全 文 ：Received 27 Oct. 2003 Accepted 16 Mar. 2004
Supported by the National Natural Science Foundation of China (30270258) and Encouraging Foundation for Outstanding Youth Scientists
of Shandong Province（03BS120）
* Author for correspondence. Tel: +86 (0) 532 2032952; Fax: +86 (0) 532 2032276; E-mail: < tangxx@ouc.edu.cn>.
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (6): 682-690
Effects of CO2 Enrichment on Photosynthesis, Lipid Peroxidation and
Activities of Antioxidative Enzymes of Platymonas subcordiformis
Subjected to UV-B Radiation Stress
YU Juan1, TANG Xue-Xi1*, ZHANG Pei-Yu1, 2, TIAN Ji-Yuan1, 3, CAI Heng-Jiang1
(1. College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China;
2. College of Life Sciences, Qufu Normal University, Qufu 273165, China;
3. Qingdao Brewery No.2, Qingdao 266100, China)
Abstract: When the CO2 concentration was kept at 360 mL/L (ambient) and 5 000 mL/L (elevated), the
effects of UV-B stress on photosynthesis, lipid peroxidation and antioxidative enzymes of marine microalgae
Platymonas subcordiformis (Wille) Hazen were examined. The experiment indicated that: (1) UV-B alone
significantly decreased dry weight, photosynthetic rate, chlorophyll a (Chl a) and carotenoid (Car.) contents;
CO2 enrichment alone enhanced dry weight and photosynthetic rate, but Chl a content and Car. content
had no major difference compared with those of ambient UV-B and ambient CO2; the dry weight and
photosynthetic rate of P. subcordiformis grown under the combination of UV-B and CO2 had no major
difference compared with that under ambient UV-B and ambient CO2; while the Chl a content and Car.
content significantly decreased compared to those of P. subcordiformis grown under ambient UV-B and
ambient CO2. (2) Both UV-B alone and CO2 enrichment alone significantly decreased soluble protein
content, when UV-B and CO2 were in combination, the soluble protein content was higher than that of UV-
B alone. Changes in soluble protein content of algae grown in high CO2 could be largely due to a decline in
Rubisco protein. (3) UV-B alone significantly increased the rate of O2
-. production, H2O2 and malondialdehyde
(MDA) content, CO2 enrichment alone significantly decreased the rate of O2
-. production, H2O2 and MDA
contents, when UV-B and CO2 were in combination, the rate of O2
-. production, H2O2 and MDA contents
were significantly lower than those of UV-B alone. The results suggested that CO2 enrichment could
reduce oxidative stress of reactive oxygen species to P. subcordiformis, and reduce the lipid peroxidation
damage of UV-B to P. subcordiformis. And (4) UV-B alone significantly increased SOD, POD, CAT, GR and
GPX activities, CO2 enrichment alone significantly decreased the activities of SOD, POD and GR, while the
CAT and GPX activities were decreased a little but not changed significantly compared to ambient UV-B and
ambient CO2. The SOD, POD, CAT, GR and GPX activities of P. subcordiformis grown under the combination
of UV-B and CO2 were much lower than those of P. subcordiformis grown under UV-B alone. The results
indicated that CO2 enrichment showed a protective effect against the oxidative damage of UV-B-induced
stress. Therefore, elevated CO2 can be favor of enhancing the capacity of stress resistance.
Key words: CO2 enrichment; UV-B radiation; Platymonas subcordiformis ; photosynthesis; lipid
peroxidation; antioxidative enzymes
Atmospheric levels of CO2 are expected to double dur-
ing the 21st century (Long et al., 1993). The possible effect
and its mechanism by elevated CO2 on global climate, eco-
logical environment, biological diversity and agriculture pro-
duction have become the hot point in the concerned
research. Under the condition of high concentration of CO2,
the photosynthetic and physiological characteristics had
significant changes, for example, the affinity for CO2 in pho-
tosynthesis and the CO2 compensation points were much
lower, compared with bubbled by air of low CO2 (Raven,
1991). Hanagata et al. (1992) reported that growth rate of
Chlorella was increased with increasing gas flow when
culture medium was bubbled with air of high CO2.
In addition to CO2, chlorofluorocarbons (CFCs), CH4
and N2O are also increasing with industrialization. The in-
crease of these trace gases is expected to deplete the strato-
spheric ozone column with a subsequent increase in the
amount of solar UV-B radiation reaching the earth
(Teramura, 1983a). In contrast to CO2, enhanced UV-B ra-
diation has been shown to reduce microalgae growth rate
(Davidson et al., 1994), to damage the photosynthesis ap-
paratus (Cullen and Neale, 1994), or to change nutrient
YU Juan et al.: Effects of CO2 Enrichment on Photosynthesis, Lipid Peroxidation and Activities of ntioxidative Enzymes of
Platymonas subcordiformis Subjected to UV-B Radiation Stress 683
metabolism (Garde and Gustavston, 1999).
Both CO2 and UV-B radiation are expected to increase
simultaneously with future changes in global climate. To
date, many studies have been done on the effects of
elevated UV-B radiation and CO2 on terrestrial plants
(Teramura et al., 1990; Hao et al., 2000). Hao et al. (2000)
found that CO2 enrichment increased the growth, total
weight and plant height of tomato. The results were similar
to the results of Teramura et al. (1990) and Staaij et al.
(1993), who found that UV-B radiation reduced elevated
CO2-induced effect. Rao et al. (1995) found that elevated
CO2 can ameliorate the oxidative damage of O3 induced to
wheat. Therefore, elevated CO2 can enhance the capacity
of plants to resist stress-induced oxidative damage (Ren et
al., 2001).
About half of the primary biomass production on our
planet is based on aquatic ecosystems, equaling the com-
bined productivity of all terrestrial ecosystems (Siegenthaler
and Sarmiento, 1993). Most of the aquatic productivity is
performed by phytoplankton (Demeter et al., 1995). The
marine phytoplankton represents the single most impor-
tant ecosystem on our planet and produces about the same
biomass as all terrestrial ecosystems being taken together.
Another important role of marine phytoplankton is to serve
as a sink for atmospheric carbon dioxide. To date, however,
each factor has been determined separately with microalgae,
but no study has been done on the effects of combination
of UV-B radiation and CO2 enrichment to marine microalgae.
Therefore, it is unclear whether elevated atmospheric CO2
will result in increases in photosynthesis and growth if
solar UV-B radiation increases concurrently.
Oxidative stress is potentially experienced by all aero-
bic life when exposed to UV-B radiation. Toxic oxygen spe-
cies such as superoxide anion (O2
-. ), hydroxyl radical (OH.),
hydrogen peroxide (H2O2) and singlet oxygen (1O2) which
have been hypothesized as the agents of photodamage
and photobleaching can be formed (Halliwell and
Gutteridge, 1989). In order to balance and control the risk of
oxygen toxicity during photosynthetic process, the plant
cell has developed antioxidative defense system. The
antioxidative defense system consists of low molecular
weight antioxidants such as ascorbate, glutathione, a-
tocopherol, and carotenoids, as well as several enzymes
such as superoxide dismutase (SOD), catalase (CAT), per-
oxidase (POD), glutathione reductase (GR) and glutathione
peroxidase (GPX) (Bowler et al., 1994). SOD converts O2
-.
radicals into H2O2 and O2. The antioxidants like ascorbate
and glutathione participate in both enzymic and non-enzy-
mic H2O2 degradation (Foyer et al., 1994). POD and CAT
are the major systems for the enzymic removal of H2O2 in
plants. CAT dismutates H2O2 into water and O2, whereas
POD decomposes H2O2 by oxidation of co-substrates such
as phenolix compounds and/or antioxidants (Campa, 1991).
GR is capable of metabolizing H2O2 by utilizing ascorbate
and glutathinoe (Foyer et al., 1994). GPX can scavenge H2O2
by oxidizing reduced glutaghione (GSH) to oxidized
glutaghione (GSSG) (Sheng, 2003).
In the current study, P. subcordiformis was subjected
to elevated CO2, elevated UV-B and CO2 /UV-B radiation in
combination. The objectives of the experiments described
here were (a) to further examine the effects of CO2 enrich-
ment on photosynthetic apparatus, (b) to examine the
changes of reactive oxygen species (ROS) and (c) to ana-
lyze the changes of CO2 enrichment on activities of
antioxidative enzymes of P. subcordiformis subjected to
UV-B radiation.
1 Materials and Methods
1.1 Algal species and culture conditions
The marine microalgae Platymonas subcordiformis
(Wille) Hazen was offered by Marine Microalgae Research
Center, Ocean University of China and cultured in Erlenm-
eyer flasks with f/2 medium (Guillard and Ryther, 1962). Sa-
linity of the seawater was (30.0±1.0)‰ and the initial pH
of the culture was 8.0±0.1. Cultures were grown at (20±1)
℃ under a 14/10 dark/light cycle of 50 mmol photon.m-2.s-1
illumination.
1.2 UV-B radiation treatment and CO2 concentrations
UV-B radiation was provided by two UV-B tubes (Philips
TL 40 W/12 UV) covered by a film of cellulose acetate (0.12
mm) to remove all radiation below 280 nm. In order to mini-
mize the change of the filter properties of the film, the cellu-
lose acetate was preburnt for 48 h at a distance of 1 m from
two UV-B lamps. The spectral irradiance was measured with
UV spectroradiometer (Beijing Normal University), only a
thin layer of the algal suspension was used to ensure ad-
equate UV-B penetration. UV-B lamp provided a UV-B
fluence that approximated the UV-B radiation received un-
der present stratospheric ozone conditions (control) and
that anticipated at Qingdao with 25% stratospheric ozone
depletion under clear sky conditions during the summer
day. In this experiment, the doses of UV-B radiation were 8.8
kJ.m-2.d-1 UV-BBE (ambient UV-B) and 13.8 kJ.m-2.d-1
UV-BBE (enhanced UV-B), respectively. The different irra-
diances were obtained by adjusting the heights of the lamps
above the tops of the microalgae to maintain a fixed dis-
tance of 0.50 and 0.35 m for the ambient and elevated UV-B
treatments, respectively. Daily UV-B radiation was supplied
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004684
throughout an 8 h period (09:00-17:00) during the
experiment, and cellulose acetate filters were changed
weekly to avoid aging effects on the UV-B spectral trans-
mission through the filters. All experiments were carried
out on triplicate.
CO2 concentrations of 360 mL/L (ambient) and 5 000
mL/L (elevated) were maintained throughout the experiment
by using a CO2 injection system (provided by Qingdao
Heli Industrial Gas Center) and the CO2 concentrations were
continuously monitored by CO2 infrared gas analyzer (CID,
USA). The gas flow rate was controlled at 300 mL/min by
using air flowmeter (LZB-3, Qingdao Huayi Meter Factory).
The following measurements were carried out on the
seventh day of the treatment of UV-B and CO2.
1.3 Measurement of dry weight
Dry weight was determined after drying algal pellet for
72 h at 80 ℃.
1.4 Chlorophylla (Chl a) content and carotenoids (Car.)
content assay
Referring to the method of Jensen (1978), samples were
collected on glass filters (Whatman GF/F). Then the filter
was transferred to a 10-mL centrifuge tube with methanol
and kept overnight at 4 ℃. The pellets were extracted again
until colorless. Measurements were performed with a
spectrophotometer, and the equations given by Jensen
(1978) were used to calculate the Chl a and Car. contents.
1.5 Measurement of photosynthetic rate
Photosynthetic rate was conducted according to Gao et
al. (1994).
1.6 The rate of O2
-. production
The rate of O2
-. production was carried out according to
the method of Wang (1990).
1.7 Determination of H2O2 content
Hydrogen peroxide levels were determined according
to Sergiev et al. (1997). Microalgae were homogenized in
ice bath with 5 mL 0.1% (W/V) TCA. The homogenate was
centrifuged at 12 000 g for 15 min and 0.5 mL of the superna-
tant was added to 0.5 mL 10 mmol/L potassium phosphate
buffer (pH 7.0) and 1 mL 1 mol/L KI. The absorbance of
supernatant was read at 390 nm. The content of H2O2 was
given on a standard curve.
1.8 Determination of the malonyldialdehyde (MDA) con-
tent
For the measurement of lipid peroxidation in microalgae,
the thiobarbitric acid (TBA) test, which determines MDA
as an end product of lipid peroxidation (Heath and Packer,
1968), was used. The absorbance of supernatant was read
at 532 nm and the value for non-specific absorption at 600
nm was subtracted. The amount of MDA-TBA complex
(red pigment) was calculated from the extinction coefficient
155 mmol.L-1.cm-1.
1.9 Soluble protein content and SOD, POD, CAT, GR,
and GPX activity assay
The algae cultures were harvested by centrifugation at
12 000 g for 10 min. The resulting pellet was resuspended in
50 mmol/L potassium phosphate buffer (pH 7.8) containing
0.5 mmol/L ethylenediaminetetraacetic acid (EDTA),
sinicated for 3 min, and centrifuged at 1 000 g for 10 min.
Antioxidative enzyme activities were assayed in the
supernatant. All steps in the preparation of the enzyme
extract were carried out at 0-4 ℃. An aliquot of the extract
was used to determine its soluble protein content by the
method of Bradford (1976) utilizing bovine serum albumin
as standard.
SOD activity was determined based on its capacity to
inhibit reduction of nitroblue tetrazolium (NBT) by super-
oxide radicals generated by xanthine-xanthine oxidase
(Beauchamp, 1971). One SOD unit was defined as the
amount of extract that gave 50% inhibition of NBT
reduction.
POD activity was assayed according to the method of
Dias and Costa (1983) with some modifications. The reac-
tion was started by adding 0.15 mL of 100 mmol/L H2O2 and
the optical density at 470 nm was recorded in a spectropho-
tometer Shimadzu against an identical mixture to which no
H2O2 was added, using the extinction coefficient of 26.6
mmol.L-1.cm-1.
CAT activity was assayed by measuring the initial rate
of disappearance of H2O2 by the method of Kato and
Shimizu (1987). The decrease in H2O2 was followed as de-
cline in optical density at 240 nm, and the activity was calcu-
lated using the extinction coefficient (40 mmol.L-1.cm-1)
for H2O2.
GR activity was determined by following the oxidation
of NADPH at 340 nm (extinction coefficient 6.2 mmol.L-1.
cm-1) as described by Rao (1992).
GPX activity was assayed according to the method of
Flohe (1974). One GPX unit was defined as the decrease of
1 mmol GSH per minute subtracting non-enzymic reaction
at 37 ℃ (pH 7.8).
1.10 Statistic analysis
Standard deviation (SD) of each treatment was
calculated. The significant differences were determined by
the Students t-test. It was considered significantly when
P＜0.05 (marked with*), extremely significantly when
P＜0.01 (marked with **), not significantly when P＞0.05.
YU Juan et al.: Effects of CO2 Enrichment on Photosynthesis, Lipid Peroxidation and Activities of ntioxidative Enzymes of
Platymonas subcordiformis Subjected to UV-B Radiation Stress 685
2 Results
2.1 Photosynthetic characteristics and soluble protein
content
High CO2 increased dry weight and photosynthetic rate
of P. subcordiformis by 11.9% (P＜0.05) and 72.6% (P＜
0.01), respectively. High UV-B significantly decreased dry
weight and photosynthetic rate of P. subcordiformis by
15.0% (P＜0.05) and 21.4% (P＜0.05), respectively. The
dry weight and the photosynthetic rate of P. subcordiformis
grown under the combination of high CO2 and high UV-B
had no major difference compared to P. subcordiformis
grown under ambient CO2 and ambient UV-B (Table 1). High
UV-B significantly decreased Chl a content and Car. con-
tent by 13.7 % and 14.7%, respectively, while Chl a and Car.
contents of P. subcordiformis grown under high CO2 was a
little lower than those of P. subcordiformis grown under
ambient CO2 and ambient UV-B, and the difference was not
significant (P＞0.05). The Chl a content and Car. content
of P. subcordiformis grown under the combination of high
CO2 and high UV-B were significantly lower than those of
P. subcordiformis grown under ambient CO2 and ambient
UV-B.
High UV-B significantly decreased soluble protein con-
tent by 22.5% (P＜0.05). High CO2 significantly decreased
soluble protein content by 18.7% (P＜0.05). Soluble pro-
tein content of P. subcordiformis grown under the combi-
nation of high CO2 and high UV-B significantly decreased
by 15.7 % compared with that of P. subcordiformis grown
under ambient CO2 and ambient UV-B.
2.2 The rate of O2
-. production, H2O2 content and MDA
content
UV-B alone enhanced the rate of O2
-. production by
23.6%（P＜ 0.01）. CO2 alone decreased the rate of O2-.
production by 17.3%（P＜ 0.05），while UV-B and CO2
in combination increased the rate of O2
-. production by 4.5%
and there was no major difference compared with ambient
CO2 and ambient UV-B（P＞0.05）(Fig.1A). UV-B alone
increased H2O2 content by 45.0%（P＜0.01）, CO2 alone
decreased H2O2 content by 9.6% compared with ambient
CO2 and ambient UV-B（P＜ 0.05）. UV-B and CO2 in
combination decreased H2O2 content by 29.3% (P＜0.05)
compared to UV-B alone (Fig.1B), indicating that CO2 en-
richment could ease the UV-B-induced damage and have
antiradiation effect.
UV-B alone increased MDA content by 56.4%（P＜0.01）.
Table 1 Changes in photosynthetic characteristics and soluble protein content of Platymonas subcordiformis with supplemental UV-
B radiation, CO2 or CO2 and UV-B radiation in combination
CO2 concen- UV-B radiation Dry weight Photosynthetic rate Chl a content Car. content Soluble protein
tration dose daily (mg/L) (10-8 mL O2. (10-8 mg/cell) (10-9 mg/cell) content
(mL/L) (kJ.m-2.d-1) cell-1.min-1) (10-9 mg/cell)
360 8.8 36.65± 1.76 12.24± 0.15 41.31± 0.93 58.11± 0.48 43.24± 1.15
13.8 31.16± 1.12* 9.62± 0.09* 35.64± 0.31* 49.56± 0.24* 33.53± 1.34*
5 000 8.8 41.00± 2.10* 21.13± 0.18** 40.84± 1.09 56.72± 0.30 35.35±1.62*
13.8 35.34± 1.57 11.32± 0.11 34.25± 0.21* 47.28± 0.26* 36.45±2.08*
The significance between treatments and control was determined by t-test. *, P＜ 0.05; **, P ＜ 0.01 (n=3).
Fig.1. Effects of CO2 enrichment on the rate of O2
-. production
(A), H2O2 content (B), MDA content (C) of Platymonas
subcordiformis subjected to UV-B radiation.
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004686
The MDA content decreased by 21.8% in P. subcordiformis
grown under elevated CO2（P＜ 0.05）, indicating that
lipid peroxidation was reduced. The MDA content in P.
subcordiformis grown under the combination of elevated
CO2 and UV-B was significantly lower than P.
subcordiformis grown under UV-B alone condition and the
MDA content was only 63.2% of that of UV-B alone (Fig.
1C). This means that high CO2 may have amelioration ef-
fect on UV-B-induced oxidative damage. Namely, elevated
atmospheric CO2 can enhance the capacity of P.
subcordiformis to resist stress-induced oxidative damage.
2.3 SOD, POD, CAT, GR and GPX activities
UV-B radiation alone significantly increased SOD, POD
and GR activities by 56.4%, 32.5% and 13.4%, respectively.
CO2 enrichment alone significantly decreased SOD, POD
and GR activities by 21.8%, 11.5% and 17.0 %, respectively.
The SOD, POD and GR activities of P. subcordiformis grown
under the combination of UV-B and CO2 were significantly
lower than those of P. subcordiformis grown under UV-B
alone, which suggested that elevated CO2 can ease UV-B
stress induced damage. Whereas, SOD and POD activities
of the combination of UV-B and CO2 were still significantly
higher than those of P. subcordiformis grown under ambi-
ent UV-B and ambient CO2. GR activity had no major differ-
ence compared with that of ambient UV-B and ambient CO2
(P＞0.05).
UV-B alone significantly increased CAT and GPX activi-
ties by 188.3% (P＜ 0.01) and 19.5% (P＜ 0.05),
respectively. CO2 enrichment alone decreased CAT and
GPX activities by 11.1% (P＞0.05) and 7.0% (P＞0.05).
CAT and GPX activities of P. subcordiformis grown under
the combination of UV-B and CO2 were significantly lower
than those of P. subcordiformis grown under UV-B alone,
and GPX activity had no major difference compared to that
of P. subcordiformis grown under ambient UV-B and ambi-
ent CO2 (P＞0.05), but CAT activity was still significantly
higher than those under ambient UV-B and ambient CO2 (P
＜0.05).
3 Discussion
3.1 Effects of CO2 enrichment and UV-B radiation on
photosynthetic characteristics and soluble protein con-
tent
Carbon dioxide has risen from a preindustrial concen-
tration of 270 mL/L to a current estimate of approximately
365 mL/L. As the need for fossil fuel energy increases with
increasing population, it is generally accepted that CO2
concentration will reach a mean of 600 mL/L sometime dur-
ing the 21st century. Marine microalgae are currently CO2
limited, significant increases in photosynthetic rate and dry
weight were found in this experiment in response to en-
riched CO2 concentration (Table 1). UV-B alone made a
Fig.2. Effects of CO2 enrichment on SOD (A), POD (B), GR
(C), CAT (D) and GPX (E) activities of Platymonas subcordiformis
subjected to UV-B radiation.
YU Juan et al.: Effects of CO2 Enrichment on Photosynthesis, Lipid Peroxidation and Activities of ntioxidative Enzymes of
Platymonas subcordiformis Subjected to UV-B Radiation Stress 687
decrease of Chl a content and Car. content, which was con-
sistent with Agrawal (1992). Agrawal (1992) showed that
UV-B caused a decrease in carotenoids and photosynthetic
pigment (particularly Chl a) in the green algae
Chlorococcum infusionum and C. elongatum. Under el-
evated CO2 concentration, the Chl a content and Car. con-
tent was not changed much compared with that of ambient
CO2 concentration. The Chl a and Car. content of P.
subcordiformis grown under the combination of UV-B and
CO2 significantly decreased compared with those of P.
subcordiformis grown under ambient UV-B and ambient
CO2 (Table 1).
In UV-B sensitive algae, photosynthetic capacity may
be reduced directly by the effect of UV-B radiation on pho-
tosynthetic enzymes or disruption of PSⅡ reaction
centers, or indirectly by effects on photosynthetic pigments
and stomatal function (Teramura, 1983b). Zhang et al. (1996)
showed that PSⅡ activity, the efficiency of primary con-
version of light energy of PSⅡ and the efficiency of po-
tential photosynthetic quantum conversion. Elevated CO2
not only provides more material for photosynthesis but
also increases ribulose-1,5-bisposphate carboxylase activ-
ity and enhances CO2 fixation capacity. On the other hand,
elevated CO2 inhibits ribulose-1, 5-bisposphate oxygenase
activity and reduces the production of substrate of photo-
respiration-glycolic acid, so that photorespiration inten-
sity is reduced . Therefore, the efficiency of photosynthe-
sis is enhanced (Chen, 2002).
Enhanced UV-B decreased soluble protein content
(Table 1), which is consistent with Santos et al. (1993).
There is different conclusion. Tevini et al. (1981) showed
that UV-B radiation increased soluble protein content. El-
evated CO2 decreased soluble protein content in G.
tenuistipitata (Garcia-Sanchez et al., 1994) and Gracilaria
gaditana (Andria et al., 1999), and increased the soluble
carbohydrate content of G. gaditana (Andria et al., 1999).
Elevated CO2 increased the ratio of C/N of macroalgae
(Garcia-Sanchez et al., 1994). In our experiment, CO2 en-
richment decreased soluble protein content of P.
subcordiformis (Table 1). Changes in soluble protein con-
tent of algae grown in high CO2 could be largely due to a
decline in Rubisco protein. Growth in high CO2 has been
reported to decrease Rubisco protein (Long et al., 1993). In
this experiment, soluble protein content in the combination
of UV-B and CO2 decreased more than that of the control.
3.2 Effects of CO2 enrichment and UV-B radiation on
ROS and lipid peroxidation
Hydroxyl radicals are produced in electron-transfer re-
actions during oxygen reduction metabolism. Oxidative
damage is resulted from an imbalance between the produc-
tion and removal of oxidants. Many deleterious effects to
photosynthesis by UV-B radiation could be caused by the
generation of free radicals that can accumulate in the thyla-
koids and be responsible for oxidative stress and
peroxidative reactions that destroy various components of
the photosynthesis apparatus (Rajagopal et al., 2000).
UV-B radiation enhanced the rate of O2.- production and
H2O2 content of P. subcordiformis (Fig.1A, B), suggesting
that ROS increased. The increase of lipid peroxidation prod-
uct of MDA content (Fig.1C) showed that the physiologi-
cal function of membrane was damaged to certain unfavor-
able effect.
MDA is a product of lipids peroxidation and is usually
used as an indicator in stress physiology of plants.
Figure 1C shows that MDA content decreased in P.
subcordiformis grown at elevated CO2, indicating lipid
peroxidation was reduced. That UV-B increased MDA con-
tent (Figure 1C) showed that the membrane was seriously
damaged. UV-B and CO2 in combination significantly de-
creased MDA content compared to UV-B alone. The re-
sults indicated that elevated CO2 could ease the UV-B-in-
duced damage to membrane. Whereas the MDA content of
P. subcordiformis grown under the combination of UV-B
and CO2 was still significantly higher than that under ambi-
ent UV-B and ambient CO2, which suggested that elevated
CO2 could not recover the damage of UV-B on membrane.
Doubling of the present atmospheric CO2 partial pres-
sure will alter the CO2/O2 ratio at the Rubisco fixation site,
caused a 50% decrease in the ratio of photorespiration
(Sharkey, 1988) and about 25%-60% increase of the acti-
vated state of Rubisco. Therefore, the decrease in lipid
peroxidation of P. subcordiformis at elevated CO2 in present
paper might be due to the favor carboxylation reaction
Rubisco, limited photorespiration and less photoreduction
of dioxygen. In this case, few electrons were transported to
dioxygen during photosynthesis and reduced the damage
potential of active oxygen to membrane system.
3.3 Effects of CO2 enrichment and UV-B radiation on
antioxidative enzymes
Based on the assumption that SOD activity reflects the
need for detoxification of O2
-. , one must conclude that the
rate of O2
-. production was increased in plants grown with
elevated CO2 and subjected to UV-B stress. UV-B alone
induced the activities of antioxidative enzymes (Malanga
and Puntarulo, 1995). Our experimental results were consis-
tent with this conclusion. In our experiment, CO2 enrich-
ment decreased antioxidative enzymes activities, which was
consistent with the result of Polle et al. (1997). Ren et al.
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004688
(2001) thought that elevated CO2 increased antioxidative
enzymes activities in well-watered plants, Rao et al. (1995)
indicated that the antioxidative enzymes activities of SOD,
POD, GR and AP of wheat grown under elevated CO2 had
no major difference compared to those of wheat grown under
ambient UV-B and ambient CO2. The reasons of these dif-
ferent results maybe lie in different experimental materials
and different experimental conditions. In this experiment,
the SOD, POD, CAT, GR and GPX activities of P.
subcordiformis grown under the combination of UV-B and
CO2 were lower than those of P. subcordiformis grown
under UV-B alone, which suggested that elevated CO2 can
ease the UV-B-induced damage to P. subcordiformis.
Therefore, when P. subcordiformis was grown under UV-B
radiation, CO2 enrichment was favor of enhancing the ra-
diation-resistant capacity.
Leaves from various plant species grown under elevated
CO2 concentration contained lower activities of SOD or
CAT than foliage from plants grown under ambient CO2
concentrations (Schwanta et al., 1996). These observations
suggest that plants grown under elevated CO2 exposed to
decreased intrinsic oxidative stress as compared with plants
grown under ambient CO2. UV-B radiation increased H2O2
content (Fig.1B). Therefore, UV-B radiation induced the
activities of POD, CAT, GR and GPX as the increasing re-
quirement of screening ROS. Elevated CO2 decreased H2O2
content, showing that the requirement of screening ROS
decreased. We presumed that the necessity for
detoxidication of H2O2 might be diminished in algae grown
under elevated CO2 concentration (Polle, 1996).
In conclusion, several mechanisms can be involved in a
modification of marine microalgae response to CO2 enrich-
ment by enhanced UV-B radiation. For example, a damaged
photosynthetic apparatus under enhanced UV-B, may alter
the photosynthetic response to elevated CO2 (Ziska and
Teramura, 1992). Elevated CO2 increased ribulose-1, 5-
bisposphate carboxylase activity and inhibited photores-
piration to increase flavonoid synthesis. The increased fla-
vonoid synthesis is mainly considered as a response aim-
ing at the reduction of the penetration of UV-B (Rozema et
al., 1997), therefore the protective capacity against UV-B
radiation was enhanced. On the other hand, elevated CO2
can increase the activity of photolyase (Zhao et al., 2003)
so as to enhance photo repair of UV-B-damaged DNA and
reduce the DNA injury of UV-B radiation to microalgae. On
the one hand, elevated CO2 can promote photosynthesis
and increase the growth of microalgae; on the other hand,
elevated CO2 can stimulate defense mechanism against UV-
B radiation. In this study, the results indicated that elevated
CO2 could reduce the ROS content induced by UV-B
radiation, and reduced the oxidative damage to microalgae.
The main cause of lower oxidative stress in microalgae
grown under elevated CO2 lies in elevated CO2 can increase
the ratio of p CO2/O2, increase the assimiliation of CO2,
reduce the formation of ROS as the product of O2 acting as
electron receptor, and elevated CO2 can reduce the forma-
tion of H2O2 as the product of photorespiration. On the
other hand, elevated CO2 can cause the necessity for
detoxidication to be diminished.
We have shown that algae grown under high CO2 would
better overcome the adverse impact of environmental stress
factor (UV-B radiation) that acts via generation of activated
oxygen species. Because there are multiple stress factors
(including heat shock, pathogen, toxicity, etc.) in the
environment, the effects of algae grown under the combi-
nation of elevated CO2 and UV-B are complex. Enhanced
UV-B and CO2 may be important, but as yet unpredictable
ecological consequences. Therefore, further studies about
microalgae with and without supplemental CO2 and UV-B
radiation remain to be done in the future.
References:
Agrawal S B. 1992. Effects of supplemental UV-B radiation on
photosynthetic pigment, protein, and glutathione contents in
green algae. Environ Exp Bot, 32: 137-143.
Andria J R, Vergara J J, Perez-Llorens J L. 1999. Biochemical
responses and photosynthetic performance of Gracilaria sp.
(Rhodophyta) from Cadiz, Spain, cultured under different
inorganic carbon and nitrogen levels. Eur J Phycol, 34: 497-
504.
Beauchamp C, Fridovich I. 1971. Superoxide dismutase: improved
assays and an assay applicable to acrylamide gels. Anal Biochem,
44: 276-287.
Bowler C, van C W, van M M, Lnze D. 1994. Superoxide
dismutase in plants. CRC Crit Tey Plant Sci, 13: 199-218.
Bradford M M. 1976. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal Biochem, 72: 248-
254.
Campa A. 1991. Peroxidases in Chemistry and Biology. Vol Ⅱ.
Boca Raton, FL: CRC Press. 25-30.
Chen P-P. 2002. Effects of elevated atmospheric carbon dioxide
concentration on plants. Bull Biol , 37: 20-22. (in Chinese)
Cullen J J, Neale P J. 1994. Ultraviolet radiation, ozone depletion,
and marine photosynthesis. Photosynth Res, 39: 303-320.
Davidson A T, Bramich D, Marchant H J, McMinn A. 1994.
Effects of UV-B radiation on growth and survival of Antarctic
marine diatoms. Mar Biol, 119: 507-515.
YU Juan et al.: Effects of CO2 Enrichment on Photosynthesis, Lipid Peroxidation and Activities of ntioxidative Enzymes of
Platymonas subcordiformis Subjected to UV-B Radiation Stress 689
Demeter S, Janda T, Kovacs L, Mende D, Wiessner W. 1995.
Effects of in vivo CO2-depletion on electron transport and
photoinhibition in the green algae, Chlamydobotrys stellata
and Chlamydomonas reinhardtii. Biochim Biophys Acta, 1229:
1-9.
Dias M A, Costa M M. 1983. Effect of low salt concentrations
on nitrate reductase and peroxidase of sugar beet leaves. J Exp
Bot, 34: 537-543.
Flohe L. 1974. Methods in Enzymology. New York: Academic
Press. 328-366.
Foyer C H, Descourvieres P, Kunert K J. 1994. Protection against
oxygen radicals: an important defense mechanism studied in
transgenic plants. Plant Cell Environ, 17: 507-523.
Gao S-D, Wu Y-P, Zhao X-Y. 1994. The physiological effects of
organotin on marine microalgae. Ⅱ. Effects of triphenyltin
and tributyltin on photosynthesis of Dicrateria zhanjiangensis
and Platymonas sp. Oceanol Limnol Sin , 25: 362-367. (in
Chinese with English abstract)
Garcia-Sanchez M J, Fernandez J A, Niell F X. 1994. Effect of
inorganic carbon supply on the photosynthetic physiology
of Gracilaria tenuistipitata. Planta, 194: 55-61.
Garde K, Gustavston K. 1999. The impact of UV-B on alkaline
phosphatase activity in phosphorus-depleted marine
ecosystems. J Exp Mar Biol Ecol, 238: 93-105.
Guillard R R, Rhyter H. 1962. Studies on marine phytoplankton
diatoms.Ⅰ. Cyclotella nana Hustedt and Denotula confervacea
(cleve). Gran Can J Microbiol, 8: 229-239.
Halliwell B, Gutteridge J M C. 1989. Free Radicals in Medicine
and Biology. Oxford: Clarendon Press. 277-289.
Hanagata N, Takeuchi T, Fukuju Y, Barnes D J, Karube I. 1992.
Tolerance of microalgae to high CO2 and high temperature.
Phytochemistry, 31: 3345-3348.
Hao X, Hale B A, Ormrod D P, Papadopoulos A P. 2000. Effects
of pre-exposure to ultraviolet-B radiation on responses of
tomato (Lycopersicon esculentum cv. New Yorker) to ozone
in ambient and elevated carbon dioxide. Environ Pollu, 110:
217-224.
Heath R L, Packer L. 1968. Photoperoxidation in isolated
chloroplasts. Ⅰ. Kinetics and stoichiometry of fatty acid
peroxidation. Arch Biochem Biophys, 125: 189-198.
Jensen A. 1978. Handbook of Physiological Methods. New York:
Cambridge University Press. 59-70.
Kato M, Shimizu S. 1987. Chlorophyll metabolism in higher plants.
Ⅶ. Chlorophyll degradation in senescing tobacco leaves: phe-
nolic-dependent peroxidative degradation. Can J Bot, 65: 729-
735.
Long S P, Baker N R, Rines C A. 1993. Analysing the response of
photosynthetic CO2 assimilation to long-term elevation of
atmospheric carbon dioxide concentration. Vegetatio, 104/105:
33-45.
Malanga G, Puntarulo S. 1995. Oxidative stress and antioxidant
content in Chlorella vulgaris after exposure to ultraviolet-B
radiation. Physiol Plant, 94: 672-679.
Polle A, Eiblmeier M, Sheppard L, Murray M. 1997. Responses
of antioxidative enzymes to elevated CO2 in leaves of beech
(Fagus sylvatica L.) seedlings grown under a range of nutrient
regimes. Plant Cell Environ, 20: 1317-1321.
Polle A. 1996. Terrestrial Ecosystem Response to Elevated Car-
bon Dioxide. New York: Academic Press. 299-315.
Rajagopal S, Murthy S D, Mohanty P. 2000. Effect of ultravio-
let-B radiation on intact cells of the cyanobacterium Spirulina
platensis: characterization of the alterations in the thylakoid
membranes. J Photochem Photobiol B: Biol, 54: 61-66.
Rao M V, Hale B A, Ormrod D P. 1995. Amelioration of ozone-
induced oxidative damage in wheat plants grown under high
carbon dioxide. Plant Physiol, 109: 421-432.
Rao M V. 1992. Cellular detoxifying mechanisms determine age
dependent injury in tropical plants exposed to SO2. J Plant
Physiol, 140: 733-740.
Raven J A. 1991. Physiology of inorganic C acquisition and im-
plication for resource use efficiency by marine phytoplankton:
relation to increased CO2 and temperature. Plant Cell Environ,
14: 779-794.
Ren H X, Chen X, Wu D X . 2001. Effects of elevated CO2 on
photosynthesis and antioxidative ability of broad bean plants
grown under drought condition. Acta Agron Sin, 27: 729-736.
(in Chinese with English abstract)
Rozema J, Leessen G M, van de Staaij J W M, Tosserams M,
Visser A J, Broekman R A. 1997. Effects of UV-B radiation on
terrestrial plants and ecosystems: interaction with CO2
enrichment. Plant Ecol, 128: 182-191.
Santos I, Almeida J M, Salema R. 1993. Plants of Zea mays L.
developed under enhanced UV-B radiation. 1. Some ultrastruc-
tural and biochemical aspects. J Plant Physiol, 141: 450-456.
Schwanz P, Picon C, Vivin P, Dreyer E, Guehl J M, Polle A.
1996. Responses of antioxidative systems to drought stress
in pendunculate oak (Quercus robur) and maritime pine (Pinus
pinaster) as modulated by elevated CO2. Plant Physiol, 110:
393-402.
Sergiev I, Alexieva V, Karanov E. 1997. Effects of spermine, atra-
zine and combination between them on some endogenous pro-
tective systems and stress markers in plants. Compt Rend
Acad Bulg Sci, 51: 121-124.
Sharkey T D. 1988. Estimating the rate of photorespiration in
leaves. Physiol Plant, 73: 147-152.
Sheng W . 2003. Study on glutathione peroxidase activity in yeast.
J Huaibei Coal Ind Teachers Coll , 24: 39-43. (in Chinese
with English abstract)
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004690
Siegenthaler U, Sarmiento J L. 1993. Atmospheric carbon dioxide
and the ocean. Nature, 365: 119-125.
Staaij J W M, Lenssen G M, Stroetenga M, Rozema J. 1993. The
combined effects of elevated CO2 levels and UV-B radiation
on growth characteristics of Elymus athericus. Vegetatio, 104/
105: 433-439.
Teramura A H, Sullivan J H, Ziska L H. 1990. Interaction of
elevated ultraviolet-B radiation and CO2 on productivity and
photosynthetic characteristics in wheat, rice and soybean.
Plant Physiol, 94: 470-475.
Teramura A H. 1983a. Effects of ultraviolet-B radiation on the
growth and yield of crop plants. Physiol Plant, 92: 141-146.
Teramura A H. 1983b. Effects of ultraviolet-B radiation on the
growth and yield of crop plants. Physiol Plant, 58: 415-427.
Tevini M, Iwanzik W, Thoma U. 1981. Some effects of enhanced
UV-B irradiation on the growth and composition of plants.
Planta, 153: 388-394.
Wang A-G, Luo G-H. 1990. Quantitative relation between the
reaction of hydroxylamine and superoxide anion radicals in
plants. Plant Physiol Commun (植物生理学通讯), 29: 55-
57. (in Chinese)
Zhang Q-D, Lu C-M , Feng L-J , Lin S-Q , Kuang T-Y, Bai K-Z.
1996. Effects of elevated CO2 on the primary conversion of
light energy of alfalfa photosynthesis. Acta Bot Sin, 38: 77-
82. (in Chinese with English abstract)
Zhao G-Q, Wang X-L, Yue M, Li F-M. 2003.The combined ef-
fects of enhanced UV-B radiation and CO2 on the growth and
photosynthesis of broad bean seedling. Acta Bot Boreal-Occi-
dent Sin, 23: 6-10. (in Chinese with English abstract)
Ziska L H, Teramura A H. 1992. CO2 enrichment of growth and
photosynthesis in rice (Oryza sativa). Modification by in-
creased UV-B radiation. Plant Physiol, 99: 473-481.
(Managing editor: HE Ping)