Responses of a hot spring cyanobacterium under ultraviolet and photosynthetically active radiation: photosynthetic performance, antioxidative enzymes, mycosporine-like amino acid profiling and its antioxidative potentials

Abstract

This study summarizes the response of a hot spring cyanobacterium Fischerella sp. strain HKAR-14, under simulated light conditions of ultraviolet radiation (UVR), photosynthetically active radiation (PAR), PAR + UV-A (PA) and PAR + UV-A + UV-B (PAB). Exposure to UVR caused a decline in growth and Chl a while total carotene content increased under PA and PAB. Maximum photochemical efficiency of photosystem II (F _v /F _m) and relative electron transport rate decreased significantly in PA and PAB exposure. Higher non-photochemical quenching and lower photochemical quenching values were observed in UVR-exposed samples as compared to the control. Levels of intracellular reactive oxygen species (ROS) increased significantly in PAB and PA. Fluorescence microscopic images showed an increase in green fluorescence, indicating the generation of ROS in UVR. The antioxidant machinery including superoxide dismutase, catalase and peroxidase showed an increase of 1.76-fold and 2.5-fold superoxide dismutase, 2.4-fold and 3.7-fold catalase, 1.83-fold and 2.5-fold peroxidase activities under PA and PAB, respectively. High-performance liquid chromatography equipped with photodiode array detector, electrospray ionization mass spectrometry, Fourier-transform infrared spectroscopy and nuclear magnetic resonance spectroscopy analyses reveal the occurrence of a single mycosporine-like amino acid, shinorine (λ _max 332.3 ± 2 nm, m/z 333.1), with a retention time of 1.157 min. The electrochemical characterization of shinorine was determined by cyclic voltammetry. The shinorine molecule possesses electrochemical activity and represents diffusion-controlled process in 0.1 M (pH 7.0) phosphate buffer. An antioxidant assay of shinorine showed its efficient activity as antioxidant which increased in a dose-dependent manner.

Keywords: Antioxidant; Chlorophyll fluorescence; Cyanobacteria; Mycosporine-like amino acids; Ultraviolet radiation.

Fig. 1

Phylogenetic relationship of the isolated cyanobacterium Fischerella sp. strain HKAR-14 among cyanobacteria and its analysis inferred from 16S rRNA gene sequences. The evolutionary distances of isolated cyanobacteria were computed using the maximum composite likelihood method. The sequence obtained in the present study is indicated by a black circle

Fig. 2

Photosynthetic yields (F_v/F_m) of Fischerella sp. strain HKAR-14 (A), Relative inhibition of maximum quantum yield of PSII (F_v/F_m) (B), after exposure to photosynthetically active radiation (PAR), PAR + UV-A (PA) and PAR + UV-A + UV-B (PAB) at different time intervals up to 72 h. A horizontal line over bars indicates no significant difference (P > 0.05) among treatments. The error bars denote standard deviations of means (means ± SD, n = 3). Similar letters over bar represent homogeneous mean groups (P > 0.05)

Fig. 3

Variation in non-photochemical quenching (NPQ) (A–C), photochemical quenching (qP) (D–F), and relative electron transfer rate (rETR) (G–I) of Fischerella sp. strain HKAR-14 cultures exposed to photosynthetically active radiation (PAR) (a), PAR + UV-A (PA) (b) and PAR + UV-A + UV-B (PAB) (c) irradiation for varying time periods. The error bar represents standard deviation of mean (means ± SD, n = 3)

Fig. 4

Activity of antioxidative enzymes in cultures exposed to photosynthetically active radiation (PAR), PAR + UV-A (PA) and PAR + UV-A + UV-B (PAB) irradiation for varying time periods. Superoxide dismutase (SOD) (A), catalase (CAT) (B) and peroxidase (POD) (C). The error bar represents standard deviation of mean (means ± SD, n = 3). A horizontal line over bars indicates no significant difference (P > 0.05) among treatments. Similar letters over the bars represent homogeneous mean groups (P > 0.05)

Fig. 5

A Fluorescence images after irradiation with photosynthetically active radiation (PAR), PAR + UV-A (PA) and PAR + UV-A + UV-B (PAB) showing intracellular green 2′,7′-dichlorodihydrofluorescein (DCF) fluorescence after various duration of exposure as a result of reactive oxygen species (ROS) production. DCF fluorescence at 0 h was used as a control. B Emission spectra of DCF fluorescence intensities after irradiation with PAR, PA and PAB after different duration of exposure. The error bar represents standard deviation of mean (means ± SD, n = 3). A horizontal line over the bars indicates no significant difference (P  > 0.05) among treatments. Similar letters over the bars represent homogeneous mean groups (P > 0.05)

Fig. 6

Absorption spectrum of the methanolic extract showing ultraviolet (UV) absorption maximum at 332.3 ± 2 nm indicating the presence of a UV-absorbing mycosporine-like amino acid (MAA) (A), high performance liquid chromatography (HPLC) chromatogram of the partially purified MAA, showing a typical peak at a retention time of 1.157 min (B) and absorption maximum at 332.3 nm (C)

Fig. 7

Absorption spectra showing the induction of shinorine after different durations of photosynthetically active radiation (PAR), PAR + UV-A (PA) and PAR + UV-A + UV-B (PAB) in Fischerella sp. strain HKAR-14 (A) and biosynthesis of shinorine under PAR, PA or PAB irradiation for 72 h (B). The error bars represent standard deviation of the means (means ± SD, n = 3). A horizontal line over bars indicates no significant difference (P > 0.05) among treatments. Similar letters over the bars represent homogeneous mean groups (P > 0.05)

Fig. 8

Electro spray ionization-mass spectrometry (ESI–MS) of high performance liquid chromatography (HPLC) purified fractions giving a peak with a m/z value of 333.1 confirming the identity of the collected purified fraction as shinorine (A), Fourier Transform Infrared (FTIR) radiation spectroscopy showing basic functional groups present in the purified fraction of the mycosporine-like amino acid (MAA), (B) Nuclear magnetic resonance (NMR) spectra of purified fraction of shinorine, ¹H (C) and ¹³C (d) ¹D NMR spectrum of the purified fraction were recorded in D₂O solution. NMR spectra revealed correlation with chemical shift data of shinorine

Fig. 9

Cyclic voltammetry (CV) scan of the shinorine molecule in 0.1 M pH 7.0 phosphate buffer at the scan rate of 20 mV s⁻¹ (A). CV of shinorine at different scan rates (20, 50, 100, 200, and 300 mVs⁻¹) in 0.1 M pH 7.0 phosphate buffer (B). Plot of anodic peak currents (measured from Fig. 1b) against the square root of scan rates (C). CV scans of shinorine molecule in 0.1 M of different pH buffers (5.8, 6.2, 6.6, 7.0, and 7.8) at the scan rate of 50 mV s⁻¹ (D)

Fig. 10

Free radical scavenging capacity of partially purified shinorine as determined by the 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) assay (A), ferric reducing antioxidant power (FRAP) assay (B) and superoxide radical scavenging activity (SRSA) assay (C) and 2, 2-Azinobis (3-ethylbenzothiazoline 6-sulfonate) (ABTS^·+) radical scavenging assay (d). The radical scavenging capacity of shinorine was determined and correlated with that of ascorbic acid (AA). The error bars denote standard deviations of means (means ± SD, n = 3)