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Research Article CODEN: IJPRNK Impact Factor: 5.567 Sudha Mishra, IJPRBS, 2017; Volume 6(4): 140-161

ISSN: 2277-8713 IJPRBS

INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH AND BIO-SCIENCE EFFECT OF PAR AND PAR+UV-B ON PHOTOSYNTHETIC PIGMENT AND ANTIOXIDATIVE ENZYMES ACTIVITY IN CYANOBACTERIUM SCYTONEMA GEITLERI DR. SUDHA MISHRA Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, India

Accepted Date: 30/07/2017; Published Date: 27/08/2017 Abstract: Cynobacterium Scytonema geitleri investigated for Phycobiliprotien, Chlorophyll a and protein content after PAR and PAR+UV-B exposure for 48 h duration. In all type of irradiation phycobiliprotein content was declined with maximum decline (88%) on PAR+UV-B exposure than PAR only exposure. Most affected phycobiliprotein was Phycocyanin and second was phycoerythrin. At 12 h of PAR+UV-B exposure phycobiliproteins concentration decline up to 50% in compare with the control. Chlorophyll a concentration increased on short period of PAR+UV-B exposure and then fell down up to the level of control on longer period of PAR+UVB exposure. Antioxidative enzyme activity has increases under PAR+UV-B only exposure with highest increase of APX and CAT. SOD activity was decreases drastically under PAR only exposure. Keywords: Scytonema geitleri, reactive oxygen species, phycobiliprotein, chlorophyll a, Antioxidative enzymes.

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Research Article CODEN: IJPRNK Impact Factor: 5.567 Sudha Mishra, IJPRBS, 2017; Volume 6(4): 140-161

ISSN: 2277-8713 IJPRBS

INTRODUCTION Sun energy that reached to earth surface comprises wide range of wavelength and broadly divided in to Ultraviolet, Visible and Infra red. Highly energetic Ultra Violet radiation has shorter wavelength is classified in to three- UV-A (320-390 nm), UV-B (280-320 nm), UV-C. UV-C is most energetic and absorbed by the ozone [1], UV-B mostly absorbed by the ozone whereas UV-A is hardly absorbed by stratospheric ozone layer. Very small portion of UV-B reached to earth surface but highly potential to damage bio-molecules. PAR referred as Photosynthetic Active radiation and significant for photosynthesis. Stratospheric ozone layer provides a shield to earth surface from the harmful effect of UVR. From past few decade ozone layers has been destroyed due to increase in chlorofluorocarbons (CFCs), hydrochlorofluorcarbons (HCFCs), carbon tetrachloride and methyl chloroform. This has increased the amount of UV-B that reaches the earth surface [2, 3]. UV-B is harmful for all living organism especially photosynthetic organisms because they are simultaneously exposed to UV-B during harvesting of sunlight. Cyanobacteria are photosynthetic prokaryotes appeared on earth 3.5 billion years ago and supposed to be responsible for accumulation of oxygen in early atmosphere on Earth [4]. They are cosmopolitan in distribution and play major role in Carbon, Nitrogen cycle and Oxygen evolution through photosynthesis. They are free living and also can form symbiotic alliance with other organism. Cyanobacteria can face various kind of environmental stress [5] such as desiccation and nutrient scarcity, therefore they are found in extreme of environment condition. They are major biomass producers whether in aquatic or terrestrial ecosystem. Cyanobacteria comprise more than 50% biomass in aquatic ecosystem [6]. Wide varieties of cyanobacteria are present on the earth. Like plant cyanobacteria requires a light harvesting complex to perform the photosynthesis. Phycobilisome is that structural complex present in cyanobacteria that captures the light and transfers this energy to photosynthetic reaction centres. Phycobilisomes are large macromolecular antenna complex contains phycobiliprotein that absorb the energy[7]. A PBS composed of central core of APC (λmax 650 nm) and 6-8 radiating rods of C-PC (λmax about 620 nm) and C-PE (λmax about 565 nm) present in some cyanobacteria for capture light energy more efficiently. Light energy is captured by C-PE than transferred to C-PC to APC and finally to Cholophyll a. Ultraviolet radiation can affect the cyanobacteria directly by damaging DNA and Protein and indirectly by the formation ROS. Cyanobacteria face increased ROS due to oxidative stress caused by UV-B stress [8,9]. Increased ROS can be detoxified by enzymatic and non-enzymatic antioxidants. Enzymatic antioxidants includes superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and glutathione reductase[10]. Superoxide dismutase mediates the conversion of superoxide radicals to molecular oxygen and H2O2. Catalase and ascorbate peroxidase (APX) can reduce H2O2 to water. Available Online at www.ijprbs.com

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Research Article CODEN: IJPRNK Impact Factor: 5.567 Sudha Mishra, IJPRBS, 2017; Volume 6(4): 140-161

ISSN: 2277-8713 IJPRBS

In this paper effect of UV-B, PAR and PAR+UV-B on the production of phycobiliprotein, Chlorophyll a and activity of SOD, APX and CAT in cynobacterium Scytonema geitleri has been studied. MATERIALS AND METHODS : Cyanobacterial Materials Filamentous cyanobacterium Scytonema geitleri growing as crust on the roof top of the department of Botany building was selected for our study. Liquid grown culture of cyanobacterium Scytonema geitleri in BG-11 medium without nitrogen (NO3-) at 24+ 10C under continuous illumination of 45 µE m-1 s-1 light intensity was used for present study. UVR Irradiation Homogenised suspension of organism from its exponential growth was transferred to a transparent container (20*20*8.5cm). The cyanobacteria were irradiated simultaneously under artificial radiation of ultraviolet-B (UV-B; 280-315 nm) and fluorescent light (PAR; 400-700nm). UV-C irradiation was eliminated with 295 nm cut-off filters (Ultraphan, Digefra, Munich, Germany). UV-B irradiation was provided by a Philips Ultraviolet-B TL 40 W:12 (Holland) tube with its main output at 312 nm. The irradiation was adjusted to 1.0 Wm -2. A 395 nm (Ultraphan, UV Opak, Digefra, Munich, Germany) UV filter was used to produce the PAR only waveband. PAR irradiation received by the samples was adjusted to 9.0 W m-2. Equal amount of cultures (sample) were collected after 6, 12, 24 and 48 hour of irradiation. Culture without irradiation was treated as control (0 h). Extraction of Phycobiliproteins Each sample (30 ml) centrifuged at 10000 rpm for 15 min. 15 ml of ice cold phosphate buffer (0.75 M, pH=7.0) containing 10% EDTA and pinch amount of PMSF was added to the pellate; sonicated at 150 mA for 5 min and centrifuged at 10000 rpm for 30 min. 60% ammonium sulphate was added to supernatant for the precipitation of protein and centrifuged at 10000 rpm for 20 min. Pellet was re-dissolved in 2 ml of extraction buffer and again centrifuged to obtain the supernatant containing partially purified phycobiliproteins. Estimation of Phycobiliproteins The amount of C-PC, C-PE and C-APC in the sample was calculated using simultaneous equations [11] and the extinction coefficients [12]: C-PC (mg 𝑚𝑙 −1 )=

𝑂𝐷620 -0.7OD650 7.28

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Research Article CODEN: IJPRNK Impact Factor: 5.567 Sudha Mishra, IJPRBS, 2017; Volume 6(4): 140-161 C-PE (mg 𝑚𝑙−1 )=

ISSN: 2277-8713 IJPRBS

𝑂𝐷565 -2.8(C-PC)-1.34(APC) 12.7

APC (mg 𝑚𝑙 −1 )=

𝑂𝐷650 -0.19𝑂𝐷620 5.65

Extraction and Estimation of Chlorophyll a Three ml of homogenate culture of cyanobacteria was taken and centrifuged at 10000 rpm 15 min. 3 ml of 95% methanol was added to pellet and kept in dark for 24 h. Then centrifuged at 10000 rpm for 30 min. O.D. of supernatant was taken at 663 nm. For quantitative estimation of chlorophyll a, the formula [13] with the modification that extinction coefficient[14] of 12.7 for chlorophyll a at 663 nm were used i.e. Chlorophyll a (μg ml-1) = 12.7 X OD663 X V/L Where OD663 = absorbance (optical density) at 663 nm; V = volume of methanolic extract in ml and L = Length of spectrophotometer cell i.e. 1 cm. Estimation of Protein Protein content of sample was assayed using Bradford[15] method. Accordingly 2.5 ml of Bradford reagent added to 50 µl of sample then left for 20 min for reaction and O.D. was taken at 595 nm. Fluorescence Emission Spectrum Phycobiliprotein extract was analysed to measure the emission spectrum of C-PC with a spectro- fluorophotometer (RF-1501, Shimadzu) at room temperature with an excitation wavelength of 615 nm and emission wavelength between 400 and 700 nm. Antioxidant Enzymatic Activities Assay Cell free extract was prepared with the 50 ml of homogenate culture and centrifuged. 100 mM ice cold phosphate buffer containing 2 mM EDTA, 1% PVP and 1 mM PMSF in isopropenol was added to the pellet and centrifuged. Supernatant obtained was concentrated by lyophilisation (protein concentration range 35-160 µg/ml. Lyophilized sample was maintained up to 1 ml with extraction buffer containing EDTA and PMSF only. Ascorbate Peroxidase (APX) Activity was measured using the method of Chen and Asada[16]. One ml of reaction mixture contenting 50 mM potassium phosphate (pH-7.5), 0.5 mM ascorbic acid and 0.1 mM H2O2 and cell free extract at room temperature. Oxidation of ascorbic acid was followed by the decrease in absorbance at 290 nm (extinction coefficient = 2.8 mM-1cm-1). Superoxide Dismutase (SOD) Activity was assayed spectrophotometrically by monitoring inhibition of nitroblue tetrazolium[17]. One ml of reaction mixture containing 50 mM Available Online at www.ijprbs.com

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phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM methionine, 75 µM nitroblue tetrazolium, 2 µM riboflavin and cell free extract placed under two 15W fluorescent lamps to start the reaction. After 10 min, absorbance was taken at 560 nm. One enzyme unit = volume of supernatant corresponding to 50% inhibition of the reaction [18]. Catalase (CAT) activity was determined by measuring the consumption of H2O2 (extinction coefficient 39.4 mM cm-1) at 240 nm for 5 min. Three ml of reaction mixture contains 100 mM phosphate buffer (pH 7.0) and cyanobacterial extract was taken and reaction was initiated by adding 10 µl of 30% (w/v) H2O2 [19]. Statistics Statistical error was expressed as the standard deviation of mean. All determinations were done in triplicate with fine samples. RESULTS Absorption Spectra of Phycobiliproteins and Chlorophyll a. Crude extracts of cyanobacterium S. geitleri show absorption maxima at 615 nm, 655 nm and 575 nm of C-PC, APC and C-PE respectively (Figure 1). Effect of irradiation is clearly visible on absorption spectrum of Phycobiliproteins. Absorbance of phycobiliproteins was decreases with exposure time after each type of irradiation. Samples that are receiving PAR only radiation, after 48 h f exposure C-PC, APC and C-PE absorbance was decreases. Samples irradiated with PAR+UV-B radiation up to 48 h, absorbance for PBP ws diminished ad peaks are almost lost. In Figure. 4 absorbance of peaks at 436 and 665 nm belong to Chlorophyll a and effect of different irradiations is clearly visible in absorbance spectra of chl a.

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Research Article CODEN: IJPRNK Impact Factor: 5.567 Sudha Mishra, IJPRBS, 2017; Volume 6(4): 140-161

ISSN: 2277-8713 IJPRBS

Figure 1 : Absorption spectra of phycobiliproteins extracted from Scytonema geitleri after exposure to PAR and PAR+UV-B radiation for different durations. Effect of PAR and PAR+UV-B irradiation on phycobiliproteins contents In control sample the APC, C-PC and C-PE content was found 0.058, 0.049 and 0.025 mg ml-1 respectively. Under PAR exposure (Figure 2) APC and C-PE concentration decreases (41% APC and 46% C-PE) at 6th h of exposure in comparison to the control and this concentration was maintained up to 48th h of PAR exposure. The concentration of C-PC continuously decreases up to 48th h of PAR exposure. At 48th h C-PC concentration was 0.018 mg ml-1 which is 64% decline in contrast to C-PC concentration in control. Under PAR + UV-B exposure (Figure 2) up to the 48th h continuous decreasing trend was found in the concentration of APC, C-PC and C-PE. APC concentration was declined up 43% at 6th h, Available Online at www.ijprbs.com

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63% at 12th h, 66% at 24th h and 76% at 48th h of PAR-UV-B exposure in comparisons to the control. Decreasing trend in the C-PC concentration was recorded up to 32% at 6th h, 50% at 12th h, 57% at 24th and up to 88% of decline was recorded at 48th h of PAR-UV-B exposure. Similarly C-PE concentration was declined up to 45%, 56%, 61% and 79% at 6 th, 12th, 24th and 48th h of PAR+UV-B exposure respectively. Maximum decline in the C-PC concentration was recorded at 48th h of PAR+UV-B exposure.

Figure 2 : Effects of PAR and PAR+UV-B treatment on allophycocyanin, phycocyanin and phycoerythrin contents in S. geitleri. (mean ± SD, n=3) Effect of different irradiation at each exposure time on phycobiliproteins contents After 6 h of irradiation C-PC, APC and C-PE concentration was almost stable in PAR only irradiation in compare to the control. After 6 h of PAR+UV-B irradiation concentration of C-PC, APC and C-PE was decreasing in compare to control sample. Under PAR+UV-B irradiance 43 % decline was recorded in APC concentration (Fig. 3).

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Research Article CODEN: IJPRNK Impact Factor: 5.567 Sudha Mishra, IJPRBS, 2017; Volume 6(4): 140-161

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After 12 h of irradiance, C-PE concentration was declined 40 % under PAR irradiance and almost same concentration was maintained after PAR+UV-B exposure. C-PC concentration was declined up to 37 % under PAR and 50 % declined was found in S.geitleri exposed under PAR+UV-B up to 12 h. APC concentration was declined up to 40 % under PAR and 63 % declined under PAR+UV-B exposure after 12 h of irradiation in comparison to the control (Fig. 3). After 24 h of PAR and PAR+UVB exposure C-PC concentration was declined up to 37 % under PAR only and 57 % declined was found under PAR-UV-B exposure. CPE concentration was declined up to 76 % under PAR and 61 % under PAR+UV-B exposure in compare to C-PE concentration in control sample. APC concentration was declined up to 43 % under PAR only and 66 % under PAR-UV-B exposure (Fig. 3). After 48 h of irradiation (Fig. 3), C-PC concentration was declined up to 63 % under PAR and 88% under PAR+UV-B exposure. C-PE concentration was declined up to 45 % under PAR only and 78 % decline was recorded after 48 h of PAR+UV-B exposure. Similarly APC concentration was also declined under each type of irradiation with 45 % of decline under PAR only and 76 % of decline under PAR+UV-B exposure after 48 h. Under each type of irradiation maximum declined was found in C-PC concentration followed by APC and C-PE concentration and under PAR+UV-B exposure up to 48 h almost 81% decline was recorded for total phycobiliproteins content (Fig. 3). Under PAR only exposure after 48 h total phycobiliprotein content was declined up to 38 % and in PAR+UV-B irradiated sample after 48 h total phycobiliprotein content was 81% lower than control.

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Figure 3 : Effect of PAR and PAR+UV-B treatment at 6th, 12th, 24th and 48th h on allophycocyanin, phycocyanin and phycoerythrin contents in S. geitleri (mean ± SD, n=3).

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Research Article CODEN: IJPRNK Impact Factor: 5.567 Sudha Mishra, IJPRBS, 2017; Volume 6(4): 140-161

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Figure 4 : Spectral profile of Chlorophyll a (434 nm, 664 nm) and carotenoids (474 nm) after exposure of PAR and PAR + UVB for 48 h. Chlorophyll a contents Chlorophyll a concentration in control sample was found 3.6 μg ml-1 (Figure. 5). After PAR and PAR +UV-B irradiation for 6 h, chl a concentration was maintained up to the level of control and after 12 h of PAR and PAR + UV-B irradiation, 20 % of chl a concentration increases. After 24 h and 48 h of PAR irradiation chl a concentration increases and 45 % of increase was recorded after 48 h of PAR exposure. In contrast to PAR chl a concentration started to decline after PAR+ UV-B exposure and after 48 h of PAR + UV-B exposure 11 % of decline was recorded in the chl a concentration of S. geitleri.

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Figure 5: Effect of PAR and PAR+UV-B treatment on chlorophyll a content in S. geitleri (mean ± SD, n=3). Effect of irradiation on Florescence profile of Phycocyanin Fluorescence Emission of control (untreated) sample of S. geitleri was recorded at 211.7 (Figure 6). Under PAR and PAR+UV-B exposure, decreasing trends in fluorescence emission of C-PC was recorded. Fluorescence emission was declined up to 70 % under PAR, 56 % decline under PAR and 88 % decline was recorded under PAR+UV-B exposure in contrast to the control at 48 h of irradiation. Shift in Fluorescence emission wavelength was also recorded under various irradiation and duration of irradiations. In control, a maximum Fluorescence emission wavelength was recorded at 641 nm in the control sample (0 h). Blue shift (634 nm) in fluorescence emission wavelength was recorded under PAR+UV-B exposure up to 48 h. Similarly a fluorescence emission peak was shifted at 635 nm under PAR and UV-B exposure up to 48 h. The results indicate the dismantling of C-PC structure due to the irradiation. UVR exposure first cause high molecular mass aggregates to breakdown into hexamers (αβ 6), then disintegrate in to trimers (αβ3) and finally to monomers (αβ)[20][21]. Similarily shifting in PC emission peaks was observed in the Tubingen strain of Phormidium uncinatum[22].

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Figure 6: Effect of PAR and PAR+UV-B on the fluorescence emission of phycobiliproteins (PC) when exited at 615 nm. Effect of irradiation on protein contents of crude extract of S. geitleri In control sample protein content was calculated 6.08 μg ml-1 (Figure. 7). This protein content was increases on 6 h of each irradiation. The increase was 154 % under PAR and 205 % under PAR+UV-B exposure after 6 h irradiation. After 12 h protein content started decreasing in both type of irradiation and after long exposure protein concentration became stable however its content was lower in the sample receiving PAR+UV-B irradiation.

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Figure 7: Effect of PAR and PAR+UV-B treatment up to 48 h on protein contents of crude extract of S. geitleri (mean ± SD, n=3). Superoxide Dismutase Activity (SOD) SOD activity of control (untreated) sample was found 0.074 Unit/mg proteins (Figure 8). SOD activity was continuously decreases in the samples receiving PAR only radiation. At 24th h and 48th h of PAR exposure, 60% and 94% respectively decline in SOD activity was recorded. In contrast to PAR, a continuous increase in the SOD activity was recorded in samples receiving UV-B exposure only. At 12th and 24th h of UV-B exposure 3-5% increase was recorded, whereas at 48th h of exposure, 80% increased activity was recorded in comparison to the control sample. Samples receiving PAR+UV-B exposure, increasing trend was found in SOD activity, but less than the UV-B only exposure. At 6th and 12th h of exposure, SOD activity was increases 2-3%, at 24th and 48th h, 12% and 36% increase was recorded in comparison to control. SOD activity was increases under UV-B and PAR+UV-B exposure, highest under UV-B, however, under PAR SOD activity was decreases. Catalase (CAT) Activity In control sample Catalase activity was found 0.00193 Unit/mg proteins (Figure 8). Under PAR and PAR+UV-B exposure Catalase activity was increases throughout the exposure time. At 24 th and 48th h of exposure, Catalase activity was more than two fold higher than control. Samples receiving UV-B only exposure, Catalase activity was highest in compare to PAR only and Available Online at www.ijprbs.com

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PAR+UV-B exposure. At 6th, 12th, 24th and 48th h of UV-B exposure Catalase activity was 4 fold, 6 fold, 9 fold and 7 fold higher respectively with compare to control (0 h, Untreated). Catalse activity increases up to 24 h of UV-B exposure and then it fell down at 48 h of exposure. Ascorbate Peroxidase (APX) Activity In control APX activity was recorded 0.423 Unit/mg proteins (Figure 8). APX activity was continuously increases irrespective of the irradiation and exposure time. At 6 th and 12th h of PAR exposure, APX activity increases 22% and 68% respectively and 24 th and 48th h of PAR exposure APX activity was more than two fold in comparison to control sample. Under PAR+UVB exposure up to six and twelve h, APX activity increases 36% and 95% and at 24th and 48th h APX activity was more than two fold higher than control (0 h).

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Figure 8: Effect of PAR and PAR + UVB radiation on enzymatic activities of ascorbate peroxidase in S. geitleri (mean ± SD, n=3).

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DISCUSSIONS Effect of UVR on photosynthetic pigments As per the finding through the present experiments, total phycobiliprotein was decreases in all type of radiation with highest (81%) decrease was found in PAR+UV-B exposure than under PAR (48%) and in UV-B only irradiated sample 38% loss was recorded. Among three phycobiliproteins, C-PC was found most affected by the irradiation and 88% of loss was recorded in samples receiving PAR+UV-B radiation at 48th h of exposure. On short period of exposure (6-12 h) C-PC loss was lower than the C-PE and APC but on longer (24 & 48 h) period of exposure C-PC loss was higher than the C-PE and APC. Photosynthetic apparatus is not only damaged by UV-B but also by the PAR. The results obtained in the present experiments shows that PAR+UV-B is more affecting the PBP than the PAR and UV-B alone. Chlorophyll a concentration was increases on short period (6 h) of UV-B exposure in S. geitleri then it fell down on 12th h of UV-B exposure up to the concentration found in control sample and this concentration was maintained on longer duration of UV-B exposure. Chlorophyll a content is affected by the radiation for the short duration and on longer exposure cyanobacterium is able to maintain the chlorophyll a content up to the level of control. PAR, UV-B and PAR+UV-B is also affects fluorescence property of C-PC. A decreasing trend in the fluorescence emission of C-PC was observed. On longer exposure fluorescence emission lowest in PAR+UV-B, than to PAR and under UV-B emission was lowered in comparison to control but higher than that of PAR/+UV-B. It suggests on longer exposure of UV-B radiation, cyanobacterium S. geitleri is able to accumulate C-PC providing protection against the oxidative damage developed by the UV-B by absorbing the excess light. Phycobiliprotein is reported to play non-enzymatic antioxidant in cyanobacteria. Results obtained shows that UVR is affecting the phycobiliprotein, Cholorophyll and protein quantitatively and qualitatively, which can leads to impaired photosynthetic activity that finally cause the decreased biomass production of cyanobacteria. Likewise many other properties of cyanobacteria i.e. nitrogen fixation is also affected. While harvesting solar energy cyanobacteria are simultaneously exposed to UVR and affected by the potentially damaging UV-B radiation. Therefore, phycobiliproteins besides their accessory light harvesting property also helping cyanobacteria to withstand/ protection from oxidative stress develop by UV-B and high light stress. UV-B exposure decreases the photosynthetic pigments; this may be due to the generation of ROS. Pigments are highly sensitive to oxidation and peroxidation reactions [23, 24, 25, 26]. Chlorophyll, Phycobiliprotein and quinine which are chromophoric compounds absorb UV-B radiation and photosensitize the generation of ROS that will lead to loss of pigment due to bleaching [27] [28]. Algal response to UV-B and decrease in photosynthetic pigment has been reported [29] [30] [31]. D1 protein of Photosystem II identified as main target of UV-B radiation leading to photoinhibition in Available Online at www.ijprbs.com

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photosynthetic microorganism[32,33]. Photosystem I is almost insensitive to UV-B damage. Carbon fixation (Rubisco enzymes) is also affected by the UV-B radiation in some cyanobacteria. Overall the UV-B radiation is affecting growth, survival, metabolism and productivity of the Cyanobacteria and UV-B is combined with PAR, its damaging effect is enhanced. When cyanobacterium Oscillatoria boryana and Phormidium tenue was tested to see the effect of UV-B (5 Wm-1) on growth, protein and photosynthesis pigment content and it was found that longer duration of UV-B exposure lower the growth of culture and photosynthetic pigments contents (APC, C-PE, C-PC, carotenoids and Chl a). Phormidium tenue have higher survival capacity than Oscillatoria boryana to longer period of UV-B exposure [34]. In Anabaena variabilis PCC 7937 photosynthetic activity is severely inhibited by both high PAR and UVR after immediate exposure (10 min) due to damage in photosynthetic apparatus however damage is reversible after two h of irradiation [35]. Cyanobacterium Phormidium corium when exposed to UV-B and PAR at room temperature, decrease in photosynthetic activity and increased oxidative damage in terms of Fv/Fm ratio and lipid peroxidation of cell membrane was reported [36]. In Scytonema javanicum, a desert-dwelling soil microorganism, UV-B radiation adversely affect the photosynthesis and causes DNA damage due to increased ROS in cell; the level of damage varies with the intensity and duration of UV-B exposure. Exogenous chemicals like Nacetylcysteine and glyphosate reported to protect DNA damage and reduced the production reactive oxygen species in cyanobacterium Scytonema javanicum after UV-B exposure [37]. Antioxidative Enzyme Activity SOD, CAT and APX activity was measured after PAR and PAR+UV-B irradiation. In control sample APX activity was highest, SOD activity was next to APX and CAT activity was lesser than SOD and APX. Samples irradiated with PAR only radiation up to 48 h, APX and CAT activity was increases more than two folds and SOD activity was diminish up to 94% in comparison to control. Under PAR+UV-B exposure same increasing trend was found for all three enzymes, however for SOD less increase was recorded in compare to APX and CAT activity. Sample irradiated with PAR+UV-B radiation, APX and CAT activity increases much higher than SOD activity in comparison to control. Under PAR+UV-B irradiation up to 48 h, CAT activity was highest (7.6 fold) and APX activity was second highest (2.4 fold). SOD activity was lowest in compare to APX and CAT but higher than control. During oxidative stress ROS stimulate the activity of several antioxidative enzymes such as Superoxide Dismutase, Catalase, and Ascorbate Peroxidase (APX). High activity of these enzymes help cyanobacteria in stress tolerance [38]. There have been reported that SOD activities change dramatically in response to conditions that favour the formation of superoxide [39]. It is reported that O2- is the first ROS generated as a consequence of UV-B stress [40]. It has been reported that SOD plays an important role in protecting cyanobacteria from UV-B Available Online at www.ijprbs.com

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stress. The results obtained in our experiments also show increase in SOD activity under UV-B stress. In order to diminish the oxidative stress caused by ROS, cyanobacteria has developed efficient enzymatic and non-enzymatic scavenging systems. Non-enzymatic molecules consist of carotenoids, tocopherols, ascorbic acid and reduce glutathione and antioxidant enzymes such as Superoxide dismutase, Catalase, Glutathione Peroxidase and enzymes involve in ascorbate – glutathione cycle e.g. Ascorbate peroxidase, mono-dehydroascorbate reductase, dehydroascorbate reductase & glutathione reductage. In our experiments, we found that SOD, CAT and APX activity is higher in UV-B radiation to mitigate the cell damage from oxidative stress in S. geitleri. Among these, Catalase activity was highest under UV-B radiation. SOD activity was lowest in sample irradiated with PAR up to 48 h. At 48 h of irradiation, Catalase activity was higher in PAR+UV-B (112%) and 3.3 fold higher in UV-B only radiation in compare to PAR only irradiated sample of S. geitleri. Maintenance of high level of antioxidant enzyme is correlated with the tolerance against the different kind of stress mainly oxidative stress. SOD and CAT combined activity is important to reduce the oxidative stress. As per results obtained in present experiments, Catalase and APX is the main antioxidant enzymes in S. geitleri protecting against oxidative damage by converting hydrogen peroxide in to water and oxygen. After treatment under UV-B and PAR, cyanobacterium Phormidium corium shows little activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX)[36]. UV-B exposure induced the 2-5 fold higher antioxidant enzyme activity (SOD, CAT and APX) in two Anabaena species[41]. Phycocyanin and Allophycocyanin also reported to have antioxidative activities in scavenging free radicals and inhibiting lipid peroxidation in Nostoc commune with PC having stronger antioxidative activity than APC [42]. CONCLUSIONS From the above finding it is concluded that Phycobiliprotiens, Chlorophyll a and protein content decreases when UV-B radiation combined with PAR which means decrease in photosynthetic activity of cyanobacterium. Antioxidative enzyme activity has increases und PAR+UV-B exposure with highest increase of APX and CAT, which convert hydrogen peroxide to water and oxygen providing protection to Scytonema geitleri from the damaging effect of hydrogen peroxide. SOD activity was also enhanced under PAR+UV-B exposure but less than APX and CAT. SOD activity was decreases drastically under PAR only exposure. REFERENCES 1. Caldwell, M.M., Teramura, A.H. & Tevini, M. The changing solar ultraviolet climate and the ecological consequences for higher plants. Trends Ecol. Evol. 1989; 4: 363-367.

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