669
Smirnoff N, Wheeler GL (2000) Ascorbic acid in plants: bio-
synthesis and function. Crit Rev Plant Sci 19:267–290
Wang LJ, Liu YJ, Ma K, Wang JZ, Liu XN (1998) Effect of NaCl
treatment on free radical metabolism of fig (Ficus cartica L.)
calli. Adv Horti 2:235–241
Willekens H, Chamnogopol S, Davey M, Schraudner M,
Langebartels C (1997) Catalase is a sink for H2O2 and is
indispensable for stress defense in C3 plants. EMBO J
16:4806–4816
Wise RR, Naylor AW (1987) Chilling-enhanced photooxidation:
evidence for the role singlet oxygen and endogenous an-
tioxidants. Plant Physiol 83:278–282
123