J Appl Phycol (2011) 23:283–292
DOI 10.1007/s10811-010-9621-8
Photosynthetic and biochemical characterization of pigments
and UV-absorbing compounds in Phormidium tenue
due to UV-B radiation
Rupali R. Bhandari & Prabhat Kumar Sharma
Received: 12 January 2010 / Revised and accepted: 18 October 2010 / Published online: 20 November 2010
# Springer Science+Business Media B.V. 2010
Abstract We report the effect of UV-B radiation (0.8±
0.1 mW cm−2) and UV-B radiation supplemented with lowintensity PAR (∼80 μmol photons m−2 s−1) on the
photosynthesis, photosynthetic pigments, phosphoglycolipids, oxidative damage, enzymatic antioxidants, and UVabsorbing compounds in Phormidium tenue, a marine
cyanobacterium. UV-B radiation resulted in a decline in
photosynthesis and photosynthetic pigments leading to
lower biomass. P. tenue synthesized UV-absorbing compounds like mycosporine-like amino acids (MAAs) and
scytonemin in response to UV-B radiation. Quantity of
MAAs and scytonemin was higher when UV-B was
supplemented with low-level PAR. UV-B treatment also
resulted in quantitative changes in phosphoglycolipids of
the membrane. The UV-B treatment resulted in a slight
increase in the level of peroxidation of cell membrane and
very little increase in the activity of superoxide dismutase
(SOD). Results indicate that UV-B affected photosynthesis
and that the main protective system was the synthesis of
MAAs and scytonemin-like compounds rather than antioxidant enzymes such as SOD.
Keywords Chlorophyll fluorescence . Cyanobacteria .
Lipids . MDA formation . Mycosporine-like amino acids .
Pigments . Phycobilisomes . Scytonemin . Superoxide
dismutase . UV-B
This paper was presented at the 7th Asia Pacific Congress on Algal
Biotechnology, New Delhi, 2009.
R. R. Bhandari : P. K. Sharma (*)
Department of Botany, Goa University,
Goa 403 206, India
e-mail: prabhat_k_sharma@rediffmail.com
Introduction
The springtime stratospheric ozone (O3) layer over the
Antarctica is thinning by as much as 50% (Yip 2000),
resulting in increased mid-ultraviolet (UV-B 280–320 nm)
radiation reaching the surface of the earth and ocean. UV
radiation can cause a broad spectrum of genetic and
cytotoxic effects in aquatic organisms. However, these
responses are partly offset by various protection strategies
such as morphological adaptations (Ma and Gao 2009),
avoidance, screening, photochemical quenching, and repair
(Roy 2000). One of the important concerns with ozone
depletion is the potential contribution to global greenhouse
gas warming by reducing the sink capacity for atmospheric
carbon dioxide through UV inhibition of marine primary
production (Takahashi et al. 1997). Multiple harmful effects
of UV-B on marine primary producers have been reported
and include the direct influences on molecular targets such
as nucleic acids, proteins, and pigments (Franklin and
Forster 1997) and depression of photosynthesis that
ultimately results in cell death (Bhandari and Sharma
2007).
High energetic UV-B radiation has greater potential for
cell damage caused by both direct effects on DNA and
proteins and indirect effects via the production of reactive
oxygen species (ROS; Vincent and Neale 2000). There are
several targets for the potentially toxic ROS including
pigments, lipids, DNA, and proteins; moreover, damage to
photosynthetic apparatus is also partially mediated by ROS
(He and Häder 2002). Morphology, cell differentiation,
survival, growth, pigmentation, motility and orientation,
nitrogen metabolism, phycobiliprotein composition, protein
profile, DNA, and CO2 uptake have been reported to be
affected by UVR (Ma and Gao 2009; Lesser 2008).
284
Phormidium tenue (Meneghini) Gomont (strain GU 3) was
isolated from marine habitats of Goa, India. The species
was isolated and identified at Biological Oceanography
Division, National Institute of Oceanography, Dona Paula,
Goa. Phormidium tenue is filamentous, with filaments
unbranched, parallel, and sheathed. Trichomes are isopolar,
more or less straight, uniserial, composed of cylindrical up
to slightly barrel-shaped cells, constricted or unconstricted
at the cross walls. Cell content is usually blue-green,
thylakoids situated perpendicularly to the cell wall. Heterocytes and akinetes are absent, reproduce by means of
fragmentation.
The cultures were routinely grown in autoclaved ASN III
medium according to Rippka et al. (1979). Culture was
maintained in 100-mL conical flasks filled to 40% of their
volume and kept on a shaker set to a temperature of 30±2°C
under cool white fluorescent tubes providing approximately
80 μmol photon m−2 s−1 PAR at the culture level with a 12h photoperiod. The alga was allowed to grow for 30 days to
obtain the balanced growth phase based on biomass
(logarithmic phase based on growth, Vonshak 1985). The
balanced growth phase (31–33 days) was determined on the
basis of Fv/Fm ratio, chlorophyll content, fresh weight, and
dry weight measurements (Fig. 1).
Cyanobacterial culture (1 mL) of the respective growth
phase was taken out from well-stirred subcultured conical
flask and centrifuged for 10 min at 3,000×g. The excess
Fresh weight (g mL-1)
0.2
0.15
0.1
0.05
0
0
Dry weight (g mL-1)
0.007
5
10
15
20
25
30
35
40
45
50
b
0.006
0.005
0.004
0.003
0.002
0.001
0
0
Chlorophyll conc. (µg mL-1)
Materials and methods
a
0.25
5
10
15
20
25
30
35
40
45
50
c
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
Fv/Fm ratio
An important physiochemical adaptational mechanism
against biologically harmful UV radiation involves the
biosynthesis and accumulation of photoprotective UV
screening compounds like mycosporine-like amino acids
(MAAs) and scytonemin. Widespread occurrence of MAAs
in cyanobacterial strains isolated from various habitats
exposed to strong radiation has been reported supposedly to
protect the cell by absorbing incoming UV radiation
(Garcia-Pichel et al. 1993). MAAs are small, watersoluble molecules of imino carbonyl derivatives of cyclohexanone with absorption maxima between 280 and
360 nm. Scytonemin, a water-insoluble molecule, occurs
in the extracellular mucilaginous sheath surrounding cyanobacterial cells and also considered to be a photoprotective compound (Garcia-Pichel and Castenholz 1991).
In this study, we investigated the effect of UV-B and
UV-B radiation supplemented with low-intensity PAR
(80 μmol photons m−2 s−1) on photosynthesis, photosynthetic pigments, phosphoglycolipids, lipid peroxidation,
antioxidant enzymes such as superoxide dismutase, and
UV-absorbing compounds like MAAs and scytonemin in
the marine cyanobacterium, Phormidium tenue.
J Appl Phycol (2011) 23:283–292
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
5
10
15
20
5
10
15
20
25
30
35
40
45
50
25
30
35
40
45
50
d
0
Days
Fig. 1 Determination of balance growth phase based on fresh weight
(a), dry weight (b), chlorophyll measurement (c), and chlorophyll
fluorescence measurement (Fv/Fm ratio) (d) in P. tenue
water in the pellet was drained by gently petting it with
blotting paper and fresh weight was taken on a digital
balance. The pellet of cyanobacterial cells used for fresh
weight measurement was dried in an oven at 70°C for 36 h
and dry weight was measured.
The UV-B source (Vilbour-Lourmat, France T-6M
source with a λmax at 312 nm) was fitted in a BOD
chamber. Cyanobacterial culture in an open Petri plate
(without lid) was directly exposed to UV-B radiation (0.8±
J Appl Phycol (2011) 23:283–292
0.1 mW cm−2) from the top in the BOD chamber at 30°C
up to 6 h while keeping the algal culture continuously
stirred using a small magnetic flea to avoid the shading of
the culture. UV-B radiation was measured using a UV-B
radiometer procured from the same supplier (VLX-312 with
a λmax at 312 nm). The level of UV-B radiation that the
cells received was 0.8±0.1 mW cm2. UV-B radiometer
sensor was placed exactly at the same position where the
Petri plate with the culture was placed during the experiments. The distance between the UV-B source and culture
was maintained to provide a specific dose (0.8 mW cm−2)
of the UV-B radiation. For certain experiments, white light
of 80 μmol photons m−2 s−1 PAR was supplemented using
light source with fiber optics at a 60° angle to the culture
during the UV-B treatment.
The Fv/Fm ratio, an indicator of photosynthetic efficiency,
was measured according to Sharma et al. (1998) using
chlorophyll fluorometer PAM 101 with actinic light PAM
102 (Walz, Germany). Culture was dark adapted for 10 min
prior to measurement at room temperature. The dark-adapted
culture was exposed to a modulated light with an intensity of
3–4 μmol photons m−2 s−1 to measure initial fluorescence
(F0). This was followed by an exposure to a saturating pulse
of white light of 4,000 μmol photons m−2 s−1 to provide the
maximum fluorescence (Fm). Variable fluorescence (Fv) was
determined by Fv = Fm − F0 and the Fv/Fm ratio was
calculated.
For pigment analysis, cyanobacterial cells were collected
after centrifugation of culture at 8,000×g for 15 min and 0.1 g
of cells was extracted in 1 mL of 80% (v/v) methanol in a
homogenizer at 4°C under dim light, followed by centrifugation at 6,000×g for 10 min at 4°C. The samples were
filtered through a 0.2-μm filter prior to HPLC analysis.
The pigments were separated by HPLC according to
Sharma and Hall (1996) using reverse phase column
(Waters Spherisorb ODS 25 μm×4.6 mm×250 mm) and a
PDA detector (Waters 2996). Twenty microliters of the
filtered sample was injected into the HPLC. The gradient
for separation was 0–100% ethyl acetate in acetonitrile/
water (9:1) over 25 min with flow rate of 1.2 mL min−1.
The quantity of pigments was calculated from peak area
value using β-carotene as external standard. Identification
of pigments was carried out using retention time against
standards and using the spectral profile of individual peaks
using PDA detector in the range of 400–700 nm.
Cell samples were concentrated by centrifugation for
15 min at 6,000×g and 0.1 g of pellet was resuspended in
5 mL of 20 mM sodium acetate buffer, pH 5.5. Cells were
broken using sonicator (Bandelin UW 2200, Germany) at
50% power with nine cycles for 1 min. Phycobilisomes
were precipitated by incubating with 1% streptomycin
sulfate (w/v) for 30 min at 4°C and collected by
centrifugation at 8,000×g for 30 min at 4°C. The amount
285
of phycocyanin, allophycocyanin, and phycoerythrin were
calculated according to Liotenberg et al. (1996).
Total lipids were extracted according to Turnham and
Northcote (1984). Freshly harvested algal pellet (8 g) was
boiled in 5 mL of isopropanol for 2 min to inhibit the lipase
activity. This isopropanol solvent was dried using nitrogen
gas and the algal pellet was homogenized in chloroform/
methanol (1:2, v/v) containing 0.01% BHT as an antioxidant agent to make the final volume to 15 mL. Lipid extract
was centrifuged for 5 min at 2,000×g to get rid of cell
debris, and to the supernatant, 0.8 mL distilled water was
added followed by 5 mL chloroform and 5 mL of 0.88%
KCl in a separating funnel to make the ratio of chloroform/
methanol/water 1:1:0.9. The mixture was shaken vigorously
for 5 min and allowed to separate for 30 min. The solvent
phase was collected in screw-capped vials and concentrated
under nitrogen gas. The dried lipid extract was redissolved in
5 mL chloroform and was used for qualitative and quantitative
determination of different class of lipids.
Total sugars in glycolipids were determined in total lipid
extract by the phenol–sulfuric acid method according to
Kushwaha and Kates (1981). The absorbance of the orange
color was read at 490 nm against a reagent blank. For
calibration, standard glucose was used.
Phospholipid was estimated by determining the amount
of phosphorus in the total lipid extract according to the
Bartlett method by Christie (1982). A blank and series of
standard samples was analyzed simultaneously which was
measured at 830 nm. The amount of phosphorus in the
unknown sample was read from the curve, prepared at the
same time.
Lipid peroxidation was determined by the production
of TBA-MDA adduct formation according to method
described by Sharma et al. (1998). Algal culture was
harvested by centrifuging at 8,000×g for 15 min. The algal
pellet was homogenized in a tissue homogenizer and
redissolved in fresh culture medium with a ratio of 1:5
(w/v). Resuspended algal culture (5 mL) was again
centrifuged and algal pellet was homogenized in 0.5%
TCA. The homogenate was made up to 5 mL and
centrifuged at 8,000×g for 15 min. The supernatant was
collected and used for measuring the peroxidation of
membrane lipids. One milliliter of the supernatant was
added to the test tube containing 2.5 mL freshly prepared
(0.5%) TBA in (20%) TCA and allowed to incubate for
30 min at 90°C in a water bath. After incubation, it was
allowed to cool at room temperature and centrifuged for
2 min at 1,000×g to settle the debris and non-specific
precipitation. The optical density was taken at 532 nm
(Schimadzu, UV-250). Peroxidation of lipids was measured using an extinction coefficient of 155 mM−1 cm−1.
Superoxide dismutase was assayed according to
Boveris (1984). Wet pellet of alga (0.5 g) was extracted
286
Results
Figure 2a shows the effect of UV-B radiation and UV-B
radiation supplemented with low-intensity PAR on morphological changes in P. tenue. Decrease in fluorescence
intensity, indicative of chlorophyll degradation and breakage of filaments, was observed due to the treatments
(Fig. 2a). The decrease in chlorophyll fluorescence and
breakage of filaments resulted in a linear decrease in Fv/Fm
(Fig. 2b). The decrease in Fv/Fm is due to the decrease in F0
as well as in the Fm (Fig. 2b, inset). Six hours of UV-B
treatment as well as UV-B radiation supplemented with
low-intensity PAR resulted in a decrease of ∼65% in Fv/Fm
compared to the respective control (Fig. 2b).
a
Control
UVB
UVB+L
10 mm
10 mm
10 mm
b
Fv/Fm ratio
in 5 mL of 50 mM sodium dihydrogen phosphate buffer
(pH 7.8) using tissue homogenizer. The extract was
centrifuged for 10 min at 6,000×g and supernatant was
used for the SOD assay. One hundred microliters of tissue
extract was added to 2.6 mL of the assay buffer
containing 6 mM EDTA in 10 mM sodium carbonate
buffer (pH 10.2) and 300 μL of 4.5 mM epinephrine.
Absorbance was recorded at 480 nm. A set of standard
with epinephrine but without tissue extract was also
assayed separately to calculate the activity. Protein
concentration of the supernatant was determined according to Lowry et al. (1951).
Extraction and purification of MAAs was carried out
according to the method of Sinha and Häder (2000).
Cyanobacterial cells (0.1 g) were homogenized using 20%
(v/v) aqueous methanol (HPLC grade) and incubated at
45°C for 2.5 h. The pellet was removed and supernatant
was evaporated to dryness. The dried supernatant was
dissolved in 0.2% acetic acid and analyzed using HPLC
(Waters Spherisorb ODS 25 μm×4.6 mm×250 mm),
detection program (Waters 2996 PDA detector) at
280 nm with a flow rate of 1.0 mL min−1 using isocratic
mobile phase of 0.2% acetic acid. Spectra of MAAs were
measured using a PDA detector (Waters, 2996) at the
wavelength range of 200–700 nm.
The cyanobacteria were homogenized in acetone (100%)
and kept for extraction overnight in a refrigerator at 4°C.
The resulting suspension was centrifuged and the supernatant was filtered through a 0.2-μm pore filter. Further
analysis of the samples was done by HPLC (Waters)
according to Sinha et al. (1999). Wavelength range for
detection was 240–500 nm with a flow rate of
1.5 mL min−1, and a mobile phase of solvent A (double
distilled water) and solvent B (acetonitrile/methanol/tetrahydrofuran, 75:15:10, v/v/v) was used with a 0- to 3-min
linear increase from 30% solvent B to 3–23 min at 100%
solvent B.
J Appl Phycol (2011) 23:283–292
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Control
UV-B
UV-B+L
Control
1
2
Fo
12
10
10
3
4
Duration (h)
UV-B
Fm
18
12
13
UV-B+L
5
6
Fig. 2 Effect of UV-B radiation and UV-B radiation supplemented
with low-intensity PAR treatment up to 6 h on morphological changes
(a) and Fv/Fm ratio (b) in P. tenue. Bar represents the mean ± SD, n=6
Photosynthetic pigments observed in P. tenue were
chlorophyll a, β-carotene, alloxanthin, and myxoxanthophylls (Table 1). As a result of UV-B alone as well as UV-B
supplemented with low-intensity PAR treatments, an
overall decrease in pigment content was observed. After
6 h of UV-B treatment, chlorophyll a decreased by 74%,
myxoxanthophylls decreased by 81%, and β-carotene
decreased by 86% as compared to control (Fig. 3). UV-B
radiation supplemented with low-intensity PAR resulted in
a greater decline in all the pigments as compared to UV-B
radiation alone (Table 1).
UV-B treatment resulted in a 13% decrease in phycocyanin
within 1 h, followed by 30% and 55% decrease after 3 and 6 h,
respectively, compared to control. Allophycocyanin and
phycoerythrin content also showed a linear decrease due to
the UV-B treatment (Fig. 4a). UV-B radiation supplemented
with low-intensity PAR treatment also gave more or less the
same results (Fig. 4b). It was observed that phycocyanin
content was much higher than the content of allophycocyanin (59%) and phycoerythrin (16%).
UV-B treatment decreased glycolipids to 55% and
phospholipids to 64% compared to the control. However,
UV-B radiation supplemented with low-intensity PAR for
the same duration did not cause much of a decrease in
glycolipids (8%) compared to the control. Percent decrease
in phospholipids due to UV-B radiation supplemented with
low-intensity PAR was more or less the same as observed
for UV-B treatment (Fig. 5a).
J Appl Phycol (2011) 23:283–292
287
Table 1 Effect of UV-B and UV-B supplemented with low visual light for 6 h on photosynthetic pigments (μg g−1 fresh weight) in P. tenue
Sr. no.
Pigments
Retention time
Absorption spectra (nm)
Amount (in μg g−1 fresh weight)
Control
450,
430,
430,
410,
448,
475,
454,
660
504,
480,
506
480
0.2681
1.2912
1.8196
0.0102
0.5088
534, 608, 666
600
UV-B+L
0.0584
0.2355
0.4688
0.0152
0.0688
0.0494
0.0849
0.2093
0.0157
0.0195
0.100
0.090
Chlorophyll a
0.080
Control
Alloxanthin
0.070
β-carotene
0.060
AU
Fig. 3 HPLC profiles of
photosynthetic pigments
extracted from P. tenue at
445 nm after exposure to UV-B
radiation and UV-B radiation
supplemented with low-intensity
PAR treatment for 6 h. Spectral
profile of photosynthetic
pigments extracted from P. tenue
is shown in inset of the figure
11.383
13.313
15.030
19.1
19.82
0.050
Myxoxanthophyll
0.040
0.030
0.020
0.010
0.000
-0.010
0.00
5.00
10.00
15.00
Minutes
0.100
475.3
20.00
25.00
30.00
430.5
0.090
UV-B
660.6
506
8
506.8
0.080
0.070
Chlorophyll a
Myxoxanthophyll
0.060
AU
Myxoxanthophyll
Alloxanthin
Chlorophyll a
Phycobilins
β-carotene
0.050
616.6
0.040
582
4
582.4
598.2
0.030
0.020
450.00
500.00
550. 00
nm
536.0
645.9660.6
677.7
702.2
600.00
650.00
450.00
500.00
700.00
550.00
nm
600.00
650.00
700.0
0.010
0.000
-0.010
0.00
5.00
10.00
15.00
20.00
Minutes
25.00
30.00
0.100
453 5
453.5
0.090
UV-B+L
481.4
448. 6
0.080
β-carotene
Alloxanthin
0.070
0.060
AU
1.
2.
3.
4.
5.
UV-B
0.050
0.040
641.0
0.030
0.020
450.00
500.00
550.00
nm
600.00
682.6
650.00
700.0
54 9. 4
0.010
45 0. 00
0.000
-0.010
0.00
5.00
10.00
15.00
Minutes
20.00
50 0. 00
550. 00
nm
25.00
638. 6
60 0. 00
65 0. 00
30.00
70 0. 0
288
0.030
J Appl Phycol (2011) 23:283–292
a
0.025
Allophycocyanin
a
Glycolipids
Phospholipids
50
Phycoerythrin
40
mg mL-1
0.020
mg mL-11
60
Phycocyanin
0.015
30
20
0.010
10
0.005
0
Control
0.000
Control
1
3
Time(h)
7
b
Phycocyanin
0.025
Allophycocyanin
Phycoerythrin
mg mL-1
0.020
UV-B and light
b
UV-B
UV-B+L
6
MDA Formation (µmol)
0.030
UV-B
Treatments
6
0.015
0.010
5
4
3
2
1
0.005
0
0.000
Control
1
3
Control
6
UV-B treatment led to a linear increase in the peroxidation
of cell membrane measured as MDA formation (Fig. 5b).
MDA formation increased by 7% after 1 h of UV-B
treatment, which was further increased to 22% after 3 h
and 35% after 6 h of treatment as compared to control. UV-B
radiation supplemented with low-intensity PAR upto 3 h did
not show any significant increase in the MDA formation. A
6-h UV-B radiation supplemented with low-intensity PAR
treatment, however, increased the MDA formation by only
19% compared to control (Fig. 5b), which was 16% less than
that observed with UV-B alone treatment.
A mere 3% increase in the activity of superoxide
dismutase was observed as a result of 6 h UV-B treatment
as compared to control. UV-B radiation supplemented with
low-intensity PAR treatment did not show any increase in
the SOD activity (Fig. 5c).
HPLC analysis of P. tenue showed the presence of
MAAs with an absorption maximum of 294 nm (shinorine;
Fig. 6a, inset). A 6-h exposure of UV-B radiation resulted
in a 21% increase in MAA as compared to the control. UVB radiation supplemented with low-intensity PAR resulted
in a further increase in the MAA content to 65% as
compared to the control.
HPLC analysis also showed the presence of scytonemin
with λmax at 237, 273, and 334 nm (Fig. 6b, inset).
Exposure of P. tenue to UV-B radiation for 6 h increased
0.006
µmol SOD mg-11min-1
Fig. 4 Effect of UV-B radiation (a) and UV-B radiation (b) supplemented with low-intensity PAR treatment up to 6 h on phycocyanin,
allophycocyanin, and phycoerythrin in P. tenue. Mean ± SD, n=3–6
1
3
6
Time (h)
Time (h)
c
UV-B
UV-B+L
0.005
0.004
0.003
0.002
0.001
0
Control
3
Time (h)
6
Fig. 5 Effect of UV-B radiation and UV-B radiation supplemented
with low-intensity PAR treatment up to 6 h on glycolipid and
phospholipid content (a), lipid peroxidation (b), and superoxide
dismutase activity (c) in P. tenue. Mean ± SD, n=4
the scytonemin content by 27% as compared to control.
UV-B radiation supplemented with low-intensity PAR
further increased the scytonemin content to 31% as
compared to control (Fig. 6b).
Discussion
Our results indicate that UV-B radiation affected photosynthesis and, thereby, growth and productivity of the cyanobacterium. The results obtained indicate direct damage to key
components within the photosystem since Fm declined
(Jansen et al. 1996) as well as loss of photosynthetic
pigments since F0 declined. Decrease in the F0 is an
indicator of a decrease in the excitation energy reaching the
J Appl Phycol (2011) 23:283–292
289
a
b
0.010
4.00
Control
3.50
0.70
0.50
AU
3.00
2.50
0.40
0.30
0.20
AU
AU
0.006
0.80
Control
0.60
AU
0.008
0.026
0.024
0.022
0.020
0.018
0.016
0.014
0.014
0.012
0.010
0.008
0.006
0.004
0.002
0.000
-0.002
-0.004
240.00 260.00 280.00 300.00 320.00 340.00 360.00
nm
0.004
2.00
0.10
0.00
1.50
0.002
Shinorine
250.00 300.00 350.00 400.00 450.00
nm
1.00
Scytonemin
0.50
0.000
0.00
0.00
1.00
2.00
3.00
Minutes
4.00
5.00
0.00
0.010
10.00
20.00
Minutes
30.00
10.00
20.00
Minutes
30.00
4.00
UV-B
3.50
UV-B
0.008
3.00
2.50
AU
AU
0.006
0.004
2.00
1.50
0.002
1.00
0.50
0.000
0.00
0.00
1.00
2.00
3.00
Minutes
4.00
0.00
5.00
0.010
4.00
UV-B+L
3.50
0.008
3.00
0.006
2.50
AU
AU
% Peak area
Shinorine
Scytonemin
UV-B+L
Control
100
100
UV-B
121
127
UV-B+L
165
131
2.00
0.004
1.50
0.002
1.00
0.50
0.000
0.00
0.00
1.00
2.00
3.00
Minutes
4.00
5.00
Fig. 6 HPLC profile of (a) MAAs and (b) scytonemin extracted from
P. tenue after exposure to UV-B radiation and UV-B radiation
supplemented with low intensity PAR treatment for 6 h. Absorption
0.00
10.00
20.00
Minutes
30.00
spectra in inset shows spectra for MAAs at 330 nm and for
scytonemin at 300 nm. Table in the inset shows the % change in
MAA and scytonemin on peak area basis
290
photosynthetic reaction center II, largely due to the loss of
pigments in the light-harvesting complex II, while a decrease
in Fm is an indicator of damage to the PS II reaction center
itself (Krause 1988). Both photoinhibition of photosynthesis
and photobleaching of pigments were used as indicators of
UV-B-induced photodamage to the cells. UV-B radiation is
known to cause strong photoinhibition in photosynthetic
microorganisms (Renger et al. 1989; Holm-Hansen 1996),
and the main target has been identified as the PS II D-1
protein in several organisms. As a result of the impaired
photochemical reactions of photosynthesis due to the UV-B
treatment, ROS are generated, which may lead to bleaching
of surrounding pigments, damage to D1 protein, and
peroxidation of lipid membranes (Sharma 2002; Bischof et
al. 2002; He and Häder 2002; Hideg et al. 1994). Decreases
in Fv/Fm, photochemical quantum yield, pigments, and
growth in various algal species under UV-B radiation have
been observed by others (Fouqueray et al. 2007; Marcoval et
al. 2007; Navarro et al. 2007; Gao et al. 2007; Wu et al.
2008). Morphological changes such as reduced trichome
length and the differentiation of heterocyst have also been
reported (Gao et al. 2007). We also observed breakage of
filaments and loss of chlorophyll in P. tenue under UV-B
treatment (Fig. 2a).
The decrease in photosynthetic pigments due to UV-B
treatment in P. tenue could be due to the generation of
reactive oxygen species, which could also affect pigment
content as pigments are highly sensitive to oxidation and
peroxidation reactions (Sharma 2002). We observed
increased oxidation of photosynthetic pigments due to
the UV-B and UV-B radiation supplemented with lowintensity PAR treatment. Our results also showed a slight
increase in lipid peroxidation of cell membrane after the
UV-B treatment, again indicating oxidative damage to the
organism under UV-B treatment in this study. The
chromopheric compounds such as chlorophyll, phycobiliproteins, and quinones absorb UV-B radiation and photosensitize the generation of ROS, leading to the loss of
pigmentation due to bleaching (Carreto et al. 2001; Xue et
al. 2007). Lipids along with pigment–protein complexes
are also some of the oxidative targets attacked by the
elevated ROS, and lipid peroxidation occurs especially at
sites where polyunsaturated fatty acids occur in high
concentrations. Ultraviolet radiation has been shown to
be very effective in inducing lipid oxidation of biological
membranes (Kochevar 1990), polyunsaturated fatty acids
(Yamashoji et al. 1979), and phospholipid liposomes (Pelle
et al. 1990); however; the extent of lipid peroxidation in
our study was considerably less. Decrease of photosynthetic pigments in various algal species under UV-B
exposure has been also reported by others (De Lange et
al. 2000; Cabrera et al. 1997; Germ et al. 2002). It is
evident from this study that UV-B promoted the produc-
J Appl Phycol (2011) 23:283–292
tion of ROS and induced oxidative stress, leading to the
oxidation of pigment and lipid molecules.
Quantitative changes in phospho- and glycolipids
observed with UV-B treatment in this study may also
be due to oxidative damage caused to cell membrane or
damage caused to enzymes involved in lipid synthesis,
though not studied here. A significant decrease in the
level of sulfoquinosyl diacylglycerol and phosphatidylglycerol in the cyanobacterium, Spirulina platensis, in
response to environmental factors has also been reported
earlier (Funteu et al. 1997).
UV-B treatment resulted in a slight increase in the
activity of superoxide dismutase, which metabolizes superoxide radicals. The increase in SOD provides protection
against photodamage; however, the increase observed in
this study is not significant. This may indicate that a slight
increase in SOD activity may not play an important role in
P. tenue’s defense mechanism as compared to the protection
offered by the production of MAAs and scytonemin which
results in screening of UV-B radiation. Accumulation of
SOD had been reported in cyanobacterium in desiccated
field to reverse the effect of oxidative stress imposed by
UV-B irradiation (Canini et al. 2001; Xue et al. 2006,
2007). Increased activity of SOD had been shown by Araoz
and Häder (1999) to confer increased protection from
oxidative damage by detoxifying ROS and regenerating
the reduced form of antioxidant. However, in this study, no
significant increase in SOD and limited peroxidation of cell
membrane (MDA formation) was observed.
Photosynthetic organisms possess features that limit UVB exposure and reduce the amount of damage sustained.
Carotenoids, MAAs, and scytonemin all absorb wavelengths in the ultraviolet region and are suggested to act
as natural sunscreens and/or antioxidant (Karentz et al.
1994). In this study, it was shown that P. tenue synthesizes
a single MAA, i.e., shinorine, a bisubstituted MAA
containing both a glycine and a serine group (Sinha et al.
2003) with absorption maximum at 294 nm, as a result of
UV-B and UV-B radiation supplemented with low-intensity
PAR treatment (Fig. 6a). The presence of shinorine has
been reported in other cyanobacteria (Sinha et al. 2007;
Singh et al. 2008) and has been suggested to provide
protection against UV-B radiation (Cockell and Knowland
1999). Sinha et al. (1999) had shown that MAAs prevent
three out of ten photons from hitting cytoplasmic targets
and provide protection as a UV sunscreen pigment. The
presence of high concentrations of MAAs in this study may
indicate an important protective process by absorbing UV
radiation.
Here, we also report the induction of scytonemin in P.
tenue due to UV-B and UV-B radiation supplemented with
low-intensity PAR treatment. There is strong evidence for
the role of scytonemin as ultraviolet-shielding compound,
J Appl Phycol (2011) 23:283–292
the accumulation of which has been reported in several
cyanobacterial isolates mostly exposed to high irradiances
(Sinha et al. 1999; Portwich and Garcia-Pichel 2000). In
cyanobacterial cultures, as much as 5% of the cellular dry
weight may be accumulated as scytonemin. The correlation
between UV protection and scytonemin presence has been
established under solar irradiance, and it is reported that as
much as 90% of the incident UV-B radiation is screened
due to the presence of scytonemin in the cyanobacterial
sheaths (Brenowitz and Castenholz 1997). Once synthesized, it remains highly stable and carries out its screening
activity without further metabolic investment from the
cell.
The results indicate oxidative nature of damage to
P. tenue under our experimental conditions, as indicated
by damage to photosynthesis and peroxidation of lipids.
We also showed that in response to UV-B radiation, there
was a very little increase in the activity of SOD, but
greater increase in UV-absorbing compounds like MAAs
and scytonemin which probably provide primary protection against UV-B induced damage to the cell organelles
and components from the full impact of deleterious effect
of UV radiation, whereas SOD (antioxidant enzyme) may
not be an efficient system in P. tenue, as reported in crop
plants.
Acknowledgment The authors thank the Department of Science &
Technology, New Delhi (grant no. SF/FT/LS-015/2007 to RB), and
Council of Scientific Institute Research, New Delhi (grant no. 38
(1169)/07/EMR-II to PKS) and Dr. T. Jagtap, National Institute of
Oceanography, Goa for the Phormidium sp. culture.
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