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

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.

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

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

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)

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.

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.

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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