Characterization of UV-screening compounds, mycosporine-like amino acids, and scytonemin in the cyanobacterium Lyngbya sp. CU2555

Authors

  • Rajesh P. Rastogi,

    1. Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
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  • Aran Incharoensakdi

    Corresponding author
    1. Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
    • Correspondence: Aran Incharoensakdi, Laboratory of Cyanobacterial Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok-10330, Thailand. Tel.: +66 2 218 5422; fax: +66 2 218 5418; e-mail: aran.i@chula.ac.th

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Abstract

Ultraviolet-screening compounds from the cyanobacterium Lyngbya sp. CU2555 were partially characterized and investigated for their induction by UV radiation, stability under different abiotic factors, and free radical scavenging activity. Based on the high-performance liquid chromatography coupled with diode array detector and ion trap liquid chromatography/mass spectrometry analysis, the compounds were identified as palythine (UVλmax: 319 nm; m/z: 245), asterina (UVλmax: 330 nm; m/z: 289), scytonemin (UVλmax: 384 nm; mw: 544), and reduced scytonemin (UVλmax: 384 nm; m/z: 547). This is the first report for the occurrence of palythine, asterina, and an unknown mycosporine-like amino acids (MAA), M-312 (UVλmax: 312 ± 1 nm), in addition to scytonemin and reduced scytonemin in Lyngbya strains studied so far. Induction of MAAs and scytonemin was significantly more prominent upon exposure to UV-A + UV-B radiation. Both MAAs and scytonemin were highly resistant to some physicochemical factors such as UV-B, heat, and a strong oxidizing agent and exhibited strong antioxidant activity. These results indicate that the studied cyanobacterium may protect itself from deleterious short-wavelength radiation by synthesizing photoprotective compounds in response to harmful UV radiation.

Introduction

Cyanobacteria are the most primitive group of Gram-negative photosynthetic oxygen evolving prokaryotes that probably appeared on the Earth during the Precambrian era (between 2.8 and 3.5 × 109 years ago) when the ozone shield was absent (Tomitani et al., 2006), thus creating an oxygenic environment for the evolution of existing life forms (Fischer, 2008). They are ubiquitous in nature ranging from hot springs to the Arctic and Antarctic regions and are major biomass producers in both aquatic and terrestrial ecosystems (Häder et al., 2011). In recent years, cyanobacteria have gained much attention as a valuable source of biofuels (Parmar et al., 2011) and several industrially important bioactive secondary compounds of medicinal and agricultural values (Rastogi & Sinha, 2009). Mycosporine-like amino acids (MAAs) and scytonemins are well-known UV-absorbing/UV-screening compounds for their pivotal role in photoprotection of cyanobacterial cells (Rastogi et al., 2010a). MAAs are small (< 400 Da), colorless, water-soluble compounds composed of cyclohexenone or cyclohexenimine chromophores conjugated with the nitrogen substituent of an amino acid or its imino alcohol. More than 22 MAAs have been reported from taxonomically diverse organisms ranging from heterotrophic bacteria, lichens, cyanobacteria, fungi, microalgae/macroalgae as well as several animals (Sinha et al., 2007). The presence of MAAs in animals is supposed to be either accumulated via the food chain or synthesized by their symbiotic algal partner. Strong UV absorption maxima (310–362 nm), high molar extinction coefficients (ε = 28 100–50 000 M−1 cm−1), and resistance to several abiotic stressors support their photoprotective role. These compounds are capable of effectively dissipating absorbed radiation as heat without producing reactive oxygen species (ROS) (Conde et al., 2007).

Contrary to MAAs, scytonemin is exclusively synthesized by cyanobacteria (Garcia-Pichel & Castenholz, 1991). It is a yellow brown, lipid-soluble dimeric compound located in the extracellular polysaccharide sheath (Fig. 1) of some cyanobacteria and acts as a passive sunscreen in protection against UV radiation (Proteau et al., 1993). Scytonemin has been reported to exist in oxidized (Mw 544 Da) or reduced (Mw 546 Da) form that depends upon the redox and acid–base conditions during the process of extraction (Garcia-Pichel & Castenholz, 1991). Three new derivatives of scytonemin pigments such as dimethoxyscytonemin, tetramethoxyscytonemin, and scytonin have been reported from Scytonema sp. (Bultel-Poncé et al., 2004). Purified scytonemin has a maximum UV absorption at 384 ± 2 nm, although it also absorbs significantly at 252, 278 and 300 nm (Sinha et al., 1999; Rastogi et al., 2013). Different analytical techniques have been used to analyze the secondary bioactive compounds from cyanobacteria; however, high-performance liquid chromatography (HPLC) and mass spectrometry (MS) have been used extensively to analyze the MAAs (Whitehead & Hedges, 2002) and scytonemin (Squier et al., 2004).

Figure 1.

Cell morphology of Lyngbya sp. CU2555 observed under a light microscope. (A) dry Lyngbya mats (shown by arrow) on the bark of a rain tree. (B) filament of Lyngbya sp. inside the brown sheath. (C–D) filament of Lyngbya migrating out of its sheath opened at the ends. (E) Lyngbya filament with and without sheath. (F) empty sheath with orange-brown-colored scytonemin (shown by arrow).

Several abiotic factors such as different wavelength bands of UV radiation, desiccation, and nutrients and salt concentration have been found to affect the production of MAAs and scytonemin in cyanobacteria (Fleming & Castenholz, 2007, 2008; Singh et al., 2008; Rastogi et al., 2010b; Mushir & Fatma, 2012; Rath et al., 2012). Both MAAs and scytonemin may act as antioxidants, preventing cellular damage resulting from UV-induced production of ROS (Takamatsu et al., 2003; De la Coba et al., 2009; Matsui et al., 2012). Scytonemin is highly stable against different stressors and performs its UV-absorbing/UV-screening activity without any further metabolic investment. Moreover, due to their potential UV-absorbing/UV-screening capacity as well as several medicinal properties, MAAs and scytonemin may be biotechnologically exploited by pharmaceutical and cosmetic industries (Rastogi & Sinha, 2009). Biosynthesis of UV-absorbing compounds with photoprotective function is very limited or not known in the cyanobacterium, Lyngbya sp. Hence, the main objective of this study was to explore the UV-absorbing/UV-screening compounds of Lyngbya sp. under photosynthetically active radiation (PAR) as well as UV radiation and to evaluate their stability and photoprotective ability against damaging effects of UV radiation.

Material and methods

Experimental organism and growth conditions

The experimental organism Lyngbya sp. CU2555 (Fig. 1) was isolated from the bark of a rain tree Albizia saman (Jacq) Merr, Bangkok, Thailand. Based on morphological characteristics, this strain was identified with the help of standard taxonomic keys and monographs (Desikachary, 1959). It is a filamentous, nonheterocystous sheathed cyanobacterium. The morphology of the cyanobacterium was observed using a light (Seek model SK-100; Fig. 1) and scanning electron microscopy (JEOL model JSM-5410LV, Japan; Fig. 2). The surface of the Lyngbya mat is of dark brown color due to the high scytonemin content in the extracellular polysaccharide sheaths (Garcia-Pichel & Castenholz, 1991) (Fig. 1A). Axenic cells were screened and cultured in a liquid culture medium (Rippka et al., 1979) at 23 ± 2 °C with continuous white fluorescent light at 8 Wm−2. The initial experiments for analysis of UV-absorbing compounds were performed with both the microbial mats of Lyngbya sp. directly obtained from nature and axenic cultures to clarify the purity of the Lyngbya mats in terms of the presence of UV-absorbing compounds.

Figure 2.

Scanning electron microscopic structure of Lyngbya filament enclosed in a fairly firm sheath (A) and showing the disk-shaped cells (B and C) inside the sheath (D).

Extraction and partial purification of MAAs and scytonemin

The MAAs were extracted in 100% HPLC-grade methanol by overnight incubation at 4 °C. After extraction, aliquots were centrifuged (8000 g, 5 min), and supernatants were transferred to new Eppendorf tubes and subjected to evaporation at 38 °C using a vacuum evaporator (Cat. No. 797001, Labconco Corp., Missouri, MO). The residues were redissolved in 600 μL sterile double-distilled water followed by the addition of 100 μL chloroform with gentle vortexing. After centrifugation (8000 g, 5 min), the photosynthetic pigment-free uppermost water phase was transferred carefully into new Eppendorf tubes and filtered through sterilized microcentrifuge syringe-driven filter (13 mm, PVDF, 0.22 μm, Jet Biofil) to obtain the partially purified MAAs.

Scytonemin was extracted in methanol/ethyl acetate (1 : 1, v/v) by overnight incubation at 4 °C. After centrifugation (8000 g, 5 min), supernatants were evaporated in a vacuum evaporator and redissolved in 500 μL of 100% methanol. Subsequently, the samples were filtered as described above prior to the HPLC analysis.

Spectroscopic analysis

Spectroscopic analysis between 200 and 800 nm was performed using a UV/Vis spectrophotometer (U-2910, 2J1-0012, Hitachi, Tokyo, Japan). The raw data were transferred to a microcomputer, and peaks were analyzed with the software provided by the manufacturer.

HPLC and MS analysis of MAAs and scytonemin

The partially purified MAAs and scytonemin were further analyzed using a Shimadzu HPLC system equipped with photodiode array (PDA) detector (SPD-M10A VP), Inertsil® ODS-3 RP-18 column (5 μm, 250 × 4 mm), and guard (Inertsil ODS-3, 5 μm, 4.0 × 10 mm). Both MAAs and scytonemin samples (50 μL) were injected into the HPLC column through an autoinjector (Shimadzu, SIL-10AD VP). The column oven (CTO-10A VP) temperature was set at 40 °C. In case of MAAs, the mobile phase was 25% aqueous methanol + 0.1% acetic acid (HPLC grade) and run isocratically at a flow rate of 1.0 mL min−1. The detection wavelength of MAA was at 330 nm, and PDA scan wavelength was from 200 to 400 nm.

In case of scytonemin, the elution was at a flow rate of 1.5 mL min−1 using the mobile phase of solvent A (ultra pure water) and solvent B (acetonitrile–methanol–tetrahydrofuran, 75 : 15 : 10, v/v). The 30-min gradient elution program was set with 0–15 min linear increase from 10% solvent A to 100% solvent B and 15–30 min at 100% solvent B. The detection wavelength was at 380 nm, and the PDA scan wavelength was from 200 to 800 nm. The concentration of scytonemin was quantified using a set of trichromatic equations as described earlier (Garcia-Pichel & Castenholz, 1991). The solvent was degassed using the degasser (Shimadzu, DGU-14A) equipped in the HPLC system. Absorption spectra of UV-absorbing compounds were recorded each second directly on the HPLC-separated peaks. The fraction containing the pure MAAs or scytonemin was collected and lyophilized (Cat. No. 7670530; Labconco Corp.). The pure lyophilized MAAs and scytonemin were further characterized by liquid chromatography–electrospray ionization mass spectrometry.

Liquid chromatography–mass spectrometry (LC-MS) analysis was performed using a Shimadzu LCMS-2020 (Serial No. 0101548) equipped with a gas nebulizer probe. Separation was achieved using Shimadzu Inertsil ODS3 cartridge columns (5 μm, 2.1 × 150 mm), protected by an ODS3 guard column (2.1 × 33 mm). The MS analysis was performed in positive electrospray ionization (ESI) mode with a scan range from m/z 100 to 600. Data were processed using the labsolutions software (Shimadzu Corporation, Kyoto, Japan). Identification of MAAs and scytonemin was made from their online UV/Vis spectra and mass spectra as described earlier (Whitehead & Hedges, 2002; Squier et al., 2004).

Induction experiment

For the induction of MAAs and scytonemin, cultures were exposed continuously for 72 h to artificial UV-A, UV-B, and PAR in sterile glass Petri dishes (90 mm diameter). Three different cutoff filter foils such as 395 (Ultraphan, UV Opak Digefra, Munich, Germany), 320 (Art. Nr. 10155099, Folex, Dreieich, Germany), and 295 nm (Ultraphan, Digefra, Munich, Germany) were used to obtain the desired radiation regimes of PAR (400–700 nm), PAR + UV-A (315–400 nm), and PAR + UV-A + UV-B (280–315 nm), respectively. Equal amounts of cells were removed at desired time intervals of 12, 24, 48, and 72 h of continuous exposure and analyzed for MAA and scytonemin induction separately. The irradiances effectively received by the samples were 0.78, 1.5, and 20 W m−2 for UV-B, UV-A, and PAR, respectively. The light source used in this study was the same as mentioned earlier (Rastogi & Incharoensakdi, 2013). All experimental cultures were irradiated at a constant temperature of about 23 °C to avoid excess heating of the cells and were shaken several times during exposure to avoid self-shading effects.

Stability of MAAs and scytonemin

Stability of MAAs and scytonemin was investigated under three different abiotic stress conditions such as UV-B radiation, heat, and strong oxidizing agent H2O2 (0.25%). The partially purified MAAs and scytonemin were treated with the above-mentioned different stress conditions for an hour after which their remaining contents were analyzed in the HPLC system. The effects of heat was assayed by incubating MAAs and scytonemin in an incubator shaker (Innova 4080, New Brunswick Scientific Co., New Jersey) at 60 °C.

Free radical scavenging capacity

The radical scavenging capacity of extracted MAAs and scytonemin was measured using a DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay as described by Kulisic et al. (2004) with slight modifications. Briefly, the test samples (100 μL) of different amounts of MAAs (0.175, 0.35, and 0.70 mg in methanol) and scytonemin (0.75, 1.5, and 3.0 mg in methanol) were mixed with 1400 μL of 0.1 mM DPPH (in 80% methanol) solution. The mixtures in glass vials were gently shaken and incubated in dark at room temperature for an hour. The decolorization of DPPH radical was visually monitored, and a decrease in absorbance of the reaction mixture was measured at 517 nm. A mixture of methanol and DPPH solution was used as a negative control, while ascorbic acid was used as a positive control. Antioxidant capacity for each concentration of MAAs and scytonemin was expressed as percentage radical scavenging as described earlier (Kulisic et al., 2004).

Statistical analysis

The results are presented as mean values of three replicates (mean ± SD; n = 3). One-way analysis of variance (anova) was applied to evaluate the significance of the data (spss 15.0, Chicago, IL). A multiple comparison Tukey's test was applied to assay the differences between treatments. The level of significance was set at 0.05.

Results and discussion

Analysis of MAAs and scytonemin

The UV-sunscreening compounds, MAAs and scytonemin, were isolated and partially characterized in the cyanobacterium Lyngbya sp. CU2555. Figure 3 shows the absorption spectrum of crude methanolic extracts (Fig. 3A) as well as partially purified MAAs in aqueous solution (Fig. 3B) with a UV absorbance at around at 327 ± 2 nm (between 290 and 350 nm). HPLC analysis of partially purified MAAs recorded three prominent peaks at 2.7 min, 4.3 min, and 5.4 min (Fig. 4A) with UV absorption maxima (UVλmax) at 320 nm (a), 330 nm (b), and 312 ± 1 nm (c), respectively (Fig. 4B). In the present study, only major fractions of MAAs with UVλmax at 320 and 330 nm were collected and subjected to LC-MS analysis. Electrospray ionization liquid chromatography–mass spectrometry (ESI-LCMS) analysis of HPLC-purified MAA showed a prominent ion peak of protonated molecule ([M + H]+) at m/z 245.05 (Fig. 5A) and 289 (Fig. 5B), which is consistent with the results of ESI-LCMS analysis for MAAs elucidated in phytoplankton (Whitehead & Hedges, 2002). Based on the online UV/Vis absorption spectra and ESI-MS analysis (Whitehead & Hedges, 2002), the compounds were tentatively identified as palythine (UVλmax: 319 nm; m/z 245; Fig. 5A) and asterina-330 (UVλmax: 330 nm; m/z 289; Fig. 5B). The compound designated as M-312 in the present study with UVλmax at 312 ± 1 nm could not be identified. It is possible that this compound may be a glycosylated MAA. Matsui et al. (2011) have reported a glycosylated MAA with an absorption maximum at 312 nm (with a small shoulder at 340 nm) from a natural colony of Nostoc commune. Contrary to other MAAs, the presence of palythine and/or asterina is reported mainly in a limited number of microalgae/macroalgae (Sinha et al., 2007). The present study is the first report for the occurrence of three MAAs, palythine, asterina as well as M-312 in the cyanobacterium Lyngbya sp.

Figure 3.

UV/Vis absorption spectrum of Lyngbya sp.: (A)100% methanolic extract showing the peaks for mycosporine-like amino acid (327 ± 2 nm), chlorophyll a (435 and 665 nm), and carotenoids (471 nm); (B) Absorption spectrum of partially purified MAA prepared for HPLC analysis.

Figure 4.

HPLC chromatogram (A) of partially purified MAAs from Lyngbya sp., showing the typical peaks at retention times of 2.7 min (a), 4.3 min (b), and 5.4 min with absorption maximum at 320 nm (palythine), 330 nm (asterina-330), and 312 ± 1 nm (M-312), respectively (B).

Figure 5.

Mass spectra of HPLC-purified MAAs, palythine (A) and asterina (B), showing a prominent peak at m/z 245.05 and 289, respectively.

In contrast to the presence of a low concentration of MAAs, a high concentration of scytonemin was found in the studied cyanobacterium. HPLC chromatograms of scytonemin from Lyngbya sp. showed the presence of two prominent peaks at retention times of 3.25 and 4.48 min (Fig. 6A) with a similar absorption maximum at 384 nm (Fig. 6B and C), which is consistent with the results of Proteau et al. (1993) and Squier et al. (2004). ESI-LCMS analysis showed a prominent ion peak of a protonated molecule ([M + H]+) at m/z 545.05 (Fig. 7A) and 547.1, respectively (Fig. 7B). Online UV/Vis spectra and ESI-LCMS analysis revealed the presence of scytonemin (UVλmax: 384 nm; m/z 545; Fig. 7A) and its reduced counterpart (reduced scytonemin: UVλmax: 384 nm; m/z 547; Fig. 7B). The existence of prominent ions (m/z 545.05 and 547.1) in the mass spectrum of Lyngbya sp. is consistent with the online LC/MS spectra of cyanobacterial mat extract showing scytonemin at m/z 545.2 and reduced scytonemin at m/z 547.2 (Squier et al., 2004). Moreover, due to the unavailability of commercial standards, the preparation of which is very costly and time-consuming, MAAs are usually identified based on their retention time and their characteristic UV absorption spectra obtained online via HPLC and DAD analysis (Carreto et al., 2005). However, reverse-phase HPLC coupled with online PDA (HPLC-PDA) and MS detection can be an invaluable contribution in the identification of an individual MAA or scytonemin. In the present study, we have extracted the UV-absorbing compounds from both the axenic cultures of Lyngbya sp. isolated from their mats as well as directly from field-grown Lyngbya mats, and similar results were found, indicating the existence of a microbial mat (Fig. 1A) purely dominated by Lyngbya sp. (Fig. 1B), which is in agreement with other microbial mats dominated by Lyngbya sp. (Balskus et al., 2011).

Figure 6.

HPLC chromatogram (A) of partially purified scytonemin from Lyngbya sp. showing the typical peaks for reduced scytonemin (a) and scytonemin (b) at retention times of 3.25 min and 4.48 min, respectively. (B) online UV/Vis spectra showing maximum absorbance at 384 nm for both reduced scytonemin (B) and scytonemin (C).

Figure 7.

Mass spectrum of HPLC-purified scytonemin (A) and reduced scytonemin (B) showing a prominent peak at m/z 545.05 and 547.1, respectively.

Induction of MAAs and scytonemin

Induction of MAAs and scytonemin was investigated under three different cutoff filters as described above. Figure 8 shows the induction of the synthesis of MAAs as influenced by PAR and UV radiation. Absorption spectra of MAAs after 42 h of irradiation clearly indicate that contrary to PAR and PAR + UV-A radiation, induction of MAAs, palythine (Fig. 8A), asterina (Fig. 8C), and M-312 (Fig. 8E) was greatly increased under PAR + UV-A + UV-B radiation. Once it became evident that UV radiation has a major effect on the induction of all three MAAs, we further investigated the induction of the synthesis of these MAAs by time course experiments for 12 h up to 72 h (Fig. 8B, D and F). There was no significant difference in the concentration of MAAs (palythine, asterina, and M-312) among exposed samples covered with 395-nm cutoff filters for different times up to 72 h (Fig. 8B, D and F). There was no significant change in palythine concentration between 12 and 24 h, as well as between 48-h- and 72-h-exposed samples covered with 320-nm cutoff filters (Fig. 8B). However, a slight increase in the palythine concentration due to UV-A under this condition was noticeable after 48 h. Induction of palythine was always significantly higher in the 295-nm cutoff filter-covered samples after 12 h of exposure and further continued with a drastic increase at 72 h of exposure (Fig. 8B). Contrary to palythine, induction of asterina was significantly higher under 320-nm cutoff filters after 24 h of exposure; however, a significantly increased concentration was found in the samples covered with 295-nm cutoff filters after 24, 48, and 72 h of exposure (Fig. 8D). There was no significant difference in asterina concentration after 12- or 24-h exposure in samples covered with either 320- or 295-nm cutoff filters (Fig. 8D). Moreover, the induction of the synthesis of asterina under 295-nm cutoff filters was more or less at levels comparable with those samples irradiated under 320-nm cutoff filters after 24-h and 48-h irradiation, respectively (Fig. 8D). There was also a significant induction in the unknown MAA, M-312 after 12 h of exposure, and it continued during the whole irradiation period of 72 h in samples covered with 320- or 295-nm cutoff filters (Fig. 8F). However, the induction of the synthesis of M-312 was higher in the samples covered with 295-nm cutoff filters than those that were covered with 320-nm cutoff filters.

Figure 8.

Induction of MAAs, palythine (A and B), asterina (C and D), M-12 (E and F), and scytonemin (G and H) under PAR (P), PAR + UV-A (PA), and PAR + UV-A + UV-B (PAB) radiation and their concentration after different durations (12–72 h) of irradiation (B, D, F and H) under 395-, 320-, and 295-nm cutoff filters, respectively. The up arrow (↑) denotes the absorption spectra of the control, P, PA, and PAB irradiated samples for 42 h from bottom to top, respectively (A, C, E and G). The error bars denote standard deviations of means (means ± SD, n = 3).

Biosynthesis of MAAs as influenced by PAR and UV radiation has not been determined in the cyanobacterium Lyngbya sp. In the present study, we observed the marked induction of MAAs under UV (particularly UV-B) radiation (Fig. 8). The induction of MAAs was dependent on dose and wavelength of light. Induction of different MAAs by PAR and UV radiation has been reported in some other cyanobacteria (Rastogi et al., 2010b). Increased biosynthesis of MAAs in organisms exposed to high levels of solar radiation has been described to provide protection as UV-absorbing pigments (Garcia-Pichel et al., 1993). MAAs have been reported to prevent three of 10 photons from hitting cytoplasmic targets in cyanobacteria (Garcia-Pichel et al., 1993). Moreover, the MAA concentrations determined in this study by means of their specific absorption in UV region indicate that palythine probably acts as a major UV-B sunscreen in Lyngbya sp. In comparison with palythine and M-312, asterina could be induced considerably by both UV-A and UV-B irradiation, but the induction by UV-B was highly prominent in comparison with the other wavelength ranges (Fig. 8D).

Similar to MAAs, induction of scytonemin was also greatly affected under UV stress (Fig. 8G). Figure 8H clearly shows the dose-dependent induction of scytonemin synthesis under different cutoff filters. Contrary to UV radiation, induction of scytonemin was very low in the samples covered with 395-nm cutoff filters, suggesting that PAR is less effective for induction of the synthesis of scytonemin in the cyanobacterium Lyngbya sp. A significant induction in scytonemin content from its initial value (0 h sample) was found under 320- or 295-nm cutoff filters after 12 h of exposure, and this increase in scytonemin concentration further continued during the following 72 h of exposure (Fig. 8H). There was no significant difference between 12 and 24 h, 48 and 60 h, as well as 60-h- and 72-h-exposed samples covered with 320-nm cutoff filters. Similarly, there was also no significant difference in the concentration of scytonemin after 48 up to 72 h of exposure in samples covered with 295-nm cutoff filters (Fig. 8H). Moreover, in comparison with PAR, the concentration of MAAs as well as scytonemin was always significantly higher in the samples covered with 295-nm cutoff filters during the whole irradiation period. This indicates that UV radiation is more effective than PAR and plays an important role in the induction of the synthesis of UV-absorbing compounds in the cyanobacterium Lyngbya sp.

Moreover, the biosynthesis of scytonemin is also greatly affected under different abiotic stresses (Dillon et al., 2002). UV radiation plays a vital role in inducing the synthesis of scytonemin. Under natural conditions, increasing UV radiation was found to stimulate the synthesis and accumulation of high scytonemin concentrations in the microbial mat of Lyngbya sp. (Karsten et al., 1998). It has been demonstrated that osmotic stress and elevated UV-A exposure in combination with temperature or photooxidative stress induce the synthesis of scytonemin (Dillon et al., 2002). Scytonemin is synthesized in response to UV-A exposure and accumulates within the extracellular sheath or slime of the cyanobacteria, forming a stable, protective layer that can absorb about 90% of incident radiation (Garcia-Pichel & Castenholz, 1991). Overall, the presence of three MAAs and scytonemin in Lyngbya sp. may provide protection against UV-A and UV-B radiation and ensure the cyanobacterium to continue its normal metabolic function by absorbing/screening the lethal doses of UV radiation during harsh environmental conditions of ambient solar intensities.

Stability of MAAs and scytonemin

In the present study, stability of MAAs and scytonemin was investigated under three different abiotic stress conditions as mentioned above (Fig. 9). After UV and heat treatment, the UV-absorbing compounds, palythine, asterina, and M-312 as well as scytonemin were only partially degraded with a slight difference from their initial values. In contrast, the contents of MAAs and scytonemin were lower under 0.25% H2O2 stress than those under UV and heat stress (Fig. 9). Furthermore, in the presence of an oxidizing agent, asterina decreased distinctly (Fig. 9C and D), while other MAAs decreased comparatively little, suggesting that palythine (Fig. 9A and B) and M-312 (Fig. 9E and F) are relatively potent antioxidants. Overall, our results indicate that MAAs as well as scytonemin distributed in the cellular system plays an important role in protecting the cells against harsh environmental conditions.

Figure 9.

Stability of MAAs, palythine (A and B), asterina (C and D), M-312 (E and F), and scytonemin (G and H) under different abiotic stress conditions of UV-B (0.78 W m−2), heat (60 °C), or strong oxidizing agent (0.25% H2O2) for 1 h. (A, C, E, and G) are absorption spectra, whereas (B, D, F, and H) are relative contents of the compounds. The error bars denote standard deviations of means (means ± SD, n = 3).

Moreover, the stability of UV-absorbing compounds may be critical for their ultimate utility as photoprotective function. There are few reports on the stability of MAA and scytonemin against various physicochemical stressors. The stability of four different MAAs was studied under various physicochemical treatments such as temperature, strong UV radiation, various solvents, and pH, and these compounds were highly resistant to most of the treatments even up to 24 h (Gröniger & Häder, 2000). Whitehead & Hedges (2005) showed the photostable nature of some MAAs including palythine in both distilled and seawater in the presence of photosensitizers. In vitro absorption properties of MAAs, porphyra-334, and/or shinorine were unaffected when irradiated with UV-B or subjected to heat treatment (75 ± 2 °C) for up to 6 h, indicating the highly stable nature of MAAs against the environmental stress factors (Sinha et al., 2000). The characterization of the excited states of palythine in aqueous solutions was studied and confirmed that palythine is highly photostable in air-saturated aqueous solutions (Conde et al., 2007).

High stability of scytonemin can be evident by the fact that about 84% of the scytonemin screen in desiccated Nostoc punctiforme cells remained intact even after almost 2 months of constant exposure to UV-A radiation (Fleming & Castenholz, 2007). Overall, the MAAs and scytonemin in this cyanobacterium seem to be environmentally quite stable against UV, heat, and oxidation stress, because its absorption properties were not so much affected.

Radical scavenging activity of MAAs and scytonemin

Figure 10 shows the radical scavenging activity detected under different concentrations of partially purified MAAs (palythine + asterina + M-312; Fig. 10A) and scytonemin (Fig. 10B) from Lyngbya sp. The total antioxidant activities of different concentrations of MAAs and scytonemin were determined by the decolorization of DPPH radicals. Decolorization of DPPH was directly monitored visually as well as with a spectrophotometer. The activity of different concentrations of MAAs and scytonemin was compared with that of ascorbic acid used as a standard. A decrease in DPPH radicals, represented as an increase in radical scavenging, was found at all concentrations of MAAs in comparison with their negative control value (Fig. 10A), indicating that the MAAs in Lyngbya sp. act as the strong radical scavenger. The antioxidation rate was 14.5%, 53.0%, and 68.9% at concentrations of 0.115, 0.230, and 0.460 mg mL−1 MAAs, respectively, while 96.1% antioxidation rate was found at 0.230 mg mL−1 of ascorbic acid. However, the individual MAAs from Lyngbya sp. were not evaluated for their antioxidant activity and need further investigation.

Figure 10.

Free radical scavenging capacity of MAAs (A) and scytonemin (B) as determined by the DPPH assay. Percentage radical scavenging capacity of MAAs and scytonemin was determined and correlated with that of ascorbic acid. [(A) 1, negative control; 2, 0.115; 3, 0.230; and 4, 0.460 mg mL−1 MAAs; 5, 0.230 mg mL−1 ascorbic acid. (B) 1, negative control; 2, 0.5; 3, 1.0; and 4, 2.0 mg mL−1 scytonemin. 5, 0.230 mg mL−1 ascorbic acid].

Recently, De la Coba et al. (2009) have isolated the MAAs palythine and asterina-330 from the red alga Gelidium corneum and showed dose-dependent strong antioxidant activity of these MAAs in terms of scavenging of hydrosoluble radicals. Thus, MAAs may act as antioxidants to prevent cellular damage resulting from UV-induced production of ROS. MAAs are capable of effectively dissipating absorbed radiation as heat without producing ROS (Conde et al., 2007). Recently, the presence of the glycosylated MAAs with radical scavenging activity has been reported in the cyanobacterium Nostoc commune (Matsui et al., 2011). Daniel et al. (2004) reported that a cream containing 0.005% MAAs (porphyra-334 + shinorine) can neutralize UV-A effects as efficiently as a suntan cream with 1% synthetic UV-A filters and 4% UV-B filters, indicating that MAAs have potential applications in commercial products (Bandaranayake, 1998). Misonou et al. (2003) have observed that MAAs can block the production of both 6–4 photoproduct and cyclobutane pyrimidine dimer formation.

The occurrence of scytonemin has been reported in a number of cyanobacteria; however, its role in photoprotection has not been extensively studied (Takamatsu et al., 2003; Matsui et al., 2012). In the present investigation, we have studied the photoprotective function of scytonemin in terms of its radical scavenging capacity (Fig. 10B). Similar to MAAs, the sunscreening pigment scytonemin also exhibited the radical scavenging activity. The dose-dependent antioxidant activity of scytonemin was 12%, 33%, and 57% at concentrations of 0.5, 1.0, and 2.0 mg mL−1, respectively (Fig. 10B). Thus, the MAAs from Lyngbya sp. showed strong antioxidant activity, while the antioxidant activity of the cyanobacterial pigment scytonemin was relatively moderate in the DPPH assay. In agreement with previous reports, our present study strongly supports the role of MAAs and scytonemin as an active antioxidants that can prevent cellular damage caused by UV-induced oxidative stress. Overall, the presence of MAAs and scytonemin might help Lyngbya sp. to cope with oxidative stress under intense solar radiation. Furthermore, the UV-absorbing compounds MAAs and scytonemin may be of great value in the development of novel sunscreens and a source for future biotechnological research.

Conclusions

The biosynthesis of UV-absorbing/UV-screening compounds is an important mechanism to prevent UV-induced photodamage in cyanobacteria. HPLC-PDA and ESI-LCMS analysis revealed the presence of three MAAs, palythine, asterina-330, and M-312 as well as a dimeric indole phenolic pigment scytonemin and its reduced counterpart (reduced scytonemin) in the cyanobacterium Lyngbya sp. In vivo experiments indicated that MAAs as well as scytonemin can be induced by UV radiation. The high stability against different environmental stress factors as well as strong photoprotective capacity indicates that these compounds may be biotechnologically exploited in the pharmaceutical and UV-protecting cosmetic industries.

Acknowledgements

R.P.R. is thankful to the Graduate School, Chulalongkorn University, and Faculty of Science for Post-Doctoral Research Grant. A.I. thanks the Commission on Higher Education, Thailand, and the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University for the National Research University Project Grant (FW0659A) and the Chulalongkorn University Centenary Academic Development Project Grant (RES560530052-FW), respectively. We are also thankful to Prof. Dr R. P. Sinha, Banaras Hindu University, India, for providing UV cutoff filters. The authors declare that they have no conflict of interest.

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