Botanical Studies (2009) 50: 25-34.
*
Corresponding author: E-mail: tmlee@mail.nsysu.edu.tw.
INTRODUCTION
Heavy metals constitute an environmental pollutant
with toxicity to biota. Since the late 19th century, heavy
metals have accumulated in the environment as a result of
mining and industrial activities. Because cadmium (Cd)¡X
largely used for plastics manufacturing, Ni-Cd batteries,
and the electroplating of steel¡Xis persistent and bio-
accumulated through the food chain, Cd contamination
and toxicity have become of particular concern in recent
years. The effects of Cd toxicity on plants are well studied
(Sanitadi Toppi and Gabbrielli, 1999). Cd was found to
produce oxidative damage to lipids and nucleic acids
(Sandalio et al., 2001; Romero-Puertas et al., 2002, 2003;
Lee and Shin, 2003; Watanabe et al., 2003). The d
amage
caused by reactive oxygen species (ROS) is known as
oxidative stress.
In response, plants have developed
defense systems via non-enzymatic and enzymatic
scavenging of cellular ROS to cope with oxidative stress
(Noctor and Foyer, 1998; Asada, 1999; Okamoto et al.,
2001a, b; Pinto et al., 2003). The water-soluble ascorbate
(AsA) and glutathione (GSH) and the water-insoluble
£\-tocopherol and carotenoids are the non-enzymatic agents
that scavenge ROS (Noctor and Foyer, 1998; Smirnoff
and Wheeler, 2000; Munne-Bosch and Alegre, 2002).
To scavenge ROS enzymatically, O
2
¡E-
, which can first be
converted to H
2
O
2
by the action of superoxide dismutase
(SOD; EC 1.15.1.1), and then ascorbate peroxidase
Effects of cadmium on the regulation of antioxidant
enzyme activity, gene expression, and antioxidant
defenses in the marine macroalga Ulva fasciata
Tzure-Meng WU
1,3
, Yi-Ting HSU
1,2,3
, and Tse-Min LEE
1,2,3,
*
1
Institute of Marine Biology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
2
The Kuroshio Research Group of the Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung
80424, Taiwan
3
The Center of Biotechnology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
(Received September 10, 2007; Accepted June 19, 2008)
ABSTRACT.
This study examined the antioxidative responses of the marine macroalga Ulva fasciata Delile
to cadmium (Cd) stress. Exposure to 0, 5, 10, 20 and 50 £gM CdCl
2
for 4 days did not affect growth, 2,3,5-tri
phenyltetrazolium chloride reduction ability, H
2
O
2
production, or lipid peroxidation. The Cd contents in thalli
increased linearly as CdCl
2
concentrations increased from 0-20 £gM CdCl
2
and declined slightly at 50 £gM
CdCl
2
. This means that long-term exposure to Cd did not produce oxidative damage to the macroalga although
Cd accumulated. Ascorbate (AsA) and dehydroascorbate (DHA) concentrations increased as Cd concentrations
increased while AsA/DHA ratios increased with a peak at 10 £gM. Glutathione (GSH) and oxidized GSH
concentrations and GSH/oxidized GSH ratios decreased as Cd concentrations increased. Cd did not affect Mn
superoxide dismutase (MnSOD; EC 1.15.1.1) activities or transcripts. Cd at 50 £gM increased FeSOD activities
and UfFesod1 (a gene of FeSOD isoform) transcripts but did not affect UfFesod2 transcripts. Among isoforms
of the SOD gene, only UfFesod1 was responsible for the increase of SOD activity by Cd. The activities of
ascorbate peroxidase (APX; EC 1.11.1.11) and catalase (CAT; EC 1.11.1.6) increased as Cd concentrations
increased, but their transcripts were not affected by Cd, suggesting that the induction of APX and CAT
activities by Cd was not under transcriptional control. Glutathione reductase (GR; EC 1.6.4.2) activities and
transcripts increased as Cd concentrations increased. The present results indicate that the increase in the AsA
pool, the consumption of GSH, and the induction in the activities of FeSOD, APX, GR and CAT are used by
U. fasciata to prevent the occurrence of oxidative damage under Cd stress. The increases in the activities of
FeSOD and GR by Cd can be attributed to enhanced gene expression.
Keywords: Antioxidant enzyme; Antioxidant; Cd; Gene expression; Ulva.
Abbreviations: APX, ascorbate peroxidase; AsA, ascorbate; CAT, catalase; GR, glutathione reductase; GSH,
glutathione; ROS, reactive oxygen species; SOD, superoxide dismutase.
BIOChEmISTRy
pg_0002
26
Botanical Studies, Vol. 50, 2009
(APX; EC 1.11. 1.11) and glutathione reductase (GR; EC
1.6.4.2) in the ascorbate-glutathione cycle are responsible
for the removal of H
2
O
2
(Asaka, 1999). APX utilizes
AsA to reduce H
2
O
2
, and AsA is in turn oxidized. Then,
the oxidized AsA is regenerated back to AsA via GSH
oxidation, and GSSG as the product of GSH oxidation
is reduced to GSH by GR via the utilization of reducing
equivalents from NAD(P)H. H
2
O
2
can be degraded to H
2
O
by catalase (CAT; EC 1.11.1.6) (Willenkens et al., 1997)
or peroxidase (POX; EC 1.11.1.7) (Asaka and Takahashi,
1987).
The antioxidative responses to Cd stress are different
between algal species. Cd increased H
2
O
2
concentrations,
lipid peroxidation, and the activities of APX, POX and
CAT in the marine microalga Nannochloropsis oculata,
but decreased the activities of SOD and GR (Lee and
Shin, 2003). In the marine red macroalga Gracilaria
tenuistipitata, Cd increased CAT activity but did not affect
SOD or APX activities (Collen et al., 2003). In the marine
dinoflagellate Gonyaulax polyedra, acute exposure to Cd
generated oxidative stress in chloroplasts while under
chronic exposure, the antioxidant system was able to
provide protection (Okamoto et al., 2001a, b).
Few studies on the regulation of genes and antioxidant
enzymes in macroalgae exposed to heavy metals are
available. Ulva fasciata Delile is a marine chlorophyte
abundant in the intertidal regions of Taiwan, which are
often subjected to heavy metal pollution from river runoff
and sewage outlets. This study was planned to determine
whether oxidative stress occurs in U. fasciata in response
to Cd and to examine how the antioxidant defense system
is regulated under Cd stress. After 4 days of exposure
to 0, 5, 10, 20, or 50 £gM CdCl
2
, the growth rate and
2,3,5-triphenyltetrazolium chloride (TTC) reduction
ability were determined, and the concentrations of H
2
O
2
and the contents of thiobarbituric acid reacting substance
(TBARS) as indicators of oxidative stress were examined.
The concentrations of ascorbate and glutathione and the
activities of SOD, APX, GR, and CAT were determined.
In the attempts to investigate whether Cd can induce the
gene expression of antioxidant enzymes, the genes of
SOD, APX, GR, and CAT were first cloned, and transcript
abundance in response to Cd stress was examined.
mATERIALS AND mEThODS
Algal culture and heavy metal treatment
Ulva fasciata Delile (15-25 cm high) were collected
from Hsitzu Bay, Kaohsiung, Taiwan. Following
collection, whole algae were extensively washed with
natural seawater to remove attached sands, and rhizoidal
portions were removed to avoid microbial contamination
in the following culture. Thalli were pre-incubated at 25¢XC
for 14 days in the 35. artificial seawater (ASW; 403.5
mM NaCl, 10 mM KCl, 10 mM CaCl
2
, 30 mM MgSO
4
and 10 mM Tris-HCl, pH 8.0) containing N (NH
4
+
and
NO
3
-
)- and P (PO
4
3-
)-free Provasoli nutrient solutions
(Provasoli, 1968). NaHCO
3
, NaNO
3
and Na
2
HPO
4
were
then added in the final concentrations of 3 mM, 400 £gM
and 20 £gM, respectively. The photoperiod was 12 h : 12
h, and the photon irradiance (400-700 nm) was 100-150
£gmol photon.m
-2
.s
-1
in the absence of algae, achieved by
cool-fluorescent lamps (FL40D, China Electric Apparatus
Ltd., Taiwan).
After 14 days of pre-incubation, thalli of 1 g wet weight
(w. wt.) were cultured in a 500-ml beaker containing 300
ml of 35. ASW. Carbon and N and P nutrients were
provided by adding NaHCO
3
, NaNO
3
, and Na
2
HPO
4
in
the final concentrations of 3 mM, 400 £gM and 20 £gM,
respectively, and other nutrient elements were provided by
adding N- (NH
4
+
and NO
3
-
) and P- (PO
4
3-
) free Provasoli
nutrient solution (Provasoli, 1968) in ASW. CdCl
2
was
added in ASW to the final concentrations of 0, 5, 10, 20
and 50 £gM. The incubation temperature was 25¢XC, and
the photoperiod was 12 h. The photosynthetically active
radiation (400-700 nm) was 150 £gmol photon.m
-2
.s
-1
in the
absence of algae. ASW was changed everyday, and after
4 days thalli were sampled for wet weight determination.
Milli-Q water was used for the preparation of chemicals
and ASW.
Thallus segments were divided into two parts: the first
part was immediately used for TTC reduction ability assay,
and the second part was fixed in liquid nitrogen and stored
in -70¢XC for analyses of lipid peroxidation, antioxidant
contents, enzyme activity, and RNA extraction.
Determination of cadmium contents
The measurement of Cd contents was modified from
the method of Fuhrer (1982). The organic compounds of
thallus segments were obtained by incubating 0.03 g d. wt.
thalli at 550¢XC for 24 h. Then, ashes were digested with
200 £gl of 65% HNO
3
and 200 £gl of 35% H
2
O
2
at 72¢XC for
at least 10 h. After nitric acid digestion of dry ashes, the
crucibles were washed with 4 ml of Milli-Q water. The
solutions were used for determination of Cd contents by
the Z-2000 Series atomic absorption spectrophotometer
(Hitachi, Tokyo, Japan) at 228.8 nm. Cd concentrations
were estimated from the standard curve of 0 - 1.5 £gg
l
-1
CdCl
2
. Cd contents (£gg g
-1
d. wt.) were calculated as
follows: A
228.8
(£gg l
-1
) ¡Ñ 4 (ml) ¡Ò 0.03 (g d. wt.).
Determination of daily specific growth rate
The initial wet weight (w. wt.) was determined as WW
0
.
After growing for 4 days, the thalli in the beaker were
weighed as a value of WW
4
. The daily specific growth rate
expressed as a percentage wet weight increase per day was
calculated using the equation %¡Pd
-1
= 100 ¡Ñ (WW
4
-WW
0
)/
WW
0
)/4. To determine the percentage dry weight increase
per day, thallus dry weight (d. wt.) was estimated from
lyophilized samples (DW
4
). The initial dry weights (DW
0
)
were estimated from the wet weight/dry weight ratio of
initial thalli. The daily specific growth rate expressed as a
percentage of dry weight increase per day was calculated
using the equation %¡Pd
-1
= 100 ¡Ñ (DW
4
-DW
0
)/DW
0
)/4.
pg_0003
WU et al. ¡X Cd and antioxidant defense in
Ulva
27
Determination of TTC reduction ability
In the attempt to determine the cellular activity, thallus
segments of approximately 0.05 g w. wt. were freshly
sampled and incubated at 25¢XC 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
triple washing with 10 ml of 35. ASW, intracellular
insoluble formanzan was extracted with 5 ml of 95%
ethanol at 80¢XC for 20 min. Ethanol extract was collected,
and the thallus segments were extracted again with 5 ml
of 95% ethanol. Ethanol extracts were combined. After
making up to 10 ml with 95% ethanol, its absorbance
was determined at 530 nm. The A
530
values of Cd-treated
thallus segments were calculated as a percentage of the
Cd-free control.
Determination of TBARS contents and h
2
O
2
concentrations
Thallus segments of approximately 0.1 g w. wt. were
ground to powder in liquid nitrogen before 1 ml of 5%
(w/v) trichloroacetic acid (TCA) was added. The mixture
was centrifuged at 12,000 g for 10 min at 4¢XC, and the
supernatant was collected for the determination of lipid
peroxidation and H
2
O
2
concentrations. The extent of
lipid peroxidation was estimated from thiobarbituric
acid reacting substance (TBARS) contents as determined
according to Health and Packer (1968). TBARS contents
were calculated based on A
532
-A
600
with the extinction
coefficient of 155 mM
-1
¡Pcm
-1
.
Thallus H
2
O
2
contents were determined based on the
decomposition of H
2
O
2
by peroxidase as described by
Okuda et al. (1991). KOH (4 M) of 11.5 £gl was added
to 0.2 ml supernatant to adjust the pH to 7.5, and the
mixture was centrifuged at 12,000 g for 1 min under
4¢XC. The supernatant was applied to a 1-ml column of
Amberlite IRA-410, and residual H
2
O
2
was washed out
by 0.8 ml of distilled water. The contents of H
2
O
2
in
the eluate were determined within 10 min post column
elution. For the determination of H
2
O
2
in the eluate, 0.4
ml of 12.5 mM 3-dimethylaminobenzoic acid (DMAB),
0.4 ml of 10 mM 3-methyl-2-benzothiazoline hydrazone
(MBTH), and finally 0.02 ml of 0.25 unit ml
-1
horseradish
peroxidase (Sigma, MO, USA) were added for the
detection of absorbance at 590 nm for 3 min. A series of
H
2
O
2
concentrations was prepared for the determination
of the H
2
O
2
standard curve as in the above method. The
concentrations of thallus H
2
O
2
were estimated from the
H
2
O
2
standard curve.
Determination of ascorbate and glutathione
concentrations
Thallus segments of approximately 0.25 g w. wt. were
ground in liquid nitrogen, and then 2.5 ml of 5% (w/v)
trichloroacetic acid (TCA) was added. After centrifugation
at 12,000 g for 10 min at 4¢XC, the supernatant was
collected for determination of ascorbate and glutathione
concentrations.
The measurement of total AsA (AsA + DHA) and AsA
concentrations was modified from the method of Hodges
et al. (1996). Total AsA concentrations were determined
in a 1-ml mixture containing 200 £gl TCA extract, 50 mM
potassium phosphate buffer (pH 7.4), 3 mM EDTA, and 1
mM dithiothreitol (DTT). After incubating the mixture at
25¢XC for 10 min, 100 £gl of N-ethylmaleimide, 400 £gl of
0.61 M TCA, 400 £gl of 0.8 M orthophosphoric acid, and
400 £gl of £\, £\¡¦-bipyridyl were added. Finally, 200 £gl of
FeCl
3
was added, and the mixture was incubated at a 40¢XC
water bath for 1 h, and the absorbance was detected at 525
nm. To determine AsA concentrations, the chemicals and
procedure were the same as above, except that DTT and
N-ethylmaleimide were replaced by distilled water. Total
AsA and AsA concentrations were estimated from the
standard curve of 0-40 nmole L-AsA. DHA concentrations
were calculated by the subtraction of AsA from total AsA.
Total GSH concentrations were determined by the
absorbance at 412 nm according to the method of Griffiths
(1980). K
2
CO
3
(1.25 M) of 38.7 £gl was added to 0.3-ml
TCA extract to adjust the pH to 7.0, and the mixture
was centrifuged at 12,000 g for 1 min under 4¢XC, and
the supernatant was collected. For the determination
of total GSH, 0.1 ml of supernatant was added to the
reaction mixture (0.5 ml of 200 mM sodium phosphate
buffer [pH 7.5], 0.1 ml of 50 mM Na
2
EDTA, 0.1 ml of
2 mM £]-NADPH, 0.1 ml of 6 mM dithionitrobenzoic
acid [DTNB] in 0.2 M sodium phosphate buffer (pH
7.5), and 0.1 ml of 0.5 unit ml
-1
glutathione reductase
[Sigma, MO, USA]), and then the reaction was measured
at 412 nm for 3 min under 30¢XC. After the removal of
reduced GSH by adding 2 £gl of 1 M 2-vinylpyridine in
0.1 ml of supernatant and incubation at 25¢XC for 1 h,
the oxidized GSH concentrations were determined as
described above. A standard curve was prepared based
on 0-20 nmole oxidized GSH (Sigma, MO, USA). The
GSH concentrations were calculated by the subtraction
of oxidized GSH concentrations from total GSH
concentrations.
Determination of enzyme activity
The enzyme extraction was modified according
our previous study (Shiu and Lee, 2005). For APX,
lyophilized thalli of 0.0125 g d. wt. were homogenized
in liquid nitrogen, and 0.5 ml of extraction buffer (0.1
M sodium phosphate buffer [pH 6.8] containing 1% [pH
6.8] PVPP, 1 mM L-AsA, and 0.25% [v/v] Triton X-100)
was added. After centrifugation at 12,000 g for 10 min
at 4¢XC, the supernatant was used for APX assay. For GR,
lyophilized thalli of 0.0125 g d. wt. were homogenized
in liquid nitrogen, and 0.5 ml of extraction buffer (0.1 M
sodium phosphate buffer [pH 6.8] containing 1% [w/v]
PVPP, 1 mM Na
2
EDTA, and 1 mM PMSF) was added.
After centrifugation at 12,000 g for 10 min at 4¢XC, the
supernatant was used for GR assay. For SOD, lyophilized
thalli of 0.0125 g d. wt. were homogenized in liquid
pg_0004
28
Botanical Studies, Vol. 50, 2009
nitrogen and 0.5 ml of extraction buffer (0.1 M sodium
phosphate buffer [pH 6.8) containing 80 £gM AsA and 1
mM PMSF) was added. After centrifugation at 12,000
g for 10 min at 4¢XC, the supernatant was used for SOD
activity assay. For CAT, lyophilized thalli of 0.0055 g d.
wt. were homogenized in liquid nitrogen, and 0.5 ml of
extraction buffer (0.1 M sodium phosphate buffer [pH
6.8] containing 5% [w/v] PVPP, 1 mM Na
2
EDTA, 10 mM
DTT and 1 mM PMSF) was added. After centrifugation
at 12,000 g for 10 min under 4¢XC, the supernatant was
subjected to 30% ammonia sulfate precipitation of protein
and standing for 30 min for full precipitation. The mixture
was centrifuged at 10,000 g for 10 min at 4¢XC, and the
pellet was then dissolved in 150 £gl of extraction buffer,
and this was then used for the CAT assay. The soluble
protein contents were determined by the coomassie blue
dye binding method (Bradford, 1976) with bovine serum
albumin as a standard curve. As in our our preliminary
experiments, enzyme activity was determined within 1 h
after extraction to avoid activity loss.
The SOD activity was determined by the inhibition of
photochemical inhibition of nitro blue tetrazolium (NBT)
according to a method of Giannopolitis and Ries (1977).
Total SOD activity was determined in the reaction mixture
that consisted of enzyme extract, 50 mM Na-phosphate
buffer (pH 7.8), 13 mM L-methionine, 0.1 mM Na
2
EDTA,
63 £gM NBT, and 1.5 £gM riboflavin. The SOD isoforms
were identified by adding 3 mM KCN to inhibit CuZnSOD
activity (that is, the MnSOD and FeSOD that could be
detected), and 3 mM KCN and 5 mM H
2
O
2
to inhibit
CuZnSOD and FeSOD activities (that is, the MnSOD that
could be detected). CuZnSOD activity was subtracted
from total SOD activity with the activity of MnSOD and
FeSOD. FeSOD activity was subtracted from the activity
of both MnSOD and FeSOD with MnSOD activity. The
APX activity was determined at A
290
for DHA according
to the extinction coefficient of 2.8 mM
-1
cm
-1
(Nakano
and Asada, 1981). The GR activity was monitored by A
340
for £]-NADPH oxidization as GSSG reduction according
to a method of Schaedle and Bassham (1977). The CAT
activity was measured at A
420
for H
2
O
2
decomposition rate
using the extinction coefficient of 40 mM
-1
cm
-1
according
to a method of Kato and Shimizu (1987). In this study, one
unit (U) of enzyme activity is defined as 1 £gmol¡Pmin
-1
for
APX, GR, and CAT while one unit of SOD is defined as a
50% inhibition of activity of the control (without extract
added).
Cloning of mnSOD, FeSOD, APX, GR, and CAT
genes
The libraries of suppressive subtractive hybridization
(SSH) and rapid amplification of cDNA ends (RACE)
were created for cloning the genes of antioxidant enzymes
from Cu-treated thalli. Total ribonucleic acid (RNA) of
thalli treated with or without extra 50 £gM CuSO
4
(3, 6 and
9 h) was extracted using TRIZOL Reagent (Invitrogen Life
Technologies, CA, USA) according to the manufacturer¡¦s
instructions. Poly(A
+
) mRNA was isolated from total
RNA using Dynabeads
R
mRNA Purification Kit (Dynal
Biotech ASA, Oslo, Norway) following the manufacturer¡¦
s instructions. The RNA purity was determined at 260
and 280 nm, and its integrity was checked on 1.2% (w/v)
agarose/formaldehyde gel.
Because U. fasciata exhibited significant oxidative
stress upon exposure to Cu (see Figures 1 and 2 in the
Result section), the Cu
(50 £gM)-treated thalli were used
to clone genes related to an antioxidant defense system.
A cDNA library was created by SSH (Diatchenko et al.,
1999) of the RNAs of Cu
treatments between 0 and 50 £gM
using the PCR-Select. cDNA Subtraction Kit (Clontech,
CA, USA). The SSH library was done with 1 £gg poly(A
+
)
mRNA and the TA cloning technique using the pGEM
R
-T
Easy Vector System II (Promega, WI, USA) following
the manufacturer¡¦s instructions. For screening differential
expression gene fragments, probes were generated by
random priming using a DIG High Prime DNA Labeling
and Detection Starter Kit (Roche, Mannheim, Germany)
following the manufacturer¡¦s instructions. Sequencing
was done on 198 clones from forward subtractions and
57 clones from reverse subtractions. Homology searches
for sequences of selected clones were performed using a
Blastx search from the Basic Local Alignment Sequence
Tool (BLAST) (Altschul et al., 1997) at http://www.ncbi.
nlm.nih.gov/BLAST applying default parameters and
non-redundant databases. From the SSH cDNA library,
300 and 360 randomly selected expressed sequence tags
(ESTs) from forward and reverse clones, respectively,
were analyzed. Thirty-nine clones were obtained for up-
regulation by Cu
2+
and two clones for down-regulation
(Table 1 in supplement data). The results of a BLASTX
search performed on the obtained total set of ESTs showed
that one of the up-regulated DNA fragments could be
assigned to gene coding for a protein known to be APX
(SSH9). For cloning the full-length cDNA of genes from
RACE library, the SSH9 fragment was used to design the
reverse primer. FeSOD, MnSOD, CAT, and GR genes,
which were not obtained from SSH library, were cloned
directly from the RACE library using the degenerated
primers.
The SMART. RACE cDNA Amplification Kit
(Clontech, CA, USA) was used to generate the 5¡¦ and
3¡¦ fragments of cDNAs as templates for the cloning of
the full-length cDNAs of FeSOD, MnSOD, CAT, APX,
and GR genes, and the BD Advantage
TM
2 Kit (Clontech,
CA, USA) was used for all PCR procedures. For the
GR and CAT genes, we obtained the fragments by using
the following degenerate primers: GR-dF (5¡¦-GAA
TTCGGNTGYGTNCCNAARAAR-3¡¦), GR-dR (5¡¦-
AAGCTTCCWRYKGCDATYAR DAT-3¡¦), CAT-dF (5¡¦-
GAATTCGARMGNGTNGTNCAYGC-3¡¦), and CAT-dR
(5¡¦-AAG CTTGTRTARAAYTTNACNGC RAANCC-3¡¦).
Each reaction, which was performed in a total volume
of 25 £gl, contained 1¡Ñ BD advantage 2 mix (Clontech,
CA, USA), 0.6 £gM of each primer, and 5¡¦ fragments of
pg_0005
WU et al. ¡X Cd and antioxidant defense in
Ulva
29
cDNAs as templates. The following degenerated PCR
amplification program was used: 94¢XC for 5 min, 40
amplification cycles of 94¢XC for 30 s, then 53¢XC for 30
s, and 72¢XC for 30 s, followed by 72¢XC for 5 min. The
fragments were used to design primers (GR-R: 5¡¦-CGC
CGTCTGGGCTCGAGTATGTCATGT-3¡¦ and CAT-R:
5¡¦-TGACGGTAGA GAATCTGACAGCCACAGG-3¡¦)
for 5¡¦-RACE. The gene specific primer (APX-R: 5¡¦-TGA
ACGGGGTGCATGAATCCAATC-3¡¦) of the APX gene
was designed from an SSH fragment. According to these
three gene specific primers (GSPs), we made progress
toward amplifying the 5¡¦-RACE product of each gene. The
conditions for the 5¡¦-RACE of GR, CAT, and APX genes
were those specified in the manufacturer¡¦s instructions. For
the MnSOD and FeSOD genes, we used the degenerated
reverse primers (MnSOD-dR: 5¡¦-AAGCTTRTGYTCCCA
NACRTCDATNCC-3¡¦ and FeSOD-dR: 5¡¦-CTGRAARTC
MAGRTAGTARGCATGCTCCCA-3¡¦) proceeding to the
5¡¦-RACE. The amplification program was designed as
follows: MnSOD gene (94¢XC for 5 min, 5 cycles of 94¢XC
for 30 s, 70¢XC for 30 s, and 72¢XC for 3 min, 30 cycles of
94¢XC for 30 s, 65¢XC for 30 s, and 72¢XC for 3 min, and
then finally 72¢XC for 5 min) and FeSOD gene (94¢XC for 5
min, 5 cycles of 94¢XC for 30 s , 65¢XC for 30 s , and 72¢XC
for 3 min, 30 cycles of 94¢XC for 30 s , 60¢XC for 30 s , and
72¢XC for 3 min, and then 72¢XC for 5 min). Based on the
sequence of extreme 5¡¦-end of the 5¡¦-RACE product, one
specific primer was designed for direct amplification of
the full-length cDNAs in 3¡¦-RACE. All GSPs were chosen
using the PrimerSelect program of Lasergene (DNASTAR,
WI, USA). PCR products were analyzed on 2% (w/v)
agarose gels, and the bands were purified by Gel-M
R
Gel
Extraction System (Viogene, Taipei, Taiwan) and cloned
with the pGEM
R
-T Easy Vector System II (Promega,
WI, USA). After sequencing, the full-length cDNAs of
MnSOD (UfMnsod, GenBank no. EF437244), FeSOD1
(UfFesod1, GenBank no. EF437245), FeSOD2 (UfFesod2,
GenBank no. EF437246), APX (Ufapx, GenBank no.
ABB88581), GR (Ufgr, GenBank no. ABB88584),
and CAT (Ufcat, GenBank no. ABB88582) genes were
obtained.
Quantitative real-time PCR detection of gene
expression
Total RNA extracted using TRIZOL Reagent
(Invitrogen Life Technologies, CA, USA) according to the
manufacturer ¡¦s instructions was DNase I digested. Five
micrograms of each RNA sample was reverse transcribed
to complementary DNA with PowerScript Reverse
Transcriptase Kit (Clontech, CA, USA) using Oligo(dT)
18
according to the manufacturer¡¦s instructions. Real-time
PCR using SYBR Green I technology on ABI PRISM 7000
Sequence Detection System (Applied Biosystems, CA,
USA) was performed. A master mix for each PCR run was
prepared with Smart Quant Green Master Mix with dUTP
& ROX Kit (Protech, Taipei, Taiwan). Each reaction,
which was performed in a total volume of 25 £gl, contained
1X Smart Quant Probe Master Mix, 0.3 £gM of each primer,
and cDNA corresponding to 10 ng input RNA in the
reverse transcriptase reaction. The following amplification
program was used: 50¢XC for 2 min, 95¢XC for 10 min, 40
cycles at 95¢XC for 15 s followed by 60¢XC for 1 min. The
dissociation curves were performed after the PCR reaction,
and the product was analyzed by gel electrophoresis
to assess the presence of a unique final product. The
fluorescence was analyzed by ABI Prism 7000 SDS
Software using auto C
T
to determine threshold of each
gene, and the 2
-¡µ¡µCT
method was used to calculate the C
T
values. The PCR product is a DNA fragment with certain
length as predicted. After sequencing, the sequences of
PCR products were in agreement with predicted gene
fragment. The data were presented as the fold change in
mRNA abundance, normalized to an endogenous reference
gene (£\-tublin), relative to the RNA sample from U .
fasciata grown without extra CuSO
4
or CdCl
2
addition.
The results presented were the averages of biological
triplicates. The forward and reverse primers for real-time
PCR were designed 5¡¦-TGCACGCCGAAGGACATA-3¡¦
and 5¡¦-CCAAAGCCATTGTGAATCGAG-3¡¦ for
UfMnsod, 5¡¦-TGCACGCCGAAGGACATA-3¡¦
and 5¡¦-CCAAAGCCATTGTGAATCGAG-3¡¦ for
UfFesod1, 5¡¦-ATTGAGTCGGTGAG CCT-3¡¦ and
5¡¦-TGCACACAAGCGTTGTTAC-3¡¦ for UfFesod2,
5¡¦-GTTTCAGGCAGGCA GCA-3¡¦ and 5¡¦- ATTCG
CATTGTTCTGGGAATC-3¡¦ for Ufapx, 5¡¦-GATTTA
GGCCAGGCGGA-3¡¦ and 5¡¦-TCATTTCATCTGA
TCATATAACAGAACCC-3¡¦ for Ufgr, 5¡¦-GAATA
CCTTGACCAAAGTGGTT-3¡¦ and 5¡¦-GTAAGTGC
AGTCTACGTCG-3¡¦ for Ufcat, and 5¡¦-GTGGGCTA
TTAAATGGAGTATTGTT-3¡¦ and 5¡¦-ACAGATAGGGTA
TCAAAGCGAA-3¡¦ for the £\-tubulin gene
Chemicals and statistical analyses
Chemicals were purchased from Merck (Germany) or
Sigma (USA). Statistics were analyzed by SAS (SAS v
9.01, NC, USA). The present results were the mean of
three replicates with a beaker as a replicate. The effects of
Cd on daily specific growth rate, TTC reduction ability,
TBARS contents, H
2
O
2
contents, water-soluble antioxidant
contents and enzyme activities, and the relative transcript
abundance was analyzed by one-way analysis of variance
(ANOVA). The difference among means was analyzed by
Tukey¡¦s test following significant ANOVA at p < 0.05.
RESULTS
Growth rate, TTC reduction ability, h
2
O
2
concentrations, and TBARS contents
The tolerance of Ulva to Cd was evaluated by daily
specific growth rate and TTC reduction ability. Cd at
50 £gM decreased wet weight growth rate
(F = 8.33, p =
0.0032) (Figure 1A), but Cd ranging from 5-50 £gM did
not affect dry weight growth rate (F = 3.92, p = 0.0364)
(Figure 1B) or TTC reduction ability (F = 0.38, p = 0.8167)
pg_0006
30
Botanical Studies, Vol. 50, 2009
(Figure 1C). Neither H
2
O
2
concentrations (Figure 1D) nor
TBARS contents (Figure 1E) were affected by Cd (p >
0.05).
Cadmium contents
Cd contents increased significantly following CdCl
2
addition (F= 18.35, p= 0.0001) (Figure 2). They increased
linearly from 5 to 20 £gM Cd Cl
2
, but exhibited a decline
above 50 £gM.
Ascorbate and glutathione concentrations
The concentrations of total AsA (Figure 3A) and DHA
(Figure 3C) decreased by 5-10 £gM Cd and increased by
Cd . 20 £gM. Similarly, the concentrations of AsA first
decreased by 5 £gM Cd and then increased as Cd . 10 £gM,
reaching a maximum at 20 £gM Cd and a slight decline at
50 £gM Cd (Figure 3B). The AsA/DHA ratios increased
by 10 £gM Cd, followed by a linear decrease (Figure 3D).
The concentrations of total GSH (GSH + oxidized GSH)
were not affected by 5-20 £gM Cd, but decreased by 50
£gM Cd (Figure 3E). The concentrations of oxidized GSH
were not affected by 5 £gM Cd, but increased by 10 and
20 £gM Cd and decreased by 50 £gM Cd (Figure 3G).
The concentrations of GSH (Figure 3F) and the ratios of
GSH/oxidized GSH (Figure 3H) decreased linearly as Cd
concentrations increased; a drop to the bottom near zero
occurred as Cd . 20 £gM.
Activities of mnSOD, FeSOD, APX, GR and CAT
and Transcript of UfMnsod, UfFesod1, UfFesod2,
Ufapx, Ufgr and Ufcat
MnSOD activities were not affected by Cd (F = 1.66,
p = 0.2434) (Figure 4A) while FeSOD activities increased
by 50 (F = 25.10, p < 0.0001) (Figure 4B). APX activities
(F = 7.22, p = 0.0125) underwent a slight decline by 5-20
£gM Cd and increased by 50 £gM Cd (Figure 4C). GR
activities increased linearly as Cd concentrations increased
(F = 11.33, p = 0.0010) (Figure 4D). CAT activity also
increased linearly as Cd concentrations increased (F =
12.22, p = 0.0011) (Figure 4E).
The transcripts of UfMnsod (Figure 4F), UfFesod2
(Figure 4H), Ufapx (Figure 4I), Ufgr (Figure 4J), and Ufcat
(Figure 4K) were not altered by Cd while the transcript
o f UfFesod1 increased linearly as Cd concentrations
increased (Figure 4G).
DISCUSSION
The marine macroalga Ulva fasciata is tolerant to Cd
ranging from 5-50 £gM. Heavy metals are implicated in
oxidative injury involved in the formation of ROS and
their subsequent attack on proteins, lipids, and nucleic
acids, leading to loss of enzyme functions, altered
membrane fluidity, and genomic damage (Dietz et al.,
1999). Because H
2
O
2
production and TBARS contents in
U. fasciata were not increased by Cd, Cd ranging from
5-50 £gM did not produce oxidative damage to U. fasciata.
The present results showed that Cd uptake by U .
fasciata occurs in a concentration dependent manner.
Thallus Cd contents are proportional to the concentrations
of CdCl
2
added to the seawater in the range from 5-20
Figure 1. The s pe cific growth rat e (A: dry weight; B: wet
weight), TTC reduction ability (C), H
2
O
2
concentrations (D),
and TBARS contents (E) (means ¡Ó SD, n = 3) of Ulva fasciata
exposed to CdCl
2
for 4 days. Different letters indicate significant
difference at p < 0.05 by Tukey¡¦s test.
Figure 2. The contents of Cd (mea ns ¡Ó S D, n = 3) in Ulva
fasciata exposed to CdCl
2
(A, B, C, D) for 4 days. Different
letters indicate significant difference at p < 0.05 by Tukey¡¦s test.
pg_0007
WU et al. ¡X Cd and antioxidant defense in
Ulva
31
£gM. However, the Cd contents in thalli were not further
increased by 50 £gM CdCl
2
. This might be related to the
significant loss of water from thalli that occurs upon
exposure to 50 £gM CdCl
2
. The results from Figure 1A
and 1B indicated marked water loss after 4 days of such
exposure. We speculate that water was lost during the early
period after exposure to 50 £gM CdCl
2
and that the Cd
influx decreased. As a result, the contents of Cd contents
were less than in treatments with CdCl
2
concentrations .
20 £gM.
The homeostasis of an antioxidant is significantly
affected by Cd. However, glutathione and ascorbate
are differentially regulated. The ascorbate pool
was significantly enlarged by Cd. In contrast, the
concentrations of glutathione and its regeneration were
significantly decreased by Cd, reflecting the fact that
glutathione is consumed by U. fasciata to cope with Cd
stress. The decrease in glutathione concentrations has
been observed in the marine red macroalga Gracilaria
tenuistipitata (Collen et al., 2003) and also in the marine
green macroalga Enteromorpha linza (Malea et al., 2006).
It has been documented that glutathione functions as an
antioxidant, reacting non-enzymatically with O
2
¡E-
, H
2
O
2
,
and OH
¡E
to prevent macromolecule oxidation (Noctor
and Foyer, 1998; Noctor et al., 2002).Glutathione is
used for synthesis of phytochelatins, which perform the
intracellular sequestration of heavy metal ions in algae (De
Vos et al., 1992).
Present evidence shows that the activities and gene
expression of SOD isoforms are selectively regulated by
Cd. The NBT-dependent activity assay and the activity
staining on the native gel reveal that CuZnSOD does
not exist in U . fasciata. A previous study comparing
the CuZnSOD proteins of land plants and green algae
has documented that CuZnSOD proteins appear in both
charophycean algae and land plants, but other green algal
groups lack them (De Jesus et al., 1989). Our results agree
with their view. MnSOD and FeSOD were the main SOD
in this green macroalga. MnSOD, mainly occurring in
the mitochondria (Asada, 1999), were not affected by Cd,
but FeSOD, mainly occurring in the chloroplasts (Asada,
1999), were increased by Cd at higher concentrations (50
£gM), reflecting the fact that FeSOD is the metalloform
responsible for converting O
2
¡E-
to H
2
O
2
under Cd stress.
The Cd induction of SOD activity has also been observed
in the marine microalga Tetraselmis gracilis (Okamoto
et al., 1996). In the marine dinoflagellate Gonyaulax
polyedra, the activities of FeSOD and MnSOD, but
not CuZnSOD, were induced by exposure to acute Cd
(Okamoto and Colepicolo, 1998).
The coincidence between FeSOD activity and the
UfFesod1 transcript, rather than UfFesod2, indicates
that the induction of SOD activity by Cd is attributable
to enhanced expression of UfFesod1. Okamoto et al.
performed a study using the marine dinoflagellate
Lingulodinium polyedrum which showed that gene
expression of a chloroplastic FeSOD was up-regulated
by Cd, but its protein amounts were not increased under
Cd stress (Okamoto et al., 2001b). They suggested the
translation of dinoflagellate FeSOD was controlled by
other factors (Okamoto et al., 2001b). Because SOD
proteins were not determined in this study, we do not
know whether the translation of SOD in U. fasciata is up-
regulated by Cd.
Figure 3. The concentrations of total AsA
(A), AsA (B), DHA (C), As A/DHA ratio,
total GSH (E), GSH (F), oxidized GSH (G),
and GSH/oxidized GSH ratio (H) (means ¡Ó
SD, n = 3) in Ulva fasciata exposed to CdCl
2
(A, B, C, D) for 4 days. Different letters
indicate significant difference at p < 0.05 by
Tukey¡¦s test.
pg_0008
32
Botanical Studies, Vol. 50, 2009
The increases in the activities of both APX and CAT
are responsible for H
2
O
2
scavenging in U. fasciata i n
response to Cd stress. This explains why H
2
O
2
did not
accumulate in U. fasciata under Cd stress. However, APX
and CAT activities are transcriptionally up-regulated by
Cd. Possibly, the modulation of protein and/or substrate
binding and turnover kinetics instead of transcriptional
modulation is involved in the induction of APX and
CAT activity by Cd. The induction of CAT activity by
Cd has been observed in the red macroalga Gracilaria
tenuistipitata (Collen et al., 2003). In higher plants, Cd
induced oxidative stress in pea leaves, characterized by an
accumulation of lipid peroxides and oxidized proteins, and
a reduction of catalase and SOD activity (Sandalio et al.,
2001; Romero-Puertas et al., 2002, 2003). The activities
of antioxidant enzymes are up-regulated in response to
Cd in rice (Oryza sativa L.) seedlings of the Cd-sensitive
cultivar (cv. Taichung Native 1, TN1) but not in the Cd-
tolerant cultivar (cv. Tainung 67, TNG67) (Kuo and Kao,
2004). The mechanisms by which Cd induces antioxidant
responses and to what extent different plant species may
share a common defense mechanism are not yet fully
understood.
Because of the loss of water in the 50 mM CdCl
2
-
treated thalli, the involvement of Cd-induced water loss
in the up-regulation of antioxidant enzyme activity and/or
transcription cannot be ignored.
In conclusion, the antioxidant homeostasis in Ulva
fasciata is markedly altered by Cd in gaining Cd tolerance,
the enlargement of the AsA pool and the consumption of
GSH. FeSOD, APX, GR, and CAT activities were also
induced to prevent oxidative damage. Enhanced gene
expression contributed to the increases in the activities of
FeSOD and GR by Cd, but APX and CAT were not under
transcriptional regulation.
Acknowledgements. We thank Prof. Ching-Huei Kao
and Dr. Wen-Dar Huang in the Department of Agronomy,
National Taiwan University, Taipei, Taiwan, for assisting
us with Cd analysis using AA. Financial support
was provided by the National Science Council (NSC
95-2311-B-110-001), Executive Yuan, Taiwan.
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