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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