Enzymatic and non-enzymatic defense mechanisms against ultraviolet-B radiation in two Anabaena...

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Process Biochemistry 48 (2013) 796–802

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Process Biochemistry

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Enzymatic and non-enzymatic defense mechanisms against ultraviolet-Bradiation in two Anabaena species

Garvita Singha,b,c, Piyoosh K. Babelea, Rajeshwar P. Sinhab,∗, Madhu B. Tyagic, Ashok Kumara

a School of Biotechnology, Banaras Hindu University, Varanasi 221005, Indiab Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, Indiac Department of Botany, MMV, Banaras Hindu University, Varanasi 221005, India

a r t i c l e i n f o

Article history:Received 29 August 2012Received in revised form 27 April 2013Accepted 29 April 2013Available online 7 May 2013

Keywords:Ultraviolet-B (UV-B) radiationAnabaena speciesAntioxidative enzymesMycosporine-like amino acids (MAAs)

a b s t r a c t

Enzymatic and non-enzymatic defense strategies against ultraviolet-B radiation (UV-B, 280–315 nm)were studied in Anabaena doliolum and Anabaena strain L31, two of the most common strains of Indiancyanobacteria. Upon UV-B irradiation, both strains showed a 2–5-fold increase in antioxidative enzymes,such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD),as compared to non-irradiated control cultures. These enzymes scavenge damaging reactive oxygenspecies (ROS), generated by UV-B radiation inside the cells. In addition, these organisms also synthesizemycosporine-like amino acids (MAAs) which are able to carry out UV-screening and/at the same time asUV-quenching. The identification and characterization of three types of MAAs from both Anabaena specieswere performed using absorption spectroscopy, high performance liquid chromatography (HPLC), elec-tro spray ionization-mass spectrometry (ESI-MS), Fourier Transform Infrared (FTIR) spectroscopy andNuclear Magnetic Resonance (NMR) spectroscopy. Shinorine was found to be the most common MAA inboth Anabaena species while porphyra-334 and mycosporine-glycine were present only in A. doliolum. Theresults of the present investigation clearly demonstrate that both enzymatic and non-enzymatic defensemechanisms are being employed by A. doliolum and Anabaena strain L31 to counteract the damagingeffects of UV-B radiation.

© 2013 Elsevier Ltd. All rights reserved.

1. Introduction

India is among those countries that are close to the equator, thusfaces high fluxes of UV radiation with sunlight. The average latitudeof India is 20◦ north of the equator and maximum ultraviolet-B(UV-B; 280–315 nm) irradiance near the equator (solar elevationangle < 25◦) under clear, sunny skies is approximately 2.5 W m−2,which may affect the major occupation of the country, i.e., agri-culture. A significant declining trend in total ozone column (TOC)over numerous stations lying in the northern part of India is highlyalarming [1]. A decrease in ozone level due to the release of chlo-rofluorocarbons (CFCs) and mononitrogen oxide (NOx) results inan increased level of UV-B radiation reaching the Earth’s surface. Inequatorial regions, the level of UV-B radiation lies between 0.023and 0.139 W m−2, a value that may increase further in coming yearsdue to increases in anthropogenic pollutants [2,3].

Nitrogen-fixing cyanobacteria are natives of tropical agro-climatic conditions, such as those of Indian paddy fields, and mostof them contribute to the carbon (C) and nitrogen (N) economy

∗ Corresponding author. Tel.: +91 542 2307147; fax: +91 542 2368174.E-mail addresses: [email protected], [email protected] (R.P. Sinha).

of soil. These cyanobacteria are susceptible to UV-B radiation, andtherefore, any increase in abiotic stresses, such as UV-B radiation,will affect the survival of microbial cyanobacterial communities[4]. Proteins and DNA, which have absorption maxima in the UVregion, are the main targets of the deleterious UV-B radiation[4,5]. However, many of these organisms have developed sev-eral lines of mitigation strategies such as avoidance, scavenging,screening, DNA repair systems and programmed cell death (apo-ptosis) to counteract the damaging effects of UV radiation. It hasbeen shown that the detrimental effects of UV-B radiation onalgae are always mediated by free radicals, which induce oxida-tive stress [6,7]. The over-production and accumulation of reducedoxygen intermediates such as superoxide radicals, singlet oxy-gen and hydrogen peroxide can negatively affect proteins, DNAand lipids [7]. To overcome these damages, cyanobacteria havedeveloped both enzymatic as well as non-enzymatic defense mech-anisms, both at intracellular and extracellular levels, respectively.Antioxidative enzymes such as superoxide-dismutase (SOD), cata-lase (CAT), peroxidases (PODs) and ascorbate peroxidases (APXs)can scavenge reactive oxygen species [6,8]. Superoxide dismutasegenerates H2O2, removing the superoxide anion via the reaction:2O2

− + 2H+ → H2O2 + O2, and catalase is one of the main enzymesthat scavenges hydrogen peroxide. Peroxidases detoxify harmful

1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.procbio.2013.04.022


G. Singh et al. / Process Biochemistry 48 (2013) 796–802 797

H2O2 present in almost every organism [9]. APX catalyzes thereduction of H2O2 to water using the reducing power of ascorbate[10]. These enzymes counteract damage caused by UV-B stress andthus contribute to the survival of cyanobacteria under UV-B stress.

Non-enzymatic mechanisms, such as the presence of pho-toprotectants, e.g., mycosporine-like amino acids (MAAs) andscytonemin, absorb mainly in the UV-B and UV-A region of thespectrum and help these agriculturally important organisms togrow and survive in habitats exposed to intense solar radiation.MAAs are colorless, water soluble and low molecular weight com-pounds (<400 Da) with a maximum absorbance between 310 and365 nm [11,12]. Chemically, these are composed of either aminocy-clohexenone or an aminocyclohexinimine ring conjugated withthe nitrogen substituent of an amino acid or its amino alcohol.A number of MAAs such as shinorine, mycosporine-glycine, paly-thine, palythinol, asterina-330 and porphyra-334 were reported incyanobacteria [12].

The present study was undertaken with the goal to unravel thesurvival mechanisms of cyanobacteria, which were developed atthe enzymatic and non-enzymatic level, in order to counteract thedamaging effects of UV-B radiation. This study may pave a way forbetter selection of cyanobacteria as biofertilizers in areas with highUV-B radiation.

2. Materials and methods

2.1. Test organisms and growth conditions

The cyanobacteria Anabaena doliolum isolated from the rice fields of Varanasiand Anabaena strain L31, a rice field isolate (kindly provided by Dr. S.K. Apte, BARC,Trombay, Mumbai, India) were used for the present study. The identity of the iso-lates was confirmed by 16S rRNA gene sequencing, which resulted in 100% matchto accession numbers JX075257 and JX075261 (NCBI database) for A. doliolum andAnabaena strain L31, respectively. Cultures were routinely grown under axenic con-ditions in BG-11 medium (without any inorganic nitrogen source) in a culture roommaintained at a temperature of 25 ± 2 ◦C and cyclic illumination with cool white flu-orescent light at an intensity of 14.4 ± 1 W m−2 for a 14 h followed by 10 h darkness.All the experiments were performed with exponentially growing cultures.

2.2. Source and mode of UV radiation

The source of UV-B irradiation was a UV-B lamp (Cat No. 3-4408, Fotodyne Inc.,USA), emitting its main output at 312.67 nm. The lamp has negligible output below290 nm. It shows its maximum output between 300 and 320 nm and hardly emitsany visible radiation (above 390 nm). However, 295 nm cut-off filter foils (Ultra-phan; Digefra, Munich, Germany) were placed over Petri dishes to avoid any UV-Cradiation. Cultures were irradiated with UV-B rays in a specially fabricated cham-ber fitted with a UV-B lamp whose distance to the sample was adjusted to have aUV-B intensity of 1 W m−2. The intensity of UV-B rays was measured by a Black-Ray J-221, Long Wave Ultraviolet Intensity Meter (UVP, Inc., San Gabriel, CA, USA).During UV-B treatment, samples were simultaneously irradiated with cool whitefluorescent light (14.4 ± 1 W m−2). Four Petri dishes (diameter 120 mm, with openlids), each containing 100 mL of homogeneous cyanobacterial culture were exposedto UV-B radiation for the desired time intervals (3, 6, 9 and 12 h). Cultures weregently magnetically stirred to facilitate uniform exposure. Hundred mL of culturewere withdrawn after each time interval of which 100 �L was plated on agar platesto determine the percent survival. The remaining culture volume was used for theestimation of photosynthetic pigments, MAAs and antioxidant enzymes. Culturesirradiated with white fluorescent light served as control. Unless otherwise stated allthe experiments were performed with the log phase cultures having an initial dryweight of approximately 0.15 mg mL−1.

2.3. Determination of percent survival and pigment estimation

For determining percent survival, 100 �L aliquots were withdrawn at desiredtime intervals after UV-B exposure and plated on agar plates. Plates were kept inthe dark for 48 h and thereafter transferred to light in the culture room. Coloniesappearing after 15 days of growth were counted in a colony counter and percentagesurvival was calculated. Estimation of chlorophyll a and phycocyanin concentra-tions were determined as per the method of Bennett and Bogorad by measuring theabsorbance at 663 and 645 nm, respectively [13–15].

2.3.1. Determination of antioxidant enzyme activityFor assaying antioxidant enzymes, cells from control (nonirradiated) and UV-B

irradiated samples were harvested by centrifugation at 12,000 rpm for 15 min at

room temperature. Cell extracts were prepared by sonicating cells in 2 mL of extrac-tion buffer under ice-cold conditions. The extraction buffer consisted of 50 mMpotassium phosphate buffer (pH 7.5), 1 mM ethylenediaminetetracetic acid (EDTA),1% (w/v) polyvinylpyrrolidone (PVP), 0.5% (w/v) TritonX-100 with the addition of1 mM ascorbate in the APX assay. The homogenate was centrifuged at 10,000 rpm for10 min at 4 ◦C, and the supernatants were collected and used for assays of catalase(CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), superoxide dismutase(SOD, EC 1.15.1.1) and peroxidases (POD, EC 1.11.1.7). Catalase activity was deter-mined according to the method of Aebi [16]. Reaction mixtures contained 300 �Mphosphate buffer (pH 7.2), 100 �M H2O2 and 500 �L of enzyme extract. Activitywas determined spectrophotometrically by recording O2 release from enzymaticdissociation of H2O2 in darkness for 1 min. O2 released by enzymatic reaction wasestimated by measuring the decrease in H2O2 absorption at 240 nm using the extinc-tion coefficient: �ε = 39.4 mM−1 cm−1 for 1 min [16]. SOD activity was measured bymonitoring the inhibition in reduction of nitro blue tetrazolium (NBT) as describedpreviously [17,18]. Reaction mixtures consisted of methionine (200 mM), nitrob-lue tetrazolium chloride (2.25 mM), EDTA (3 mM), phosphate buffer (0.5 M, pH 7.5),sodium carbonate (1.5 M) and then enzyme extract was added to make the volume3 mL. Reaction was initiated by adding riboflavin (100 �L). Reaction mixture withoutenzyme extract was considered to be 100% (served as blank), and enzyme activitywas calculated by normalizing to the control and determining percent inhibition.Approximately 50% inhibition was considered equivalent to 1 unit of SOD activityin comparison to tubes lacking enzymes. Ascorbate peroxidase (APX) activity wasdetermined as per the method of Nakano and Asada [19], by measuring ascorbateoxidation to mono-dehydroascorbate at 290 nm, �ε = 2.8 mM−1 cm−1. The activitywas determined by measuring the decrease in absorbance at 290 nm for 1 min. Reac-tion mixtures consisted of 0.1 mM H2O2, 0.1 mM EDTA, 0.5 mM ascorbate and 100 �Lof enzyme extract. Peroxidase activity was measured by taking the absorbance at420 nm, every 20 s for 2 min. Reaction mixtures contained 0.1 M phosphate bufferpH 6.0, 5.33% pyrogallol solution (M/V), 3% H2O2 as substrate and 100 �L of enzymeextract. One unit of peroxidase activity is defined as the amount of enzyme requiredto catalyze the production of 1 mg of purpurogallin from pyrogallol in 20 s at 20 ◦Cunder assay conditions [20]. Total soluble protein was measured by the Bradfordmethod [21].

2.3.2. MAA extraction and spectroscopic analysisFor the extraction of MAAs, cells from control and UV-B irradiated samples were

harvested by centrifugation and extracted in 3 mL of 100% HPLC grade methanol bystoring overnight at 4 ◦C. The methanol extracts were centrifuged at 10,000 rpm for10 min, and the supernatant was subjected to spectroscopic analysis between 250and 700 nm wavelengths, using a double beam spectrophotometer (Shimadzu 1800-UV, Shimadzu Corp., Kyoto, Japan). The raw spectra (peaks) were analyzed using UVProbe version software (Shimadzu Corp., Kyoto, Japan).

2.3.3. Purification and estimation of MAAsAfter initial characterization of MAAs by spectroscopic analysis, methanolic

extracts were evaporated to dryness at 45 ◦C, and the dried product was dissolved in1 mL Milli Q water in a microcentrifuge tube. After adding a few drops of chloroform,the suspension was subjected to centrifugation, and the water phase was carefullytransferred into a fresh microcentrifuge tube to remove contaminating lipophilliccompounds. Finally, the resulting suspension was filtered through a 0.2 �m poresize syringe filters (Axiva Sichem Biotech., New Delhi) and further subjected to highperformance liquid chromatography (HPLC; Waters, Elstree, UK) analysis, using areverse phase semi-preparative column (symmetry prep C18, 7 �m particle size,7.8 mm × 300 mm long) connected to an asymmetry guard column, outfitted witha Waters Photodiode array detector (solvent 0.2% acetic acid in water, detection at330 nm). The sharp peak, common to both strains of Anabaena with retention timesof approximately 2.3 min was eluted and collected with the help of a fraction collec-tor attached to the HPLC unit. Quantification was performed by using peak area, andvalues are expressed as nmol g−1 dry weight. Elution was carried out at a flow rateof 1.0 mL min−1. Purified fractions of MAAs were collected on the basis of retentiontime and lyophilized for further characterization and identification.

2.3.4. Characterization of MAAs by electro spray ionization-mass spectrometry(ESI-MS), Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance(NMR) spectroscopy

Purified MAAs obtained by HPLC were used to produce protonated moleculesby ESI. Mass spectra were recorded on an Amazon SL mass spectrometer (BrukerDaltonics Inc., Billerica, MA, USA). Cone voltage of 30 V was found to induce the for-mation of (M+H)1+ with a mass range of 100–1000 m/z. Data were analyzed using thesoftware DataAnalysis 4.0 (Bruker Daltonics Inc., Billerica, MA, USA). For perform-ing FTIR, lyophilized MAAs were fused with oven dried potassium bromide (storedin dessicator) in 1:100 ratio, and a transparent disk was prepared using hydraulicpress and carefully inserted in a Perkin Elmer Infrared Spectrophotometer version10 (Perkin Elmer, Waltham, MA, USA) to record the spectra. Similarly, lyophilizedMAAs were dissolved in D2O (isotopic purity: 99.5 atoms) at a concentration of6 mg mL−1 and 1H and 13C NMR spectra were carried out with JEOL AL300 FTNMRspectrometer (JEOL Ltd., Tokyo, Japan) at room temperature.


798 G. Singh et al. / Process Biochemistry 48 (2013) 796–802

Fig. 1. Effects of UV-B radiation as a function of time (h) on percent survival (A), content of chlorophyll a (B), and phycocyanin (C) on A. doliolum and Anabaena strain L31.Results are expressed as means of three replicates. Vertical bars indicate standard deviation of the means.

2.3.5. Statistical analysisAll the experiments were conducted in triplicate. A one-way ANOVA (Analysis of

variance) was applied to confirm the significance of data according to Duncan’s [22]multiple range test (MRT) at P ≤ 0.05. SPSS-16 software was used for MRT. Unlessotherwise stated, values are the mean ± SD (n = 3).

3. Results

3.1. Effects of UV-B radiation on survival and pigment content

In order to study the impacts of UV-B radiation on survival of A.doliolum and Anabaena strain L31, we first determined the percent

survival of the two species after 3, 6, 9 and 12 h of UV-B exposureand compared the values to those of the control (untreated) cul-tures. Our results show that survival was not significantly affectedafter 3 h of exposure; however, increasing the duration of exposureproduced a progressive decrease in survival rates, which reachedvalues of 44% and 55% after 12 h of UV-B exposure in A. doliolumand Anabaena strain L31, respectively (Fig. 1A). Effects on pigmentcontent were determined by estimating chlorophyll a and phy-cocyanin concentrations. It was observed that in A. doliolum andAnabaena strain L31, chlorophyll a levels were maintained for upto 9 h of UV-B exposure and thereafter declined to approximately50% and 35%, respectively, after 12 h of continuous UV-B exposure

Fig. 2. Activity of antioxidative enzymes in cultures exposed to UV-B radiation for varying time periods. SOD (A), CAT (B), POD (C), and APX (D). Results are expressed asmeans of three replicates. Vertical bars indicate standard deviation of the means. Experimental conditions were identical for both the test organisms.



(Fig. 1B). However, pronounced effect on phycocyanin content inboth the species was observed. After 12 h of irradiation, the phyco-cyanin content was downregulated approximately 3-fold both in A.doliolum and Anabaena strain L31 (Fig. 1C).

3.2. Effects on enzymatic defense mechanisms

UV-B stress induced significant alterations in the levels of allthe studied antioxidative enzymes. We observed a 3.3-fold induc-tion of SOD activity in A. doliolum and a 2.1-fold induction inAnabaena strain L31 after 9 h of exposure, but after 12 h, the activ-ity began to decline (Fig. 2A). There was a significant increase inSOD activity in Anabaena strain L31, which reached a maximum of0.63 U mg−1 of protein after 9 h of treatment, and then, the activitydeclined after 12 h of exposure. A similar trend was noted in cata-lase activity. Catalase activity was 0.05 and 0.04 �mol min−1 mg−1

of protein in untreated control cultures of A. doliolum and Anabaenastrain L31, respectively, but increased to 0.12 �mol min−1 mg−1

of protein (2.4-fold) in A. doliolum and 0.14 �mol min−1 mg−1 ofprotein (3.5-fold) in Anabaena strain L31 following 9 h of UV-B exposure (Fig. 2B). Similarly, peroxidase activity was 0.008 UPOD mg−1 of protein in untreated control culture of A. doliolum andincreased to 0.02 U POD mg−1 of protein after 9 h (2.5-fold). Theactivity declined to 0.01 U POD mg−1 of protein after 12 h of UV-B exposure. Peroxidase activity in Anabaena strain L31 increasedto 5.4-fold (0.027 U POD mg−1 of protein) at 9 h of UV-B expo-sure but came down to 0.018 U POD mg−1 of protein after 12 h ofexposure (Fig. 2C). Interestingly, the trend of APX activity after UV-B exposure was found to be lower than the activity of SOD andPOD in both Anabaena sp. (Fig. 2D). In A. doliolum, control culturesshowed an initial activity of 0.0015 nmol min−1 mg−1 of protein andreached to 0.002 nmol min−1 mg−1 of protein (1.3-fold) at 9 h ofUV-B exposure and thereafter declined to 0.001 nmol min−1 mg−1

of protein after 12 h of exposure. Similarly, the Anabaena strain L31showed a maximum activity of 0.0018 nmol min−1 mg−1 of proteinat 9 h, which was 2.6-fold higher than the control but declined to0.001 nmol min−1 mg−1 of protein after 12 h. Though the activityof all the enzymes tested declined after 12 h of continuous UV-Bexposure to both Anabaena species, level of activity was still higherthan the untreated control culture except APX activity of Anabaenastrain L31.

3.3. Effects on non-enzymatic defense mechanisms

Spectroscopic analysis of methanolic extract revealed promi-nent absorption peaks of UV-B absorbing compounds (absorbancebetween 300 and 360 nm) suggesting the presence of colorlessMAAs in both Anabaena species. Quantitative estimation showed

Fig. 3. MAAs concentration in cultures of A. doliolum and Anabaena strain L31 afterexposure to UV-B radiation for different periods of time. Results are expressed asmeans of three replicates. Vertical bars indicate standard deviation of the means.

3.8-fold induction of MAAs after 6 h of UV-B exposure in A. doliolumwhich increased to 5.4-fold after 9 h of continuous UV-B exposure.There was 3.5-fold increase in MAAs content after 9 h in Anabaenastrain L31. However there was decline in level of MAAs contentafter 12 h of continuous UV-B exposure in both Anabaena species.Concentration of MAAs was 1600 and 1400 nmol g−1 dry wt in A.doliolum and Anabaena strain L31, respectively, after 9 h of UV-Bexposure (Fig. 3). In comparison to Anabaena strain L31, A. doliolumshowed a 5.8-fold increase in MAA levels after 9 h of UV-B stress, butin case of Anabaena strain L31, MAA did not drop below the basallevel even after 12 h of UV-B stress as found in A. doliolum. HPLCanalysis confirmed the presence of MAAs in both Anabaena species,showing a common peak at 334 nm (retention time 2.1–2.8 min).However a few other peaks also appeared in the samples of boththe species in HPLC analysis (Fig. 4A and B). Major fraction of MAAs(common peak of both the species) was collected separately andHPLC analysis was performed again. A single peak correspondingto standard shinorine was noted in the chromatogram of both thespecies (data not shown). Identity of shinorine became evidentfrom the ESI-MS analysis of the peak as a value of m/z 3321+ wasobtained from both Anabaena species (Fig. 5A). The other two addi-tional peaks with retention times of 3.3 and 3.7 min of A. doliolumwere subjected to ESI-MS analysis which gave m/z of 2471+ and3481+ (Fig. 5B and C). On the basis of retention time of HPLC and ESI-MS analysis above two peaks seem to be of porphyra-334 (retentiontime 3.3 min, MW-348) and mycosporine glycine (retention time3.7 min, MW-247). Furthermore one new peak with retention time

Fig. 4. HPLC analysis of cell extracts of A. doliolum (A), and Anabaena strain L31 (B). Common peak appearing at 2.1–2.8 min resembles to shinorine (SH) [41]. Peakscorresponding to porphyra-334 (P-334) and mycosporine-glycine (MG) were also recorded in chromatogram of A. doliolum (A). One extra peak with retention time of 14 minin Anabaena strain L31 (B) seems to be new and may be related to core functional group of MAAs.



Fig. 5. Electro spray ionization-mass spectrometry (ESI-MS) of HPLC purified frac-tions showing the peaks of shinorine (SH) (A), mycosporine-glycine (MG) (B), andporphyra-334 (P-334) (C).

14 min of Anabaena strain L31 was also subjected to FTIR analy-sis which gave strong frequency pattern with bands of (3400.13,3138.44, 2079.60, 1634, 1400, 1117.62, 980.53 and 618.54 cm−1)(Fig. 6A). Values obtained seem quite similar to the basic functionalgroup of MAAs. Further characterization and identification of thiscompound may prove very useful in view of its maximum inductionafter 9 h of UV-B exposure.

We only proceeded with the sharp common peak from bothAnabaena species with retention time of 2.1–2.8 min and m/z of3321+ (tentatively identified as shinorine) was subjected to FTIRanalysis. FTIR results of the collected fractions containing KBr pel-lets showed six prominent bands. Bands of 3448 cm−1 may be

Fig. 6. Fourier Transform Infrared (FTIR) radiation spectroscopy showing functionalgroups present in the purified unknown fraction (with retention time of 14 min)of Anabaena strain L31 (A) showing basic functional groups related to MAAs, andpurified fraction of shinorine (B).

assigned to OH group, 2927.1 cm−1 for side chain vibrations con-sisting of C H stretching and indicating the presence of (NH2

+) andbands of 1634.8 and 1385.2 cm−1 may be assigned for the pres-ence of an NH2 and carboxylic groups, respectively (Fig. 6B). NMRanalysis was also carried out to characterize the compound. Thepositions of hydrogen (1H) and carbon (13C) indicated that the puri-fied compound is indeed shinorine (Fig. 7A and B). Proton NMR(1H) analysis of the compound showed absence of aromatic andheteroaromatic protons. 13C spectra also showed an aliphatic func-tional group (60.275).

4. Discussion

In the present study attempts have been made to studythe response of two different defense mechanisms adapted bytwo common Indian soil cyanobacteria namely A. doliolum andAnabaena strain L31, under UV-B stress. Estimation of percent sur-vival under UV-B stress clearly indicated that the Anabaena strainL31 has developed a better survival strategy in comparison to A.doliolum whose percentage survival decreased to below 50% after12 h of UV-B exposure. Significant decreases in chlorophyll a andphycocyanin was evident after 12 h of continuous UV-B radia-tion exposure in both Anabaena species, although the effect wasmore pronounced on phycocyanin. Decrease in chlorophyll a con-tent may be attributed to photoreduction of protochlorophyllideto chlorophyllide under UV-B stress [23]. Additionally, bleaching ofphotosynthetic pigments by UV-B has been reported by few work-ers [5,23]. Strong inhibition of phycocyanin by UV-B has also beenreported and it seems that proteinaceous pigments may be theprimary target of UV-B [23,24].



Fig. 7. Nuclear magnetic resonance (NMR) spectra of purified fraction of shinorine. The 1H (A) and the 13C (B) 1D NMR spectrum of the purified fraction were recorded inD2O solution. NMR spectra revealed correlation with chemical shift data of shinorine [32,34].

Our study suggest the existence of two lines of defense mech-anisms under UV-B stress, one of which is enzymatic and theother being non-enzymatic mediated through synthesis of a spe-cial class of secondary metabolites namely MAAs. Induction oftranscripts of antioxidative enzyme systems by UV-B radiation hasbeen reported by several researchers [25–30]. Our findings alsoshowed multifold induction of SOD, CAT, POD and APX activity. Itis also evident that Anabaena strain L31 is more potent in induc-tion of SOD (0.63 U mg−1 of protein) after 9 h of UV-B exposureas compared to A. doliolum, which showed only 0.46 U mg−1 ofprotein. Furthermore, Anabaena strain L31 maintained high basallevels of SOD activity (0.49 U mg−1 of protein) as compared toA. doliolum (0.38 U mg−1 of protein) even after 12 h of continu-ous UV-B exposure. Most probably higher survival of Anabaenastrain L31 might be because of relatively higher levels of antiox-idant enzyme activity such as SOD, CAT and POD even after 12 hof prolonged UV-B exposure. Activity of APX under UV-B stresswas not as significant as that of CAT, SOD and POD. It has beenreported that higher plants have suppressed levels of APX activitywhich in turn induces higher level of SOD, CAT and POD activity[31]. Since we observed moderate levels of APX activity in bothAnabaena species, low levels of induction of SOD, CAT and PODactivity seems justified in view of the above reports in higherplants. This suggests the existence of a differentially regulateddefense mechanism in cyanobacteria and its absence in higherplants.

Different types of secondary metabolites, including flavonoids,are known to act as a sunscreen for UV radiations in plants, butcyanobacteria and certain other microbes synthesize novel typesof a sunscreen compounds that absorb in the UV-range and havehigh molar extinction coefficients (ε = 28,100–50,000 M−1 cm−1),suggesting a UV-screening role. Results of the present studyshowed 3.5–5-fold increases in MAA content as a consequenceof exposing cultures of both strains to UV-B radiation. The con-tent of MAAs reached 1600 nmol g−1 dry weight in A. doliolum,and 1400 nmol g−1 dry wt in Anabaena strain L31, after 9 h of UV-Bexposure. It is also interesting to note that the induction of a com-mon type of MAA namely, shinorine occurred in both the Anabaenaspecies. Most probably shinorine plays a key role in protectionagainst damages caused by UV-B. However, the role of other typesof MAAs may not be ruled out. This was evident from the fact thatinduction of two other types of MAAs such as mycosporine-glycineand porphyra-334 was noted in A. doliolum. Similarly in Anabaenastrain L31 a novel type of MAA whose identity is as yet not known,was synthesized after UV-B exposure. Attempts are under way tocharacterize and identify this novel compound.

Once it became apparent that shinorine is chiefly synthesizedunder UV-B stress in both the Anabaena sp. it was characterized indetail. ESI-MS analysis of the purified fraction of common MAA ofboth the Anabaena sp. showed a m/z value of 3321+. Furthermore,analysis of the functional group of the purified MAA (m/z as 3321+)was carried out with the help of FTIR. The spectral bands obtainedshowed similarity with published reports [32–39] and thus allowedus to confirm its identity as shinorine. Additionally, functionalgroup prediction from corresponding wave numbers confirmed thepresence of core (imino-cyclohexene ring moiety) of MAAs [32,34].The NMR spectra also revealed a satisfactory correlation with previ-ously published chemical shift data for shinorine and other similarMAAs [36,37]. All these data clearly suggest that the main sunscreen compound synthesized under UV-B stress is shinorine inboth the Anabaena sp. Two additional peaks which appeared inHPLC chromatogram of A. doliolum were also tentatively iden-tified. ESI-MS analysis suggested that these two peeks containmycosporine-glycine (RT = 3.7 min, m/z = 2471+) and porphyra-334(RT = 3.3 min, m/z = 3481+) in A. doliolum. Our findings are in agree-ment with an earlier report where the occurrence of MAAs, such asshinorine, porphyra-334 and mycosporine-glycine was reported incertain other cyanobacteria [40]. The potential of Anabaena speciesto synthesize and induce MAAs under UV-B stress may be partlyresponsible for the presence and low level of induction of antiox-idative enzymes as compared to higher plants where the existenceof MAAs has not been demonstrated so far. Occurrence of MAAsis widespread in cyanobacteria and has the potential to prevent 3out of 10 photons from hitting cytoplasmic targets [41,42]. Survivalof cyanobacteria under UV-B stress will surely depend on the con-centration of MAAs in the cell and their level of induction understress.

A comparison of UV protective mechanisms in both theAnabaena sp. clearly suggests the role of both enzymatic and non-enzymatic defense mechanisms in conferring protection underUV-B stress. To a certain extent the induction of antioxidativeenzymes such as SOD, CAT, POD and APX is responsible for thesurvival even after 12 h of continuous UV-B stress in both theAnabaena species. However, the level of induction of the aboveenzymes may vary in different species under UV-B stress and thatmay govern the degree of survival. MAAs synthesized by bothAnabaena species, both at an extracellular and at a cytosolic level,exclude harmful effects of UV-B but may not be effective for longerduration as a drastic decrease in their concentration was noticedafter 9 h of exposure. This explains the crucial role of antioxidantenzymes which maintain basal levels even after 12 h of UV-B expo-sure. This is evident from the fact that A. doliolum synthesizes



three types of MAAs and shows 5-fold induction whereas Anabaenastrain L31 shows better survival as it shows high antioxidativeenzymatic activity. It may be concluded that both enzymatic andnon-enzymatic modes of protection mechanisms are important inprotecting damages caused by UV-B stress. Our findings suggestthat Anabaena strain L31 would be better suited as a potent biofer-tilizer strain (nitrogen fixer) in agricultural areas with high UV-Birradiation.

Acknowledgements

This work was financially supported by the project grant sanc-tioned by the Department of Science and Technology, Govt. of India,New Delhi (SR/SO/PS-49/09). Thanks are due to anonymous review-ers for their valuable comments and suggestions.

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