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Journal of Photochemistry and Photobiology B: Biology 71 (2003) 35–42
www.elsevier.com/locate/jphotobiol
Role of white light in reversing UV-B-mediated effects in the N2-fixingcyanobacterium Anabaena BT2
Ashok Kumar a, Madhu B. Tyagi b, Nilima Singh a, Rashmi Tyagi a, Prabhat N. Jha a,Rajeshwar P. Sinha c, Donat-P. H€aader c,*
a School of Biotechnology, Banaras Hindu University, Varanasi 221 005, Indiab Department of Botany, Women�s College, Banaras Hindu University, Varanasi 221 005, India
c Institut f€uur Botanik und Pharmazeutische Biologie, Friedrich-Alexander-Universit€aat, Staudtstr. 5, Erlangen D-91058, Germany
Received 25 September 2002; received in revised form 2 May 2003; accepted 7 July 2003
Abstract
The effects of various irradiances of artificial UV-B (280–315 nm) in the presence or absence of visible light (photosynthetically
active radiation) on growth, survival, 14CO2 uptake and ribulose 1,5-bisphosphate carboxylase (RuBISCO) activity were studied in
the N2-fixing cyanobacterium Anabaena BT2. We tested the hypothesis whether or not visible radiation offers any protection against
UV-B-induced deleterious effects on growth and photosynthesis in Anabaena BT2. Attempts were also made to determine the ir-
radiances of UV-B where inhibitory effects could be mitigated by simultaneous irradiation with visible light. Exposure of cultures to
0.2 W m�2 or higher irradiance of UV-B caused inhibition of growth and survival and growth ceased above 1.0 W m�2. 14CO2
uptake and RuBISCO activity were found to be more sensitive to UV-B and around 60% reduction in 14CO2 uptake and RuBISCO
activity occurred after exposure of cultures to 0.4 W m�2 for 1 h. However, growth, 14CO2 uptake and RuBISCO activity were
nearly normal when UV-B (0.4 W m�2) and visible light (14.4 W m�2) were given simultaneously. Blue radiation (450 nm) was found
to be the most effective in photoreactivation against UV-B, better than UV-A or any other light wavelength band. Our results
demonstrate that the studied cyanobacterium possesses active photoreactivation mechanism(s) against UV-B-mediated damage
which in turn probably allow survival under natural conditions in spite of being continuously exposed to the UV-B component
present in the solar radiation. Continued growth of many algae and cyanobacteria in the presence of intense solar UV-B radiation
under natural conditions seems to be due to the active role of photoreactivation.
� 2003 Elsevier B.V. All rights reserved.
Keywords: 14CO2 uptake; RuBISCO activity; Visible light; Anabaena BT2; Photoreactivation; Survival; Ultraviolet radiation
1. Introduction
Depletion of stratospheric ozone and an associated
increase in UV-B (280–315 nm) radiation reaching the
Earth�s surface [1–3] have been shown to be detrimental
to various terrestrial and aquatic ecosystems [4–6]. The
damaging effects of UV-B include photobleaching of Chl
a, reduced photosynthesis, inactivation of the photo-system II reaction center and degradation of light har-
vesting proteins [5–7]. Inhibition of N2 fixation as well
as certain enzymes of nitrogen metabolism have been
* Corresponding author. Tel.: +49-9131-8528216; fax: +49-9131-
8528215.
E-mail address: dphaeder@biologie.uni-erlangen.de (D.-P. H€ader).
1011-1344/$ - see front matter � 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotobiol.2003.07.002
reported in various algae [6,8,9]. UV-B-induced damage
of DNA has also been demonstrated [10,11].
Many species of cyanobacteria show a wide variation
in tolerance to UV-B and possess a variety of defense
strategies which enable them to grow and survive in
environments receiving high UV-B fluxes. The strategies
include avoidance of brightly lit habitats, production of
UV-screening pigments/substances, quenching reactionsfor phototoxic products, such as reactive oxygen species
(ROS), and repair of UV-induced damage [12–17]. UV-
screening compounds such as scytonemin and MAAs
(mycosporine-like amino acids) have been reported from
a number of cyanobacteria [12,13,17]. An increased
production of these compounds following UV exposure
has been demonstrated [17]. A new type of glycosylated
36 A. Kumar et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 35–42
MAA from Nostoc commune which is excreted into the
medium and therefore acts as a true screen has been
reported [18].
Photoreactivation seems to be the simplest UV repair
response since under natural conditions organisms aresupplied with abundant light of the appropriate wave-
lengths. In some photosynthetic organisms it has been
demonstrated that high levels of white as well as blue
light mediate photorepair and ameliorate UV-B-induced
damage [19,20]. Blakefield and Harris [10] found a delay
of cell differentiation by UV-B radiation and its recovery
by photoreactivation and excision repair in the cyano-
bacterium Anabaena aequalis. The induction of nucleicacid lesions by UV-B and their repair have been
reported in Antarctic marine phytoplankton [21]. In
contrast, H€aader et al. [22] could not demonstrate pho-
toreactivation of UV-B-induced inhibition of motility in
Phormidium uncinatum by UV-A or visible light.
Cyanobacteria are the only O2-evolving photosyn-
thetic prokaryotes which fix atmospheric molecular ni-
trogen and add significant amounts of fixed nitrogen tothe soil [6,23]. Various N2-fixing species of this group
grow abundantly in rice fields and act as a natural
source of biofertilizer. Cyanobacteria, being photoau-
totrophic in nature, solely depend on solar radiation for
obtaining energy, and in the process they absorb UV-B
radiation reaching the Earth�s surface. The implication
is that cyanobacteria must possess efficient protection
mechanisms to counteract the damaging effects of UV-Bradiation. Our hypothesis is that lesions caused by UV-
B radiation are primarily repaired by the visible light in
a majority of the cyanobacteria and the UV-absorbing/
sunscreen compounds have secondary and/or additive
roles in the protection mechanisms. Most probably
the role(s) of MAAs and sunscreen pigments are more
effective and prevalent in those species which grow
in extreme habitats such as those exposed to highlight intensity, high temperature or are under water or
oxidative stress.
In the present study, using the cyanobacterium Ana-
baena BT2, we evaluated the role of visible radiation in
reversing the UV-B-mediated effects on growth and
photosynthesis. Our specific objectives were (i) to ex-
amine the minimal inhibitory irradiance of UV-B on
growth and survival, (ii) to determine the role of visible/monochromatic light in reversing the inhibitory effects
of UV-B and (iii) to compare our results with similar
studies conducted on other algae and higher plants.
2. Materials and methods
2.1. Test organism and growth conditions
The filamentous and heterocystous N2-fixing cyano-
bacterium Anabaena BT2 was isolated from a local rice
field in September 1998 by standard microbiological
techniques. Since then this isolate is being maintained at
the culture collection of Microbial Biotechnology Unit,
School of Biotechnology, Banaras Hindu University,
Varanasi, India. Cultures were routinely grown in mod-ified Chu-10 medium [24] in a culture room at 27� 2 �Cand illuminated by Sylvania 40 W T12 cool white fluo-
rescent lamps at an irradiance of 14.4� 1 W m�2 for a
14/10 h light/dark cycle. Unless otherwise stated, all ex-
periments were performed with log phase cultures having
an initial dry weight of approximately 0.15 mg ml�1.
2.2. Mode and source of UV-B irradiation
Artificial UV-B irradiation was provided by a UV-B
lamp (No. 3-4408, Fotodyne Inc., USA) giving its main
output at 312.67 nm. Experiments were performed in a
specially fabricated UV-B chamber equipped with an
exhaust fan to avoid overheating. The desired irradi-
ances (0.1–2.4 W m�2) were obtained by adjusting the
distance between the UV-B light source and the sample.Cultures were harvested and mildly sonicated in a
Branson Sonifier 450 (Branson Ultrasonics Corp.,
Danbury, CT) for 2–3 min to break the long filaments
into homogeneous single cell or 2–3 cell fragments. Cells
were treated with UV-B for predefined time intervals in
sterilized 75-mm glass Petri dishes (Corning) with lids
open, each containing 7.5 ml of homogeneous algal
suspension so that the depth of liquid was less than2.5 mm. The culture suspension was gently stirred
magnetically during UV-B irradiation to facilitate uni-
form exposure. The irradiance of UV-B was measured at
the top of the sample by using a Black-Ray J-221 Long
Wave Ultraviolet Meter (UVP Inc., San Gabriel, CA).
2.3. Percent survival and growth estimation
For determining the percent survival, 0.05 ml aliquots
were withdrawn at predefined time intervals during UV-
B or UV-B+ visible light (14.4 W m�2) irradiation and
plated on agar plates. Before transferring to visible light,
UV-B-treated cultures were incubated in darkness for
48 h to avoid photoreactivation. After 15 days of growth
in light, colonies appearing on agar plates were counted
in a colony counter and percent survival was calculated.Similarly, samples (2 ml) were removed at predeter-
mined time intervals during UV-B or UV-B+ visible
light irradiation and transferred into fresh liquid growth
medium to test growth. Growth was measured by esti-
mating protein content [25] at specific time intervals.
Lysozyme served as the protein standard.
2.4. NaH14CO3 uptake
NaH14CO3 uptake was estimated using the method of
Kumar et al. [23]. A 10-ml culture suspension was
A. Kumar et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 35–42 37
exposed to various irradiances of UV-B either in the
presence or absence of visible light for 1 or 2 h. There-
after cultures were transferred to visible light (14.4 W
m�2) and supplemented with 50 ll of NaH14CO3 (spe-
cific activity 962 Bq ml�1; Bhabha Atomic ResearchCentre, Trombay, Mumbai). Aliquots (1 ml) were
withdrawn after 2 h of incubation and transferred into
scintillation vials containing 0.2 ml of 50% acetic acid.
The suspension was bubbled with air for 5 min to re-
move unreacted 14CO2. A 10 ml scintillation cocktail
(Bray�s solution) [26] was added and the samples were
counted in an LKB-1209 Rack Beta Liquid Scintillation
Counter (LKB Wallac, Turku, Finland).
2.5. Estimation of ribulose 1,5-bisphosphate carboxylase
activity
Exponentially growing cultures were harvested by
centrifugation and the pellet was suspended in supple-
mented Tris buffer (STB; 5 mM Tris, 1 mM EDTA,
1 mM MgCl2 and 20 mM NaHCO3, pH 8.0) and cen-trifuged again. The resulting pellet was resuspended in
STB buffer containing 10% sucrose and 2 mg ml�1 ly-
sozyme and incubated at 35 �C for 30 min. Thereafter,
the cells were sonicated at 4 �C and centrifuged at
10,000g for 10 min. The resulting supernatant (enzyme
extract) was irradiated with UV or UV+visible light for
1 or 2 h and used for the estimation of ribulose 1,5-
bisphosphate carboxylase (RuBISCO) activity by themethod of Codd and Stewart [27]. The RuBP-dependent
NaH14CO3 fixation was measured in a reaction mixture
of 0.3 ml (final volume), which contained 33 mM Tris at
pH 8.2, 1 mM EDTA, 6.6 mM MgCl2, 17 mM
NaH14CO3, 2 mM b-mercaptoethanol, 1 mM RuBP and
0.1 ml enzyme extract. The enzyme was incubated with
all the components except RuBP for 5 min at 30 �C. Thereaction was started by the addition of RuBP and
Surv
ival
[%]
UV-B in
Fig. 1. Impact of increasing irradiances of UV-B (continuous exposure for
survival (plotted on a logarithmic scale) of Anabaena BT2. Equal numbers o
separate but identical experiments�SD.
allowed to proceed for 10 min. The reaction was ter-
minated by the addition of 0.1 ml of 50% TCA. This
mixture was incubated overnight to remove unfixed
NaH14CO3. Thereafter, 10 ml scintillation cocktail was
added and the samples were counted in an LKB-1209Rack Beta Liquid Scintillation Counter.
2.6. Photoreactivation test
UV-B irradiated cultures were distributed (7.5 ml
each) into six Petri dishes and exposed separately to dif-
ferent qualities of photoreactivating light including
UV-A (maximum output at 355 nm, 20 W bulb, Philips),fluorescent (100W cool white lamp), blue (450 nm), green
(520), yellow (580) and red (650 nm) light. Blue, green,
yellow and red light was obtained by passing fluorescent
light (from 150 W Philips Comptalux reflector lamp)
through colored glass filters (Carolina Biological Supply
Company, Burlington, USA). Petri dishes were covered
with a glass plate (1 mm thick) to block out UV radiation
present if any in the fluorescent light. The desired irra-diances of UV-A (2.2 W m�2) and visible light (14.4 W
m�2) were obtained by adjusting the distance between
light source and the sample. Irradiances (visible light)
were measured by an Illuminometer Model-5200 (Ky-
oritsu Electrical Instrument Ltd., Japan). UV-A intensity
was measured by Long Wave Ultraviolet Meter (with
maximal sensitivity at 365 nm). All experiments were
repeated three times. For all treatments at least threereplicates were analyzed and SD was determined.
3. Results
It is evident that with increasing UV-B irradiances
(0.1–2.4 W m�2) there was a gradual decrease in percent
survival (Fig. 1). Exposure of cultures to 2 W m�2 of
tensity [W m-2]
2 h) in the presence and absence of visible light (VL) on percent (%)
f cells were plated after each treatment. The values are means of three
38 A. Kumar et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 35–42
UV-B alone for 2 h resulted in complete killing. An ir-
radiance of 0.4 W m�2 elicited a 20% loss of survival if
cultures received UV-B continuously for 2 h. Once it
became apparent that UV-B radiation alone was inhib-
itory for the survival of Anabaena BT2, the effects ofvisible light on possible protection against UV-B were
evaluated. Cultures simultaneously exposed to UV-B
and visible light (14.4 W m�2) showed a considerable
increase in survival over that obtained with the UV-B
exposure alone (Fig. 1). The inhibitory effect was almost
undetectable below 0.4 W m�2 of UV-B in the presence
of visible light (Fig. 1). Similar to the data for survival,
gradual inhibition of growth of Anabaena BT2 was ob-served in liquid medium following exposure of cultures
to UV-B irradiance above 0.1 W m�2 (Fig. 2). No sub-
sequent growth took place in cultures exposed to 1 W
Fig. 2. Growth of Anabaena BT2 in liquid medium following exposure of cultu
W m�2). Treatment was given for 2 h and thereafter kept in the dark for 48 h
growth was measured by estimating protein content at regular intervals for
Table 114CO2 uptake by Anabaena BT2 after exposure to UV-B or UV-B+VL (vis
UV-B (W m�2) 14CO2 uptake (DPM lg protein�1)b
UV-B
Time (h)
1 2
0.0 6725� 67 11,850� 82
0.1 3654� 44 5095� 61
0.2 2824� 35 4266� 46
0.4 2286� 32 3673� 43
0.6 1883� 29 2607� 36
0.8 1412� 22 2014� 32
1.0 310� 6 502� 8
2.0 225� 4 316� 5a Irradiance of visible light was kept at 14.4 W m�2. Details of experimenbThe results are representative of three separate but identical experiments
m�2 or higher doses of UV-B alone for 2 h; however
appreciable growth (18% inhibition) occurred when
cultures were simultaneously exposed to 1 W m�2 UV-
B+ visible light. In fact, a detectable level of growth
(60% over the untreated control) was observed at 2.4 Wm�2 UV-B+visible light. Growth in UV-B+ visible
light-treated cultures resumed only after a lag period of
2 days (Fig. 2). The lag period in the untreated control
culture lasted for 1 day only.
Uptake of 14CO2 by the cyanobacterium was mea-
sured after treatment with UV-B alone or following si-
multaneous irradiation with visible light and UV-B.
Exposure of cultures to UV-B irradiances greater than0.2 W m�2 for 1 h caused severe inhibition (66% re-
duction at 0.4 W m�2) of 14CO2 uptake, less than 5%
uptake activity was attained above 0.8 W m�2 (Table 1).
res to UV-B alone (0.1–2.4 W m�2) or UV-B and visible light (VL; 14.4
to avoid photoreactivation. Thereafter transferred to visible light, and
10 days. Each point represents the mean�SD.
ible light)a
UV-B+VL
Time (h)
1 2
6725� 67 11,850� 82
6590� 65 11,613� 79
6523� 64 11,257� 78
6456� 64 10,783� 75
6052� 62 10,191� 73
5649� 63 8887� 70
5380� 61 7939� 68
4767� 49 6399� 65
tal conditions as provided in Section 2.
. The values represent means�SD.
Table 2
In vitro RuBISCO activity of Anabaena BT2 after exposure to UV-B or UV-B+VL (visible light)a
UV-B (W m�2) 14CO2 fixed (DPM mg protein�1 min�1)b
UV-B UV-B+VL
Time (h) Time (h)
1 2 1 2
0.0 8980� 75 12,778� 87 8980� 75 12,778� 87
0.1 4825� 48 6987� 55 8820� 72 12,480� 82
0.2 3912� 37 5290� 42 8550� 68 12,120� 76
0.4 3212� 32 4872� 35 7954� 65 11,912� 72
0.6 2180� 20 4150� 32 7562� 62 11,540� 70
0.8 1930� 18 3155� 26 6870� 61 10,870� 65
1.0 850� 8 1676� 6 6255� 58 9630� 62
2.0 375� 5 526� 5 5675� 45 8840� 58a Irradiance of visible light was kept at 14.4 W m�2. For details see Section 2.b The results are representative of three separate but identical experiments. The values represent means� SD.
A. Kumar et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 35–42 39
Cultures receiving UV-B+ visible light simultaneously
for 1 h showed only 20% inhibition in comparison to the
control (visible light alone) at as high as 1 W m�2 ofUV-B radiation. Consistent with the results of 14CO2
uptake described above, more or less similar results were
recorded for RuBISCO activity when the cells were
treated with UV-B either alone or in combination with
visible light (Table 2).
Once it became apparent that visible light exerts a
protective role against UV-B-mediated effects on
growth, 14CO2 uptake and RuBISCO activity, the ef-fectiveness of presumed photoreactivation in repairing
and restoration of UV-B-induced damages especially to
photosynthesis was further studied. A culture of Ana-
baena BT2 whose 14CO2 uptake activity was com-
pletely inactivated by exposure to UV-B (2.4 W m�2
for 1 h) was employed. Restoration of 14CO2 uptake in
visible light was tested either immediately after UV-B
Fig. 3. Test for the role of photoreactivation on 14CO2 uptake after UV-B tre
UV-B (2 W m�2) for 1 h so as to inactivate 14CO2 uptake. Such cultures wer
min. Thereafter NaH14CO3 was added and the cultures were placed again und
and subjected to 14CO2 measurements (For details see Section 2). Each poin
exposure or after holding the samples in the dark or
visible light (14.4 W m�2) for 30, 60 or 120 min. It is
evident from the data in Fig. 3 that cultures whichwere kept in the dark showed very low 14CO2 uptake
in comparison to those which were allowed to photo-
reactivate. Cultures which received photoreactivation
periods of 120 min showed almost complete restoration
and recovery of 14CO2 uptake (Fig. 3). Restoration
and recovery of 14CO2 uptake activity following ex-
posure of cultures to visible light aroused our interest
to test the involvement of particular light quality inthis process. Accordingly, photoreactivation was tested
under UV-A, blue (450 nm), green (520 nm), yellow
(580 nm) and red (650 nm) light. From the data in
Table 3 it is clear that blue light proved significantly
effective in photoreactivation followed by UV-A; other
light qualities had negligible effects on reactivation of14CO2 uptake.
atment in Anabaena BT2. Actively growing cultures were placed under
e kept in dark for 2 h or exposed to visible light (VL) for 120, 60 or 30
er visible light. At regular intervals of 30 min aliquots were withdrawn
t represents the mean�SD.
Table 3
Test of reactivation of 14CO2 uptake by Anabaena BT2 under different
lighta conditions
Light quality (wavelength) 14CO2 uptake
(DPM lg protein�1)b
Control 11,890� 82
UV-A (355 nm) 3224� 36
Blue (450 nm) 5624� 64
Green (520 nm) 2292� 32
Yellow (580 nm) 2186� 31
Red (650 nm) 1812� 29
Dark 1260� 21aDetails of experimental conditions are as mentioned in Section 2. In
brief, actively growing cultures were exposed to UV-B (2 W m�2) for 1
h so as to inactivate 14CO2 uptake activity and thereafter incubated
under different light qualities or dark for 2 h. NaH14CO3 was added in
each set and cultures were placed under visible light. 14CO2 uptake was
measured after 2 h of incubation.b The results are representative of three separate but identical ex-
periments. The values represent means+SD.
40 A. Kumar et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 35–42
4. Discussion
Earlier reports of UV-B effects on cyanobacteria and
algae are based on experiments that have been per-
formed under unnaturally high UV-B irradiances which
are not comparable with irradiances present in solar
radiation [6,8,28]. Knowing the fact that the effects ofUV-B on living organisms depend upon the irradiance,
the wave band and the duration of exposure, in the
present study moderate irradiances of UV-B that are
likely to be present in natural habitats were used so as to
obtain ecologically meaningful results. From our find-
ings it is evident that the effects of UV-B radiation on
the cyanobacterium Anabaena BT2 are irradiance-de-
pendent, there were pronounced inhibitory effects ongrowth and survival in the test organism above 0.4 W
m�2 and complete killing at 2 W m�2 in cultures con-
tinuously exposed for 2 h. Similar to our results lethal
effects of artificial UV-B radiation at irradiances ranging
from 2 to 5 W m�2 have been reported in several cya-
nobacteria [6,15,22,29].
It is pertinent to mention that living organisms never
get exposed solely to UV-radiation in natural habitatsand thus complete killing as observed under laboratory
conditions may not be expected in nature [3,14,22]. The
above presumption seems valid from our studies where
visible light-mediated reversal of UV-B effects was re-
corded. It was observed that when cultures of Anabaena
BT2 were treated with UV-B and visible light together,
growth and survival were almost unaffected up to 0.4 W
m�2 of UV-B. Surprisingly, cultures showed appreciablegrowth at as high as 1.4 W m�2 and complete killing did
not occur even at 2.4 W m�2 of UV-B radiation. To a
greater extent our results are comparable to the natural
solar UV-B radiation where the photosynthetic organ-
isms experience the effects in the presence of visible light
and show uninterrupted growth especially at low doses
of UV-B. Results obtained herein are similar to earlier
reports where inhibition of motility, pigmentation and
other vital processes in certain algae were documented at
higher UV-B irradiances (above 1.3 W m�2) present in
solar radiation [6,9,21,22].Inhibition of growth and survival of Anabaena BT2
by UV-B or UV-B and visible light could be due to a
number of known effects reported in many cyanobac-
teria and algae [6,9,19,21,30]. Although the exact target
of UV-B-induced damages is still under discussion it
may include the D1 and D2 proteins [31], DNA [32],
ribulose-bisphosphate carboxylase and membranes [33].
In addition, pigmentation, photosynthetic CO2 fixationand O2 evolution, nitrogenase activity and a few other
processes have been reported to be severely affected by
UV-B radiation [8–10,19,21]. It has been proposed that
the cellular constituents absorbing radiation between
280 and 320 nm are destroyed by UV-B in living or-
ganisms. Most probably complete killing or severe in-
hibition of growth and survival of Anabaena BT2 at 2 W
m�2 or higher irradiances of UV-B in the absence orpresence of visible light might be due to the complete
inactivation of cellular constituents or loss of photore-
activation system [6,32,34].
Results obtained in the present investigation clearly
demonstrate that visible light was capable of reversing to
a great extent the damaging effects of UV-B. UV-B ra-
diation at 1 W m�2 did not cause significant damage to
the cells in the presence of light. Protection of growth aswell as 14CO2 uptake and RuBISCO activity inAnabaena
BT2 by visible light during or after UV-B treatment may
be due to the presence of an active photoreactivation
system. Existence of an active photoreactivation system
against UV radiation-induced damage has been reported
in a number of organisms including bacteria, cyano-
bacteria, algae and higher plants [10,15,28,30,31,35–37].
In higher plants, Cen and Bornman [20] demonstratedthat under high light irradiance (700 lmol m�2 s�1) plus
UV-B radiation, bean plants appeared most resistant to
the enhanced levels of UV-B radiation while lower irra-
diances increased the sensitivity of the plants to UV-B
radiation. Several other reports also suggest that the level
of visible light plays a key role in mitigating the damages
caused by UV radiation [7,10,20,35,36]. Prokaryotic
microbes should have more effective repair mechanismssince UV radiation shows more deleterious effects on
them because these organisms have single haploid ge-
nomes with little or no functional redundancy [37].
Furthermore being smaller in size, they are devoid of
effective shading or protective pigments as observed in
many higher plants and animals [16,20,35].
That the visible light is indeed involved in restoring
growth and photosynthesis inAnabaenaBT2 is supportedby the findings of a number of workers. Karentz et al. [21]
have shown the formation of UV-B-induced photoprod-
ucts and its photoreactivation in 12 Antarctic marine
A. Kumar et al. / Journal of Photochemistry and Photobiology B: Biology 71 (2003) 35–42 41
phytoplankton species. Their study based on the com-
parison of cellular responses associated with photoen-
hanced repair and nucleotide excision repair revealed that
light-mediated repair of UV damages was an important
factor in survival. Blakefield andHarris [10] reported thatheterocyst differentiation in A. aequalis was essentially
stopped at all exposure levels of UV-B when photoreac-
tivation was prevented, even when excision repair was
active in the cells. In addition to visible light, the
involvement of UV-A in photoreactivation has been re-
ported in several bacteria, cyanobacteria and phyto-
plankton [28,36,37]. Quesada et al. [28] showed that at
specific UV-B irradiances the inhibition of growth ofAntarctic cyanobacteria depended on the ratio of UV-B
to UV-A and that growth rates increased linearly with
increasingUV-A.However, it is not clear from their study
whether UV-A is directly involved in photoreactivation
through photolyase or whether there is synthesis of shock
proteins. In another study UV-A/blue light-induced re-
activation of photosynthesis in UV-B irradiated cyano-
bacterium, Anabaena sp. has been demonstrated [36].Since we did not examine the formation of lesions/
pyrimidine dimers in DNA following UV-B treatment
the exact mode and mechanism of photoreactivation as
mediated by visible light in Anabaena BT2 remains
speculative. However, it is known that photoreactiva-
tion of photosynthesis is independent of the cleavage of
pyrimidine dimers [19,30]. Van Baalen [30] showed that
photosynthetic damage caused by UV-C could be pho-toreactivated by subsequent illumination with blue
(430 nm) light in Agmenellum quadruplicatum. Hirosawa
and Miyachi [19] have also demonstrated photoreacti-
vation by visible light of Hill reaction inactivated by
long-wavelength ultraviolet radiation (UV-A) in the
cyanobacterium Anacystis nidulans. They also demon-
strated that reactivation was completely inhibited by
3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU).The maximum level of reactivation of 14CO2 uptake
activity with blue light is in agreement with the above
reports [35,36]. There is a general consensus that the
photorepair mechanism in cyanobacteria relies primarily
on blue wavelengths. The purified photolyase from
A. nidulans showed maximum photoreactivation at
450 nm with a 75% decrease in activity at 400 nm [28].
From the results of photoreactivation studies it appearsthat Anabaena BT2 possesses a deazaflavin class of
photolyase (maximum activity at 440 nm).
In conclusion, our results show that UV-B irradi-
ances above 0.4 W m�2 alone are highly inhibitory for
growth and survival of cyanobacteria. The inhibitory
effect is greatly reduced in the presence of fluorescent
(visible) light. Our results demonstrate the differential
responses of the cyanobacterium Anabaena BT2 that isexposed to UV-B radiation either alone or in combina-
tion with visible light and emphasize the problems which
might arise in comparing the data of in situ and labo-
ratory studies. We strongly feel that there is a need for
more detailed studies in understanding the mechanism
of photoreactivation processes operative in cyanobac-
teria employing solar radiation (PAR) in field conditions
with varying doses of UV-B.
Acknowledgements
This study was partially supported by the Depart-
ment of Environment, Ministry of Environment and
Forests, Government of India (Grant No. 14/28/89/
MAB/RE). Research work in the laboratory of A.K. is
supported by grants received from the Department ofBiotechnology, Govt. of India (No.BT/PR/1239/AGR/
02/065/98).
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