Introduction

The Gram-negative prokaryotes, the cyanobacteria constitute a heterogeneous assemblage of oxygen-evolving photosynthetic living organisms having cosmopolitan distribution (Stanier and Cohen-Bazire 1977). They appeared on the Earth in the time of the Precambrian period (2.8–3.5 × 109 years ago) and gave a favorable situation to the current oxidized atmosphere (Fischer 2008; Pathak et al. 2021). Cyanobacteria are significant biomass producers (Häder et al. 2007) and possess a central position in nutrient cycling of biological systems. Secondary metabolites derived from cyanobacteria have pharmaceutical qualities (Rastogi and Sinha 2009; Rajneesh et al. 2017a; Singh et al. 2020a, b). Apart from having potential for biotechnological applications in the field of biofertilizers, fuel, food, biomedicals and mariculture (Richa et al. 2011; Singh et al. 2016; Pathak et al. 2018), cyanobacteria serve as crucial model organisms in photosynthetic experiments, partly due to their prokaryotic cellular organization (Olsson-Francis et al. 2013).

The presence of the ozone layer in the stratosphere protects living beings on the Earth from the lethal ultraviolet radiation (UVR) (Weatherhead and Andersen 2006). This layer is being depleted tremendously due to anthropogenic air pollutants such as chlorofluorocarbons, organobromides, and chlorocarbons, prompting the ozone holes (Crutzen 1992). Increased sunlight based UVR is a significant stress factor for prokaryotic as well as eukaryotic phytoplankton (Manney et al. 2011; Richa et al. 2016). Cyanobacteria are exposed to deadly doses of ultraviolet-B (UV-B, 280–315 nm) and ultraviolet-A (UV-A, 315–400 nm) radiation in their common brightly lit habitats (Rajneesh et al. 2019). Although just a little UV-B (under 1% of the gross solar radiation) reaches to the Earth's surface, it is extremely damaging for biological system as it can be absorbed by significant biomolecules such as nucleic acids, lipids and proteins ultimately having an adverse impact on living system (Karentz et al. 1991; He and Häder 2002a; Jantaro et al. 2011; Rajneesh et al. 2019). The UV-B radiation with high energy has considerable potential for cell impairment with direct consequences to DNA and proteins and indirect impacts via the generation of reactive oxygen species (ROS) (Rajneesh et al. 2019; Ahmed et al. 2021a; Singh et al. 2022). UVR effectively impacts different life processes in cyanobacteria, such as survival, growth and development, morphology, pigmentation, cell separation (differentiation), motility and orientation, phycobiliprotein composition, N2 fixation, protein profile, CO2 uptake and DNA damage (Gao et al. 2007a, b; Lesser 2008; Kannaujiya and Sinha 2015; Singh et al. 2017a, b; Pathak et al. 2019; Kumar et al. 2020).

In aquatic environments cyanobacterial blooms are a major cause of concern and occur frequently worldwide. The process of bloom formation is aggravated by enhanced eutrophication of water bodies (Shao et al. 2009; Mayer et al. 2011), leading to the deterioration of water quality as these algae produce toxins, scum, hypoxia and also result in bad tastes and odors of the water (Codd et al. 2005). Hence, controlling these algal blooms is an important area of research and naturally occurring allelochemicals could serve as better approach for controlling harmful cyanobacterial blooms compared to physical and chemical methods because of their environment friendly nature (Melzer 1999; Vanderstukken et al. 2011). Myriophyllum spicatum has significant inhibitory effects on the major bloom and toxin producing cyanobacterium Microcystis aeruginosa allelopathically (Nakai et al. 2000). Four allelochemicals which were polyphenol in nature, namely, pyrogallic acid (PA), ellagic acid, gallic acid and ( +)-catechin were identified in the culture solution of M. spicatum. Studies showed that these polyphenolic compounds exert their inhibitory effects on algae by suppressing the activities of alkaline phosphatase and attacking photosystem II (PSII) of M. aeruginosa leading to oxidative damage in the cells (Gross et al. 1996; Leu et al. 2002; Zhu et al. 2010; Wang et al. 2011). As a well-known biologically active compound with widespread occurrence in aquatic macrophytes, PA is regarded as the most promising of such compounds. However, there is a scarcity of information regading the mechanism of action of PA on cyanobacterial species together with exogenous supplements of mycosporine-like amino acids (MAAs).

Cyanobacteria are a valuable source of various natural products of medicinal and industrial importance (Sinha and Häder 2008; Rastogi and Sinha 2009; Rajneesh et al. 2017a; Singh et al. 2021) sythesising numerous secondary metabolites. MAAs, scytonemin, carotenoids and a few other UV-absorbing substances have been recognized from different groups of organisms (Pathak et al. 2017, 2020; Pandey et al. 2020; Ahmed et al. 2021a, b). MAAs absorb essentially in the UV-B (280–315 nm) and UV-A (315–400 nm) range, and this helps these organisms to develop and survive in environments exposed to intense solar radiation (Sinha and Häder 2008; Singh et al. 2010; Rastogi and Incharoensakdi 2013; Rastogi et al. 2014). The distribution of MAAs varies from tropical to polar regions in various groups of organisms (Shick and Dunlap 2002), but these compounds are typically present in organisms such as cyanobacteria and other prokaryotes and eukaryotic microorganisms such as microalgae, fungi, lichens, marine macroalgae, corals, and other marine organisms which are exposed to UVR/high-intensity radiation (Shibata 1969). Some of these organisms accumulate MAAs from their feed via the food chain (Sinha et al. 2007; Pandey et al. 2017). In terms of biosynthesis, accumulation, and metabolism of MAAs, the Nostocales is the most investigated group (Jain et al. 2017). Furthermore, until now no MAAs have been reported in the cyanobacterial orders Gloebacterales, Chroococcidiopsidales, Pleurocapsales and Spirulinales (Jain et al. 2017). Techniques, such as, ultraviolet–visible (UV–VIS) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI–MS) are quick and high-resolution separation detection systems, with high-throughput (Dunn et al. 2005). High-performance liquid chromatography (HPLC)-based profiling of MAAs in cyanobacteria is well documented (Matsui et al. 2011; Rastogi and Incharoensakdi 2014; Hu et al. 2015). NMR spin-relaxation times data after NMR spectroscopy help in the characterization of MAAs (Burja et al. 2003). Torres et al. (2006) have reported the structure and molecular formula of porphyra-334 (P-334) by the application of mass spectroscopy (MS) in conjunction with 1H and 13C NMR data. Moreover, different functional groups in P-334 were determined by FTIR analysis of the purified MAA (Richa and Sinha 2015).

MAAs show stability under various abiotic stressors, such as, heat, UV-B, H2O2, and pH (Rastogi et al. 2016), which makes them suitable to be used as a sunscreen. The wide diversity,distribution, photoprotection and physicochemical stability of MAAs make them an important class of UV-screening compounds and future investigations must be focused on biosynthesis, profiling, and characterization of these sunscreens.

In the present study we screened and characterized MAAs in two cyanobacteria, Fischerella sp. HKAR-5 and Anabaena sp. HKAR-7, from different habitats. Furthermore, we assessed the stability, antioxidant and ROS scavenging potentials of selected MAAs as well as their role in reduction of UVR-induced formation of ROS. We also studied the effects of P-334 against different doses of strong allelochemical PA in Anabaena sp. HKAR-7. Induction and screening of UV-protective compounds (MAAs) was studied in both cyanobacteria under different combination of exposure to photosynthetically active radiation (PAR), PAR + UV-A, PAR + UV-B and PAR + UV-A + UV-B radiation.

Materials and methods

Culture methods and maintenance

BG-11 (-) medium (BG-11 minus nitrogen) was used for the culture of the cyanobacteria (Rippka et al. 1979). The cultures were regularly grown under axenic conditions, in a culture room at 28 ± 2 °C, under fluorescent white light (12 W m–2). The cultures were hand-shaken 3–4 times a day for avoiding clumping and shelf shading.

Growth measurement

Growth of cyanobacteria was determined by determining the changes in chlorophyll a (Chl a) content and optical density (O.D.) at 750 nm.

UV irradiation and exogenous supplements

The test cultures were exposed to artificial UVR in a chamber designed with UV-tubes (UV-chamber). During UVR exposure, the test samples were simultaneously irradiated with cool white fluorescent light (12 ± 1.0 W m−2). The homogeneous cultures (250 mL of culture in each Petri dish with OD750 nm = 0.68 ± 0.5; path length 1 cm) were treated with UVR or PA in Petri dishes (replicate of three) for the desired time intervals. Petri dishes were covered with 395, 320 and 295 nm cut-off filter foils (Ultraphan; Digefra, Germany) to ensure PAR, PAR + UV-A, PAR + UV-B and PAR + UV-A + UV-B radiation exposure respectively. A constant temperature of 23 ± 2 °C was maintained in all experimental cultures in UV-chamber to avoid heating effects. The cultures were shaken continuously during experiments. White fluorescent light illuminated samples served as control. All the trials were carried out in the exponential phase of cultures with OD750 of ~ 0.75 to 0.85.

Percent survival

For estimation of survival percentage, 100 µL aliquots from each treated culture were withdrawn at the desired time and plated on agar plates. Treated plates were incubated in the dark for 48 h and then transferred to light in the culture room. After 12–15 days of growth colonies were counted and percentage survival was determined.

Protein estimation

Protein estimation was according to Bradford (1976). Bovine serum albumin (BSA) was utilized as the standard.

Pigments estimation

The chlorophyll (Chl a) was extracted in methanol. The harvested cyanobacterial sample was kept in 100% methanol for overnight in the dark at 4 °C and the Chl a content was determined according to Porra (2002):

$$\mathrm{Chl}\;\mathrm{a}\left({\mu \mathrm{g} }\;{\mathrm{mL}}^{-1}\right)=16.29*{A}_{665.2}-8.54*{A}_{652}$$

Carotenoid content was determined following the method of Jensen (1978) with slight change. In brief, homogenized culture suspension was centrifuged at 5000 × g for 10 min and the supernatant discarded. The macerated pellet was placed in 85% acetone and incubated overnight.Carotenoids were determined by measuring the O.D. at 450 nm. For estimation of phycocyanin (PC), cyanobacterial cells were harvested by centrifugation (5000 × g for 10 min) at room temperature and cells were washed with 50 mM phosphate buffer (pH 7.0). Thereafter, cells were resuspended in a minimal amount of the same buffer with addition of 1 mM phenylmethanesulfonylfluoride (PMSF), 10% (w/v) EDTA and 5% (w/v) sucrose. Cells were sonicated for 3–5 min and the resulting suspension was subjected to repeated freeze–thaw cycles at -20 and 4 °C, respectively. The cell debris was removed by centrifugation at 15,000 × g for 30 min and the supernatant was considered as partially purified phycobiliproteins. Spectra were recorded at 200–700 nm against phosphate buffer. The PC content was calculated using equation described by Bryant et al. (1979).

$$\mathrm{PC}\left({\mathrm{mg\;mL}}^{-1}\right)=\left\{\left({\mathrm{A}}_{620}-\left(0.7*{\mathrm{A}}_{650}\right)\right.\right\}/7.38$$

Photosynthetic activity assay

The pulse amplitude modulation (PAM) fluorometer technique was used to determine the maximum photochemical efficiency (quantum yield) of open reaction centre (RC)IIs (Fv/Fm) by using PAM fluorometer (PAM-2500, Heinz Walz GmbH, Germany). The treated cyanobacterial samples were dark-adapted for 30 min to allow complete oxidation of PSII reaction centers and the minimum (F0) and maximum (Fm) fluorescent yields of PSII in the dark-adapted state were determined and used to calculate the Fv/Fm by the formula as described by Genty et al. (1989) and (Cosgrove and Borowitzka 2011). Estimation of the maximim relative electon transport rate (rETRmax) was done from the operational PSII photochemical yield measured at different photosynthetic photon flux densities (PPFDs) (Cosgrove and Borowitzka 2011).

$${\mathrm{rETR}}_{\mathrm{max}}={\mathrm{F}}_{\mathrm{q}}^{\mathrm{^{\prime}}}/{\mathrm{F}}_{\mathrm{m}}^{\mathrm{^{\prime}}}\times 0.5\times \mathrm{PPFD}$$

where Fq′/Fm′ denotes the effective photochemical efficiency of reaction center II (RCII) in actinic light.

MAAs extraction and partial purification

Extraction and purification of MAAs was done by UV–Vis spectroscopy and HPLC as per (Rastogi and Incharoensakdi 2013) with slight modification. Briefly, irradiated cyanobacterial samples were centrifuged and pellets were extracted in 5.0 mL of 100% (v/v) HPLC-grade methanol and incubated overnight at 4 °C. Thereafter, the aliquots were centrifuged (8,000 × g for 10 min) and the supernatant (methanolic extract) was spectroscopically analyzed (UV–Vis 2900, Hitachi, Japan). The methanolic extracts were dried at 45 °C and re-dissolved in 1 mL double distilled water and again absorption spectra were recorded. The samples were filtered through 0.2 μm pore-sized small-scale centrifuge filters for HPLC examination (Waters 2998, pump L-7100, Photodiode Array, USA with Waters, Spherisorb diagnostic column, 5 μm, 4.6 × 250 mm diameter, a Licrospher RP 18 column and guard. MAAs assessment was completed by injecting the samples into the HPLC through a Waters 717 plus autosampler. The mobile phase was 0.02% acetic acid (v/v) in double distilled water at a flow rate of 1.0 mL min−1 (Richa and Sinha 2015). The MAAs was identified by comparing previously published information dependent on absorption spectra and RT. The extracted MAAs were analyzed and separated by using the HPLC system and subsequently, the partially purified MAAs were subjected to characterization, stability and antioxidant function assays.

Biochemical characterization of MAAs

Electrospray ionization-mass spectrometry (ESI–MS)

MAAs collected from HPLC were subjected to ESI–MS to produce protonated molecules. Mass spectra were recorded on an Amazon SL mass spectrometer (Bruker Daltonics Inc., Germany). Cone voltage of 30 V was found to induce the formation of (M + H)1+ with a mass range of 100–1,000 m/z. Data were analyzed using the software Data Analysis 4.0 (Bruker Daltonics Inc).

Fourier transform infrared (FTIR) spectroscopy

MAAs isolated from the HPLC system werelyophilized and combined with oven-dried potassium bromide in a 1:100 ratio. Transparent disk was prepared for FTIR examination and spectra were recorded in a Perkin Elmer Infrared Spectrophotometer version10 (USA).

Nuclear magnetic resonance (NMR) spectroscopy

The purified MAAs were dissolved in 5 mg mL−1 D2O (isotopic purity: 99.5 atoms) after lyophilization. Room temperature 1H and 13C NMR spectra were recorded with a With the assistance of theJEOL AL300 FTNMR spectrometer (JEOL Ltd., Tokyo, Japan).

CHNS analysis

The composition ratio of organic carbon, hydrogen, and nitrogen was determined in a CHNS Elemental Analyzer (EURO EA 3000, EuroVector S.P.A., Italy).

Stability of MAAs

Stability of MAAs was observed under different abiotic stressors such as UVR (PAR, PAR + UV-A, PAR + UV-A + UV-B), heat (-20, 4, 25 and 60 °C), pH (1, 3, 7, 9 and 12) and H2O2 (strong oxidizing agent) (0.1, 0.25 and 0.50%). The purified MAAs was treated with different pH and variable concentration of H2O2 for 5 h and their effects were analyzed at 1, 3 and 5 h intervals. For the heat treatment, MAAs were exposed at variable temperature until 45 days of exposure.The treated MAAs samples were assessed after 15, 30 and 45 days of exposure. In the radiation experiment, purified MAA were exposed to different combinations of radiation by using different cutoff filters of 395 nm (PAR), 320 nm (PAR + UV-A) and 295 nm (PAR + UV-A + UV-B) for 7 days in the UV-induction chamber. Samples were analyzed at of 1, 3, 5 and 7 days of exposure. A sample without any exposures served as control.

Free radical scavenging activity of MAAs

The antioxidant capacity of purified MAAs, P-334 and MG-310 at different diluted concentrations (stock 100 μg mL−1) of 0, 20, 40, 60, 80 and 100 μL was measured by performing DPPH, SRSA and RP assaya. Determination of free radical scavenging capacity of purified MAAs was done in comparison to ascorbic acid (AA).

2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay

The free-radical scavenging activities by DPPH assay were assessed according to Kulisic et al. (2004) and Rastogi et al. (2016) with slight changes. Briefly, both MAAs, P-334 and MG-310 with different doses were kept with 0.1 mM DPPH in 80% methanol for 1 h in the dark at room temperature. The sample without any MAAs concentration (only DPPH solution) served as control. The optical density of each reaction mixture was determined at 517 nm. The DPPH radical scavenging potential (%) for each concentration of test samples of MAAs were determined using the equation (Rastogi et al. 2016):

$$\mathrm{DPPH}\;\mathrm{radical}\;\mathrm{scavenging}\;\mathrm{capacity}\;\left(\%\right)=\left[\left({\mathrm A}_1-{\mathrm A}_2\right)/{\mathrm A}_1\right]\times100$$

where A1 = absorbance at 517 nm of control (DPPH solution) and A2 = absorbance at 517 nm of samples (MAAs + DPPH).

Superoxide radical scavenging activity (SRSA) assay

The SRSA assay was performed by the modified method of Li (2012) and Rastogi et al. (2016). SRSA action depends on pyrogallol autoxidation. The percentage of SRSA was determined by the equation:

$$\mathrm{SRSA }\left(\mathrm{\%}\right)=\left[\left({\Delta \mathrm{A}}_{1}\right)-\left({\Delta \mathrm{A}}_{2}\right)/\left({\Delta \mathrm{A}}_{1}\right)\right]\times 100$$

where ΔA1 = change in the absorbance (A325) of control and ΔA2 = change in the absorbance(A325) of samples.

Reducing power (RP) assay

Purified MAAs were used to determine their reducing potential by the methods of Oyaizu (1986) and Rastogi et al. (2016). Ascorbic acid was utilized as standard. Various concentrations of MAAs (P-334 and MG-310) were mixed with buffer solution (200 μL) of 0.2 M sodium phosphate at pH 6.6 containing 1% potassium ferricyanide. The mixture was incubated and then 10% trichloroacetic acid (200 μL) was added. An equivalent quantity of water was mixed with the reaction mixture and 120 μL of 0.1% ferric chloride was added. After 10 min reaction, the absorbance was recorded at 700 nm.

ROS detection by fluorescence spectrophotometry and fluorescence microscopy

The fluorimetric probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) was utilized to observe the ROS level in the cyanobacterial cells (Rastogi et al. 2010). DCFH-DA at a concentration of 5 μM was used. The fluorescence intensity of the treated samples was determined with a a fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies) with an excitation wavelength of 485 nm and an emission band at 525 nm at room temperature. To observe fluorescence the treated samples were analyzed by utilizing a Nikon eclipse Ni fluorescence microscope equipped with NIS-Elements (BR) imaging software. Fluorescence intensities of green (G) and red (R) were acquired for arbitrarily chosen regions on the cyanobacterial filaments. The ratio G/R and fluorescence intensity was observed for the chosen region by using the software provided by the manufacturer (Rajneesh et al. 2017b).

Determination of antioxidant enzyme activity

To assay the changes occurring in the concentration of antioxidant enzymes during course of experiment, cell extracts were prepared after sonication in an extraction mix containing 50 mM phosphate buffer (pH 7.5), 2.5 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM ethylenediaminetetraacetic acid (EDTA) and 1% (w/v) polyvinylpyrrolidone (PVP). The extraction suspension was sonicated (Sonic and Materials, USA) and centrifuged at 10,000 × g for 25 min at 4 °C. The supernatant was used for the analysis of enzymes.

Catalase (CAT) activity

CAT activity was determined by the method of Rao et al. (1996). Reaction mixtures contained 2.86 mL phosphate buffer (pH 7.5), 4.4 mM H2O2 and 100 μL of enzyme extract. CAT activity was observed by recording absorbance at 240 nm. CAT activity measures the extent of O2 release from enzymatic dissociation of H2O2 in darkness for 5 min.

Superoxide dismutase assay (SOD)

SOD assay measures the inhibition in the photochemical reduction of nitroblue tetrazolium (NBT). The reaction product was measured at 560 nm (Donahue et al. 1997). The reaction mixture of 2.1 mL contained 1.5 mL, 0.1 mM EDTA, 13 mM Met, 75 μm nitroblue tetrazolium and 2 μm riboflavin in 50 mM phosphate buffer (pH 7.8) and 600 μL of cell extract. Riboflavin was added last and the reaction was initiated by placing the tubes under two 15 W m−2 fluorescent lamps. The reaction was terminated after 10 min by removal from the light source.

DNA extraction and gel electrophoresis

DNA extraction was by the method of Sinha et al. (2001a). Briefly, cyanobacterial pellet was washed 2–3 times with of STE buffer (1 mL) (50 mM Tris–HCl (pH—8.0) + 50 mM NaCl + 5 mM EDTA) subsequently re-suspended in a TE (500 μL) buffer (50 mM Tris–HCl (pH—8.0) + 50 mM EDTA). Buffer containing pellet was sonicated for 5 min on ice. Then the cyanobacterial filaments were treated with proteinase K (100 μg mL−1). The extraction buffer consists of 3% (w/v) CTAB + 1% (w/v) sarkosyl + 20 mM EDTA + 1.4 M NaCl + 0.1 M Tris–HCl, pH—8.0 + 1% (w/v) 2-mercaptoethanol). Prewarmed extraction buffer (750 μL) was mixed and the sample incubated for 1 h at 55 °C in a water bath. Then formed suspension was cooled for few minutes and then chilled chloroform: isoamyl alcohol (24:1, v/v) of equivalent volume was added and gently mixed to form an emulsion. The emulsion was centrifuged (10,000 × g for 10 min) and the supernatant was transferred to sterile microcentrifuge tubes. Subsequently, ethanol and 3 M sodium acetate (pH—5.2) in ratio of 2 volumes and 0.1 volume were added, mixed gently and kept overnight at -20 °C for DNA precipitation. The the sample was centrifuged at 8,000 × g for 15 min at 4 °C and the supernatant decanted. The pellet was rinsed once with chilled 70% ethanol, dried and re-hydrated with 30 mL TE buffer (10 mM Tris–HCl (pH—8.0) + 1 mM EDTA). DNA purity was assessed by the A260/A280 ratio (pure DNA preparations have A260/A280 value of 1.8). DNA integrity was checked by sample (5 μL aliquot) resolved on agarose gel (0.8%). Equivalent measures of DNA stained with Gel Red were separated by electrophoresis on a 1.5% TBE agarose gel at 60 V for 90 min, and visualization of bands were done by utilizing the Bio-Rad Imaging System.

Malondialdehyde (MDA) content

MDA content was measured using the protocol described by Chen et al. (2009). Briefly, centrifuged cultures were homogenized with 10 mL 10% trichloroacetic acid (TCA). Then the culture was centrifuged at 6000 × g for 15 min and the supernatant (2 mL) was added to 2 mL 0.6% (w/v) thiobarbituric acid (TBA) and incubated in warm bubbling water for 12 min. The sample was again centrifuged at 10,000 × g for 10 min and the spectrum of the supernatant was recorded.

dsDNA breaks

DNA strand breaks were determined by fluorometric investigation of DNA unwinding (FADU) as descrtibed by He and Häder (2002b).

Results

UV-induced biosynthesis of photoprotective compounds (MAAs)

The cyanobacteria Anabaena sp. HKAR-7 and Fischerella sp. HKAR-5 were irradiated under different combinations of radiation i.e., PAR, PAR + UV-A, PAR + UV-B and PAR + UV-A + UV-B up to 72 h. In both cyanobacteria PAR + UV-A, PAR + UV-B and PAR + UV-A + UV-B were more effective wavebands for MAAs biosynthesis.

UV–Vis spectra and HPLC chromatogram of partially purified MAAs

Figure 1 depicts the absorption spectra of the partially purified MAAs. The HPLC chromatogram of MAAs from Anabaena sp. HKAR-7 showed a single prominent peak at RT 3.21 min with UVλmax 334 nm (Fig. 2a, c) and Fischerella sp. HKAR-5 showed three peaks at RT 1.16, 2.18 and 3.14 min with UVλmax 310, 332 and 334 nm, respectively (Fig. 2b, d).

Fig. 1
figure 1

UV–Vis absorption spectra of partially purified MAAs from Anabaena sp. strain HKAR-7 and Fischerella sp. HKAR-5

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Fig. 2
figure 2

HPLC chromatograms of MAAs and their corresponding absorption spectra in Anabaena sp. HKAR-7 (a and c) and Fischerella sp. HKAR-5 (b and d)

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ESI–MS analysis

The HPLC separated fractions of each prominent peak were subjected to ESI–MS analysis. ESI–MS analysis revealed a prominent ion peak of protonated molecules [M + H]+ at m/z 346.9 (Fig. 3a) from Anabaena sp. HKAR-7 and 333.1 (Fig. 3b), 246.4 (Fig. 3c) and 303.2 (Fig. 3d) from Fischerella sp. HKAR-5. Based on UV–Vis absorption spectra and MS, the single MAA, porphyra-334 (P-334) (λmax: 334 nm, m/z346.9) was tentatively identified from Anabaena sp. HKAR-7. However, three MAAs, shinorine (λmax: 334 nm, m/z333.1), mycosporine-glycine (MG-310) (λmax: 309.6, m/z246.4) and palythinol (λmax: 332, m/z303.2) were identified in Fischerella sp. HKAR-5 (Table 1). Among the five identified MAAs, the peak for P-334 from Anabaena sp. HKAR-7 and major peak for MG-310 from Fischerella sp. HKAR-5 were used for further characterization.

Fig. 3
figure 3

Electrospray ionization-mass spectrometry of HPLC-purified MAAs. Porphyra 334; P-334 (a), shinorine (b), mycosporine glycine; MG-310 (c) and palythinol (d) showing peaks with m/z value of 346.9 (a), 333.1 (b), 246.4 (c) and 303.2 (d) respectively

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Table 1 List of MAAs extracted from Anabaena sp. strain HKAR-7 and Fischerella sp. strain HKAR-5
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FTIR analysis

Purified and lyophilized MAAs samples were subjected to FTIR analysis to determine the presence of the functional groups. The FTIR wave motif of P-334 (from Anabaena sp. HKAR-7) demonstrated 3377.44, 2926.38, 2855.11, 2083.19, 1633.53, 1407.50, 1208.58, 1048.51 and 616.88 cm−1 bands indicating the presence of iminocyclohexene ring moiety, the center of MAAs (Takano et al. 1978) and indicated comparability with recently published reports for P-334 (Oyamada et al. 2008; Richa and Sinha 2015) (Supplementary Fig. 1a). Similarly, FTIR wave pattern of MG-310 (Fischerella sp. HKAR-5) gave frequency pattern with bands of 3403.10, 2925.26, 2854.47, 2270.23, 1743, 1628.04, 1461.60, 1074.79 and 606.29 cm−1 (Supplementary Fig. 1b) indicated the presence of MAA that resemble with MG-310 and quite similar to early published data (Singh et al. 2017a). Bands of 3377.44 cm−1/3403.10 cm−1 might be of -OH functional group, 2926.38/2925.26 cm−1 for side-chain vibrations comprising of C-H stretching and showing the existence of -NH2 functional group and 1633.53/1628.04 and 1407.50/1461.60 cm−1 bands might be assigned to the -NH2 and carboxylic functional group, respectively. However, some additional different wave frequencies were also present in the P-334 wave pattern which make it different from MG-310.

Nuclear magnetic resonance (NMR) spectra analysis

The NMR spectra of purified fraction of MAAs, P-334 are shown in Supplementary Fig. 2a and b, and for MG-310 are shown in Supplementary Fig. 3a and b. The obtained data were correlated with recently published chemical shift information for P-334 (Oyamada et al. 2008; Yoshiki et al. 2009) and MG-310 (Singh et al. 2017a).

CHN analysis

Data obtained from the CHNS analyzer also supported the percentage composition of studied MAAs, P-334 and MG-310 (Table 2).

Table 2 Percentages of C, H and N of MAAs, P-334 and MG-310 (means ± SD, n = 3)
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Stability of MAAs

In vitro stability of P-334 and MG-310 was studied under different physicochemical stressors. However, both MAAs showed resistance to most of the treatments. Among different exposure of stressors such as radiation (Fig. 4a), pH (Fig. 4b), strong oxidizing agent H2O2 (Fig. 4c) and variable temperature (Fig. 4d), higher concentrations of H2O2 (0.50%) had the most detrimental effects at later duration of incubation (5 h). Both MAAs showed similar trends of stability under different temperatures i.e. -20 °C, 4 °C, 25 °C and 60 °C. However, a significant decrease in concentration of MAAs was observed in the samples exposed to higher temperature i.e., 60 °C at 45 days exposure. In the radiation experiment, PAR + UV-A + UV-B exposure was the most effective waveband combination which resulted in a decrease in MAAs concentration after 7 days exposure. MAA samples with higher doses of an oxidizing agent like H2O2 (0.25 and 0.50%) showed the most detrimental effects with an enhanced duration of exposure (3 and 5 h). P-334 and MG-310 were quite stable in slightly acidic as well as neutral pH, however, their stability was affected by acidic (pH = 1) as well as highly basic medium (pH = 12) with a higher duration of exposure.

Fig. 4
figure 4

Stability of porphyra-334 (P-334) and mycosporine glycine-310 (MG-310) under different abiotic stressors like radiations (Photosynthetically active radiation (PAR), PAR + UV-A, PAR + UV-A + UV-B) (a), pH (1, 3, 7, 9 and 12) (b), strong oxidizing agent H2O2 (0, 0.25 and 0.50%) (c) and temperature (-20, 4, 25 and 60 °C) (d). The error bars denote standard deviations of means (n = 3). Similar letters over bar represent homogeneous mean group (p > 0.05)

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In vitro antioxidant capacity of MAAs

DPPH assay

P-334 showed a dose-dependent DPPH-scavenging activity and the anti-oxidation was 20, 31, 42, 60 and 90% and MG-310 exhibited 18, 25, 32, 52 and 84% at the doses of 20, 40, 60, 80 and 100 μL−1 MAA, respectively. However, 98% anti-oxidation was found at 100 μL−1 of ascorbic acid (Fig. 5a). IC50 estimations of P-334 and MG-310 against DPPH-radical are shown in Table 3.

Fig. 5
figure 5

Free radical scavenging capacity of MAAs, porphyra-334 (P-334) and mycosporine glycine-310 (MG-310) by 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay (a) and superoxide radical scavenging activity (SRSA) assay (b), correlated with that of ascorbic acid (AA). The error bars denote standard deviations of means (n = 3). Similar letters over bar represent homogeneous mean group (p > 0.05)

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Table 3 IC50 estimations of P-334 and MG-310 and ascorbic acid for different oxygen species derived from various in vitro antioxidant tests. Data are expressed as means of percentage inhibition ± standard deviations of means (n = 3)
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SRSA assay

UV-induced ROS may have consequences for cyanobacteria. Superoxide radicals (O2−) assume a significant role in lipid-peroxidation and thus, the biological role of MAAs in limiting the superoxide radicals (O2−) was studied. The superoxide radicals scavenging percentage was 18, 29, 45, 58 and 70% for P-334 and 14, 25, 41, 54 and 67% for MG-310 at concentrations of 20, 40, 60, 80 and 100 μL−1 MAAs, respectively (Fig. 5b). The IC50 values of P-334 and MG-310 against SRS assay are shown in Table 3.

RP assay

Significant reducing capacity of MG-310 and P-334 was observed as indicated by the dose-dependent enhancement in the absorbance value at 700 nm (Fig. 6). The current outcomes demonstrated that MAAs act as strong antioxidative agent to prevent cellular damage occurring due to UV-induced ROS production.

Fig. 6
figure 6

Reducing potentials of porphyra-334 (P-334), mycosporine glycine-310 (MG-310) and ascorbic acid with their corresponding doses of 0, 20, 40, 60, 80 and 100 μL. The error bars denote standard deviations of means (n = 3). Similar letters over bar represent homogeneous mean group (P > 0.05)

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In vivo ROS scavenging capacity of MAAs

Significant increase in levels of ROS was observed upon exposure to UV-B radiation. Significant decrease (P < 0.05) in fluorescence level of 2′,7′-dichlorofluorescein (DCF) was observed in the MAAs-treated cells exposed to UV-B radiation (Fig. 7a, b). These results were further confirmed by fluorescence pictures of Anabaena sp. HKAR-7 and Fischerella sp. HKAR-5 under various exposure conditions, for example, UV-B, UV-B + P-334, UV-B + MG-310 and UV-B + P-334 + MG-310 after 12 h. Also, a diminished level of in vivo ROS in MAA-treated samples indicated the photoprotective capacity of MAAs against UV-induced oxidative stress. ROS scavenging capacity was observed maximum in the UV-B irradiated sample treated with P-334 and MG-310.

Fig. 7
figure 7

Fluorescence images of Anabaena sp. HKAR-7 (a) and Fischerella sp. HKAR-5 (b) after 12 h exposure of UV-B, UV-B + porphyra-334 (P-334), UV-B + mycosporine glycine-310 (MG-310) and UV-B + P-334 + MG-310. Fluorescence images showing the generation of intracellular reactive oxygen species (ROS) (green DCF fluorescence)

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Role of exogenous supplement of P-334 against pyrogallic acid (PA) in Anabaena sp. HKAR-7

PA-induced growth inhibition

The changes in percent survival of studied cyanobacterium were observed under different doses of PA (0, 5, 10, 20 and 50 mg L−1) for 24 h (Fig. 8). Percent survival was inhibited under exposure of PA and the inhibition was dose and time-dependent. The number of colonies that appeared without any exposure of PA at 0 h served as control (untreated) and were considered as 100%. Cell survival (%) considerably declined in all treated samples until 24 h of exposure and a maximum decrease of 61% was observed in 50 mg L−1 PA treated culture at 24 h. Moreover, approximately 49% of cell viability was reduced in 20 mg L−1 PA sample after 24 h.

Fig. 8
figure 8

Changes in percentage survival of Anabaena sp. HKAR-7 in response to different concentrations of pyrogallic acid (0, 5, 10, 20 and 50 mg L−1) for different duration of time. The error bar represents standard deviation of mean (n = 3)

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Effects of PA and MAA (P-334) on photosynthetic pigments

Chlorophyll a

The effect of 12 h incubation under 0, 2, 4 and 8 mg L−1 PA and PA + P-334 on photosynthetic pigments in Anabaena sp. HKAR-7 is shown in Fig. 9a. The results show varied Chl a concentration under exposed stress. The Chl a concentration reduced by 24% (0.94 μg mL−1), 52% (0.57 μg mL−1) and 68% (0.40 μg mL−1) in 2, 4 and 8 mg L−1 PA treated samples respectively after 12 h. However, this decline was comparatively less i.e., 11% (1.2 μg mL−1), 26% (0.90 μg mL−1) and 53% (0.81 μg mL−1) in 2, 4 and 8 mg L−1 PA + P-334 treated samples, respectively. Samples without PA and with P-334 had enhanced Chl a (1.35 μg mL−1) and (1.42 μg mL−1) as compared to control (1.23 μg mL−1) after 12 h.

Fig. 9
figure 9

Effects of pyrogallic acid with/without MAA, porphyra 334 (P-334) on Chl a (a) carotenoids (b) phycocyanin (c) and total protein content (d) in Anabaena sp. HKAR-7. Results are expressed as means of three replicates. Vertical bars indicate standard deviation of the means (n = 3)

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Carotenoids

Carotenoids content increased by up to 183% (54.80 μg mL−1) and 164% (50.78 μg mL−1) in the 2 mg L−1 PA and PA + P-334 treated samples respectively after 12 h. Thereafter, a decrease was recorded with increased doses of PA and maximum decrease was 32% (23.28 μg mL−1) in the 8 mg L−1 PA exposed samples. Carotenoid concentration was relatively high, i.e., 35.60 μg mL−1 (without PA treated sample) and 37.12 μg mL−1 (with P-334 treated samples) as compared to the control, i.e., 30.70 μg mL−1 after 12 h (Fig. 9b).

Phycocyanin (PC)

The PC content was adversely affected by PA and declined by 29% (0.17 mg mL−1), 60% (0.096 mg mL−1) and 73% (0.062 mg mL−1) in 2, 4 and 8 mg L−1 in the 12 h PA exposed samples (Fig. 9c). Under PA + P-334 exposure it was 16% (0.21 mg mL−1), 37% (0.15 mg mL−1) and 54% (0.12 mg mL−1) in 2, 4 and 8 mg L−1 PA exposed samples for 12 h.

Total protein

Total protein contents increased under 0 and 2 mg L−1, PA and PA + P-334 treatment as compared to the control (0.40 mg mL−1). Thereafter, a decrease of approximately 20% (0.33 mg mL−1) and 42% (0.23 mg mL−1) was observed in the 4 and 8 mg L−1 PA treated samples after 12 h (Fig. 9d).

Photosynthetic activity

Maximum photochemical efficiency (quantum yield) of open reaction centre (RC)IIs (Fv/Fm) and relative electron transport rate (rETRmax) of Anabaena sp. HKAR-7 were negatively affected by PA, whereas this effect was comparatively less in samples supplemented with exogenous P-334 along with PA (Table 4). The PA treatment causes considerable decrease in Fv/Fm by 22% (0.29), 50% (0.19) and 76% (0.09) upon 2, 4 and 8 mg L−1 PA exposure, respectively. Similarly, the decline in Fv/Fm was 13% (0.33), 26% (0.28) and 44% (0.21) upon 2, 4 and 8 mg L−1 PA + P-334 exposure, respectively. The rETRmax value declined up to 32%, 53%, and 77% after 2, 4 and 8 mg L−1 PA treatment, respectively. The decline of rETRmax percentage was comparatively less in samples treated with 2, 4 and 8 mg L−1 of PA + P-334.

Table 4 Effects of different concentrations of PA with/without exposure of MAA, P-334 on maximum quantum yield (Fv/Fm) and maximum ETR in Anabaena sp. strain HKAR-7. Values represent means ± S.D (n = 3)
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Superoxide dismutase (SOD) and Catalase (CAT)

The SOD and CAT activities increased in all treated samples as compared to the control (Table 5). SOD activity increases up to 152% (0.32), 204% (0.43), 271% (0.57) in 2, 4 and 8 mg L−1 PA treatments and 123% (0.26), 147% (0.31), 195% (0.41) in 2, 4 and 8 mg L−1 PA + P-334 treatments. Similar trends were also recorded in CAT activity. CAT activity was comparatively less, i.e., 138% (0.38 U mol min−1 mg−1 protein), 168% (0.49 U mol min−1 mg−1 protein), 203% (0.59 U mol min−1 mg−1 protein) in 2, 4 and 8 mg L−1 PA + P-334 treatments.

Table 5 Effect of different concentration of PA with/without exposure of MAA, P-334 on SOD and catalase activity in Anabaena sp. strain HKAR-7. Values represent means ± S.D, (n = 3)
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Detection of intracellular ROS level

A significant amount of ROS was generated under different concentrations of PA and PA + P-334 in Anabaena sp. HKAR-7 (Fig. 10a) 0, 2, 4 and 8 mg L−1 PA had more pronounced visible changes after 12 h, whereas treatment with PA (0, 2, 4 and 8 mg L−1) along with P-334 showed comparatively low fluorescence signals in the cell filaments. DCF and Chl a auto-fluorescence derived green and red ratio (G/R) was analyzed under 0, 2, 4 and 8 mg L−1 PA and PA + P-334 (Fig. 10b) that the G/R ratio increased with an increase in PA concentration in a dose-dependent manner. The G/R ratio was maximum i.e., 1.48 (495%) in 8 mg L−1 PA, whereas it was 0.90 (298%) in 8 mg L−1 PA + P-334 treated samples after 12 h (Fig. 10c).

Fig. 10
figure 10

Fluorescence images (a), fluorescence-based Green/Red (G/R) ratio (b), reactive oxygen species (ROS) generation (c) under different doses of pyrogallic acid along with or without MAA (porphyra-334; P-334) in Anabaena sp. HKAR-7. Vertical bars indicate the standard deviation of the means (n = 3)

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The MAA was utilized as antioxidant to distinguish the ROS scavenging activity incited by H2O2. High doses of PA exposure results in enhanced ROS generation. The ROS generation reached up to 385% (345 AU) and 512% (470 AU) in 4 and 8 mg L−1 PA treated samples, respectively, as compared to the control (89 AU). Comparatively decreased DCF fluorescence level was recorded in samples exposed to 4 and 8 mg L−1 PA + P-334 and it was 263% (237 AU) and 325% (292 AU), respectively, after 12 h.

Lipid peroxidation, DNA strand breaks and gel electrophoresis

Figure 11a and b show PA-induced DNA strand breaks and lipid peroxidation with and without P-334 exposure in Anabaena sp. HKAR-7. As the concentration of PA increased, DNA strand breakage and lipid peroxidation increased as compared to the control. Lipid peroxidation increased up to 150% (0.19 μmol MDA g−1 dry wt), 240% (0.30 μmol MDA g−1 dry wt), 338% (0.44 μmol MDA g−1 dry wt), 425% (0.55 μmol MDA g−1 dry wt) in samples exposed to 0, 2, 4 and 8 mg L−1 PA, respectively. Lipid peroxidation in 0, 2, 4 and 8 mg L−1 PA along with P-334 treated samples were 133% (0.17 μmol MDA g−1 dry wt), 176% (0.22 μmol MDA g−1 dry wt), 228% (0.29 μmol MDA g−1 dry wt), 334% (0.40 μmol MDA g−1 dry wt), respectively, after 12 h. A decreased dsDNA content of 43 and 60% was recorded in the 4 and 8 mg L−1 PA treated samples, respectively. Exogenous supplements of P-334 along with PA retain the dsDNA content with some extent. A positive relationship was seen between lipid peroxidation and ROS generation with respect to doses of PA in Anabaena sp. HKAR-7. Intensity of genomic DNA bands that depict the toxicity of PA in Anabaena sp. HKAR-7 is shown in Fig. 11c.

Fig. 11
figure 11

Effect of pyrogallic acid (PA) and PA + porphyra 334 (P-334) on lipid peroxidation (a) ds DNA break (b) genomic DNA banding pattern (c) in Anabaena sp. HKAR-7 after 12 h. Vertical bars indicate standard deviation of the means. (n = 3) C: Control; 1: 4 mg L−1 PA; 2: 4 mg L−1 PA + P-334; 3: 8 mg L−1 PA; 4: 8 mg L−1 PA + P-334

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Discussion

Cyanobacteria can survive in a wide range of harsh environments such as brightly lit habitats having high UV fluxes. The present investigation compares the MAAs profile in two cyanobacteria, Anabaena sp. HKAR-7 and Fischerella sp. HKAR-5, from diverse habitats such as rice field and rock. The role of MAAs as antioxidants, their ROS scavenging and reducing potential were also investigated. Based on UV–Vis absorption spectra, HPLC and ESI–MS data, the single MAA, P-334 was tentatively identified from Anabaena sp. HKAR-7. However, three MAAs, shinorine, MG-310 and palythinol were identified in Fischerella sp. HKAR-5. Occurrence and composition of MAAs may vary within or among species (Sonntag et al. 2007). Similarily in our finding the cyanobacterium inhabiting rocks (Fischerella sp. HKAR-5) produced more diverse forms of MAAs (Shinorine, MG-310 and Palythinol) as compared to the rice-field cyanobacterium Anabaena sp. HKAR-7 (Single MAA, P-334), explain its role in survial of cyanobacterium. Possibly, Fischerella sp. HKAR-5 growing on rocks face various abiotic stresses including desiccation and UV-B radiation and this could be the reason for the synthesis of diverse forms of MAAs in this cyanobacterium. In cyanobacteria, MAAs can bind to their cell wall (Ehling-Schulz et al. 1997) or can be distributed homogeneously within the cytoplasm (Garcia-Pichel and Castenholz 1993). As effective photoprotectants, MAAs provide broad band UV filtration to several terrestrial and aquatic organisms. It was found that MAAs prevent three out of ten photons from hitting the cytoplasmic targets and high concentrations of MAAs in cells provide approximately 25% more resistance to UVR (Garcia-Pichel et al. 1993). The presence of a higher concentration of MAAs in cyanobacteria highlights their role in stress management. Our results also support the inductive role of UV-B radiation on MAAs biosynthesis. Besides, exposure of PAR + UV-A + UV-B radiation has a more effective role in MAAs synthesis in both the studied cyanobacteria.

MG and the precursor of MAAs, gadusol act as antioxidants and prevent cellular damage occuring from ROS which are generated upon UVR exposure (Coba et al. 2009). Moreover, accumulation or biosynthesis of photoprotective compounds such as MAAs protects the cellular organelles from the deleterious impacts of UVR (Singh et al. 2010, 2022). The MAA P-334 is commonly found in the red alga Porphyra as well as in a number of cyanobacteria. However, information regarding its detailed chemical characterization is scarce and its exogenous application against strong allelochemicals such as PA has been performed for the first time. The presence of P-334, shinorine and MG-310 has been documented in different genera of cyanobacteria (Garcia-Pichel and Castenholz 1993; Sinha et al. 2001a, b; Torres et al. 2006; Singh et al. 2008a, b, 2010; Khanipour et al. 2015). In contrast to other MAAs, the presence of palythinol is reported for the first time in Fischerella sp. inhabiting rock habitats.

Photoprotection, physicochemical stability, wide distribution and diversity of MAAs make them an unique group of sun-screening compounds (Rastogi et al. 2016). Similarily, we observed that, two MAAs (P-334 and MG-310) were efficient stable metabolites that showed strong resistivity against different physicochemical stressors, such as temperature, UV-B, strong oxidizing agent (H2O2) and pH. Apart from the important role as sunscreen agents, MAAs also play role in several biological processes of organisms (Mason et al. 1998; Neale et al. 1998; Bandaranayake and Des Rocher 1999; Shick and Dunlap 2002; Oren and Gunde-Cimerman 2007) and their embryos (Adams and Shick 2001), such as osmotic regulation (Oren 1997; Portwich and Garcia-Pichel 1999; Sinha and Häder 2003; Kogej et al. 2006; Singh et al. 2008a, b; Waditee-Sirisattha et al. 2014), antioxidant and ROS scavenging properties (Rastogi et al. 2016), defense against oxidative and thermal stresses (Michalek-Wagner 2001; Shick and Dunlap 2002), and desiccation tolerance (Feng et al. 2012; Olsson-Francis et al. 2013). On the basis of photophysical and photochemical studies, it was found that MAAs absorb UVR and release it almost completely as heat, without the generation of ROS (Conde et al. 2000, 2004). MAAs help in maintaining the antioxidant defense system of the skin, similar to the expression of Hsp70 through its antioxidant activity (Coba et al. 2007a, b, 2009). Similarily in our study, the different antioxidant assays also showed that the studied MAAs, P-334 and MG-310, acted as strong antioxidants that quench ROS inside the cells. MAAs also decreased ROS generation as confirmed by in vitro incubation of both MAAs separately as well with their combination as also supported by fluorescence images of in vitro incubated cyanobacterial samples. Thus it can be concluded that MAAs are efficient and stable sunscreen compounds that act as strong antioxidants and ROS quenchers. It has been found that allelopathic compounds influence several life processes in phytoplankton, for example, cell division, photosynthesis, enzyme activity, water and minerals uptake, and signal transduction (Belz and Hurle 2004; Hong et al. 2009; Inderjit and Duke 2003).

PA is used commonly in many industrial and consumer products and is widely distributed in nature (Upadhyay et al. 2010; Avase et al. 2015). Despite its beneficial properties, PA-mediated toxicity has been a major concern. It has been reported that PA showed mutagenic effects and liver, lung, kidney and gastrointestinal tract were its major target organs (Upadhyay et al. 2010). Recently, PA as a strong allelochemical attracted attention. Release of the allelochemicals (secondary metabolite) by the submerged macrophytes is considered to be a way to inhibit the growth of phytoplankton (Gross 2003; Hilt and Gross 2008). As discussed previously, it was found that the inhibitory effect of PA against M. aeruginosa may involve oxidative stress (Wu et al. 2007), photosynthesis inhibition (Dziga et al. 2007; Zhu et al. 2010; Wu et al. 2013) and interfering the expression of antioxidative gene (Shao et al. 2009). Studies showed that DNA strands and the cell membrane were two targets of ROS induced by PA, and oxidative damage was an important mechanism for the toxicity of PA against M. aeruginosa (Lu et al. 2016). It might be possible that in natural conditions cyanobacteria protect themselves from the allelopathic effect of PA by synthesizing MAAs. The present study gives an insight about the possible role of P-334 as a defense strategy against PA in cyanobacteria. It also explores the allelopathy mechanism of PA in terms of lipid peroxidation (MDA formation) and denaturation of dsDNA (DNA bands) that may be helpful for controlling the harmful bloom forming cyanobacteria population and water quality deterioration. This allelopathy mechanism of action provides great promise for treating harmful algal blooms.

We also tried to assess the antioxidant and metabolic responses of Anabaena sp. HKAR-7 to PA, and exogenous supplement of P-334 in terms of photosynthetic pigments, photosynthetic efficiency, ROS generation, antioxidative enzymes, lipid peroxidation and DNA damage. Our observations revealed that higher doses (20 and 50 mg L−1) of PA exposure strongly inhibited survival of this cyanobacterium with increased exposure time. Wu et al. (2013) also found that 4 mg L−1 of PA significantly inhibited the growth of the cyanobacterium Cylindrospermopsis raciborskii F2. Similarly, photosynthetic pigments such as Chl a and PC content were adversely influenced under different doses of PA. Samples exposed to PA along with P-334 showed fewer damaging effects in comparison to those treated by PA alone. PC was found to more sensitive in comparison to Chl a under high doses (4 and 8 mg L−1) of PA.

Carotenoids and total cellular protein content increased under low doses of PA (2 mg L−1), whereas increased doses of PA (4 and 8 mg L−1) caused detrimental effects. It is possible that higher doses of PA exposure led to enhanced cellular ROS generation and membrane disintegration subsequently leading to impairment of photosynthetic pigments and protein content. It might be possible that PA-induced generation of ROS is reduced exogenous supplement of P-334. Photosynthetic inhibition and oxidative harm were seen as significant methods of action for the allelopathic impact of PA on C. raciborskii F2 (Wu et al. 2013). In our findings, the photosynthetic inducators, Fv/Fm and rETRmax also were affected adversely in all PA treatments.

PA exposure induces enhanced ROS generation in cyanobacteria which triggers the enzymatic antioxidant defense system increasing SOD and CAT activities (Wu et al. 2013). We also observes the existence of antioxidative responses (SOD and CAT activities) in Anabaena sp. HKAR-7 after PA exposure in a dose-dependent manner. However, PA + P-334 treated samples showed comparably less SOD and CAT activity. Therefore, it can be concluded that P-334 played a role in the reduction of PA-induced generated ROS.

We attempted to recognize the extent of ROS level in vivo by utilizing an oxidant-detecting probe DCFH-DA. In our study, cyanobacterial filaments were found to change their color from red to green as the intracellular ROS generation increased due to PA exposure. This shift was comparatively less in PA + P-334 exposed samples as also indicated by the G/R ratio Wu et al (2013) similarly found that high doses of PA repressed the growth of C. raciborskii F2 and brought about a change in the oxidative system and the expression of seven key genes (Wu et al. 2013).

In the present investigation, DNA strand breaks were enhanced as the dose of PA increased. Similar patterns were also seen in lipid peroxidation in Anabaena sp. HKAR-7 as shown by MDA values. Therefore, it can be concluded that extensive ROS formation causes lipid peroxidation (MDA formation) and denaturation of dsDNA (DNA bands). PA together with exogenous P-334 reduces the detrimental effects caused by ROS, therefore, MDA formation and unwinding percentage of dsDNA was comparatively low. This explains the crucial role of P-334 as antioxidant against cellular ROS under stressed conditions.