Antioxidative responses of Nostoc ellipsosporum and

Journal of Applied Phycology https://doi.org/10.1007/s10811-018-1506-2

Antioxidative responses of Nostoc ellipsosporum and Nostoc piscinale to salt stress Maryam Rezayian 1 & Vahid Niknam 1 & Mohammad Ali Faramarzi 2 Received: 30 January 2018 / Revised and accepted: 2 May 2018 # Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract The responses of the cyanobacteria Nostoc ellipsosporum and Nostoc piscinale to salt stress during various growth stages were studied. Nostoc ellipsosporum was more NaCl tolerant and attained more biomass under salinity than N. piscinale. Accumulation of proline was detected in N. ellipsosporum under salinity stress. Malondialdehyde content decreased in both species on day 9 (mid log phase) under salt stress. A similar trend was detected in hydrogen peroxide (H2O2) content in N. ellipsosporum. Strong induction in catalase (CAT) activity was observed in N. ellipsosporum on day 9 in the presence of salt. The increase in CAT activity of N. piscinale was observed only at higher concentrations of NaCl. In contrast to N. ellipsosporum, induction in peroxidase and polyphenol oxidase (PPO) activities on day 9 was stronger in N. piscinale. Salinity enhanced superoxide dismutase (SOD) and PPO activity in N. ellipsosporum at all growth stages. Moreover, different isoforms of CAT and SOD were detected in these cyanobacteria. Apparently, selection pressure in these cyanobacteria has led to the evolution of SODs and CATs as the main antioxidant enzymes against reactive oxygen species. Phycobiliprotein content in N. ellipsosporum under all conditions was significantly higher than that in N. piscinale. NaCl at moderate concentrations significantly increased phycobiliprotein content in the middle of the log phase in both species. Moreover, contrary to N. piscinale, the size of phycobilisomes [phycoerythrin + phycocyanin / allophycocyanin] in N. ellipsosporum increased significantly under salt stress at the mid of log phase and later. The increase in size of phycobilisomes in N. ellipsosporum could help this species to withstand salt stress by enhancing energy transfer capacity. These results suggest that N. ellipsosporum cells could be better protected against salinity-induced oxidative damage by maintaining higher levels of antioxidative enzymes, proline, and phycobiliproteins than the cells of N. piscinale. Keywords Salt stress . Nostoc ellipsosporum . Nostoc piscinale . Antioxidative enzymes . Phycobiliproteins . Cyanobacterium

Introduction Cyanobacteria are a primitive group of Gram-negative and nitrogen-fixing prokaryotes and they were the first photosynthetic oxygen-evolving organisms. They appeared during the Precambrian era and provided the conditions for the evolution of aerobic life (Fischer 2008). These organisms change their physiology, biochemistry, and molecular metabolism in * Vahid Niknam [email protected] 1

Department of Plant Biology, and Center of Excellence in Phylogeny of Living Organisms in Iran, School of Biology, College of Science, University of Tehran, Tehran 14155, Iran

2

Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, P.O. Box 14155–6451, Tehran 14176, Iran

response to stresses in their habitats to maximize energy production and increase survival (Singh and Montgomery 2011). Salinity as a type of abiotic stress has been suggested as a controlling factor for blooms of cyanobacteria in estuaries (Moisander et al. 2002). Cyanobacteria are also major biomass producers both in aquatic and terrestrial ecosystems, producing various natural products of medicinal and industrial value (e.g., Cardozo et al. 2007; Häder et al. 2007; Sekar and Chandramohan 2008; Martínez-Roldán et al. 2014; Gris et al. 2017; Singh et al. 2017). Phycobiliproteins (PBPs) are water-soluble and highly fluorescent proteins which are very stable at physiological pH (Glazer 1999). They are non-toxic and non-carcinogenic and are gaining importance as natural colorants (Chaneva et al. 2007). Phycobilisomes (PBSs) are supramolecular assemblies of PBP attached to the outer surface of the photosynthetic lamellae of cyanobacteria that efficiently harvest light energy and transfer it to photosynthetic reaction centers. The major

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components of PBSs are the bilin-containing proteins: phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC). Phycocyanin, a brilliant blue-colored pigment, has greater importance because of its various biological and pharmacological properties including ROS scavenging, antiviral, anticancer, neuroprotective, hepatoprotective, radioprotective, and anti-inflammatory properties (Liu et al. 2000; Romay and Gonzalez 2000; Xia et al. 2016). Carotenoids are a class of accessory pigments that occur in all photosynthetic organisms and have many different biological functions, such as specific coloration, photoprotection, and light harvesting and serve as precursors for many hormones (Sharma et al. 2014). The mechanisms responsible for salinity tolerance in cyanobacteria have received less attention compared to that of higher plants. Cyanobacteria have a close phylogenetic relationship with plant chloroplasts and are therefore regarded as suitable model systems for studying plant responses to environmental stresses (e.g., Joset et al. 1996; Bhadauriya et al. 2007; Boonburapong et al. 2016). In order to investigate the salinity stress-induced biochemical and physiological changes and to elucidate adaptive mechanisms of two Nostoc species, the status of growth, the content of proline, protein, hydrogen peroxide, and lipid peroxidation products, and the activity of some antioxidative enzymes responsible for detoxifying ROS under NaCl salinity were analyzed. Another aim of this study was to determine the changes in the contents of biopigments (Chla, Car, and phycobiliproteins) under salt stress and their physiological implications in salt tolerance of two Nostoc species.

Materials and methods

150, 200, and 250 mM) and the cultures were sampled during three harvest times (5, 9, and 13 days after treatment).

Determination of fresh and dry mass Growth parameters including dry weight (DW) and fresh weight (FW) were determined based on the method of Leganes et al. (1987) and Zhu and Lee (1997). Aliquots of 50 mL filamentous cyanobacteria suspension were filtered through pre-weighed quantitative Whatman No. 42 filter papers. The filter papers were washed with distilled water twice or thrice and weighed in vacuo at 65 °C.

Determination of protein and proline contents A sample of 1 g of fresh microalgae was frozen at − 70 °C. The frozen sample was sonicated for 20 min in an ultrasound apparatus from Eyela (Japan). The extract was separated from the pellet and recovered by centrifugation, and stored at 4 °C in the dark until analysis. For protein extraction, the lysed cells were homogenized in 2 mL of 1 M Tris–HCl (pH 6.8) buffer containing 0.3 M sucrose using a pre-chilled mortar and pestle. The homogenates were centrifuged at 13,000×g for 30 min at 4 °C. The supernatant was used for protein content and enzyme activity assays. Soluble protein content of the enzyme extracts was determined according to Bradford (1976) using bovine serum albumin (BSA) as a standard. For proline extraction, the lysed cells were homogenized in 10 mL of 3% (w/v) aqueous sulfosalicyclic acid. Free proline content was determined according to Bates et al. (1973), using L-proline as a standard.

Determination of malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents

Algal strains and cultivation Axenic cultures of two filamentous cyanobacteria Nostoc ellipsosporum (Ghasemi et al. 2006; Moradpour et al. 2010) and Nostoc piscinale (Gharaei-Fathabad et al. 2007, 2008; Kalbasi et al. 2009) were prepared. They had been isolated from soil samples in the previous studies (Hajimahmoodi et al. 2010; Forootanfar et al. 2013). The cyanobacteria were grown and maintained on sterile BG-11 medium (Borowitzka 1988) agar slants and subcultured freshly before use in the later experiments. They were transferred to a fresh medium every 2 months. A loopful of each strain was individually inoculated into 100 mL of BG-11 medium in a 500-mL Erlenmeyer flask and incubated at 25 °C under continuous illumination of 60 μmol photons m−2 s−1. The initial pH of the medium was adjusted to 7.2 prior to sterilization by autoclave. To study the effect of salt stress on growth and physiological and biochemical parameters, both microalgae were grown on BG-11 medium containing different concentrations of NaCl (0, 50, 100,

The level of lipid peroxidation was measured in terms of thiobarbituric acid-reactive substances (TBARS), following the method of Heath and Packer (1968). The concentration of malondialdehyde was calculated using extinction coefficient of 155 mM−1 cm−1. The content of H2O2 was determined according to the method of Velikova et al. (2000). The lysed cells (2 g) were homogenized in 1 mL of 0.1% (w/v) trichloroacetic acid (TCA) on ice and centrifuged at 12, 000×g for 15 min. One milliliter of potassium phosphate buffer and 1 mL KI were added to 0.5 mL aliquot of the supernatant. The absorbance of the supernatant was recorded at 390 nm and H2O2 content was calculated using a standard curve.

Antioxidant enzyme activities For estimations of enzymes activities, the lysed cells were homogenized at 4 °C with 1 M Tris–HCl (pH 6.8) containing 0.3 M sucrose using a pre-chilled mortar and pestle. The

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homogenates were centrifuged at 13,000×g for 30 min at 4 °C. Supernatants were kept at -70 °C and used for determination of enzyme activities. Superoxide dismutase (SOD; EC 1.15.1.1) activity was determined according to Beauchamp and Fridovich (1971), measuring inhibition in the photochemical reduction of nitroblue tetrazolium (NBT) spectrophotometrically at 560 nm. The reaction mixture contains 50 mM potassium phosphate buffer (pH 7.8) with 0.1 mM ethylene diamine tetraacetic acid (EDTA), 75 μM NBT, 13 mM methionine, 2 μM riboflavin, and 100 μL protein extract. Reactions were carried out for 12 min at a light intensity of 300 μmol photons m−2 s−1. The non-irradiated reaction mixture served as control and was deducted from absorption at 560 nm. One unit of SOD was defined as the amount of enzyme which caused 50% inhibition of NBT reduction under the assay condition, and the results were expressed as units per milligram of protein. SOD isoforms were examined in 10% acrylamide gel using the procedures of Laemmli (1970). Gels were incubated in 0.2 M Tris–HCl (pH 8.0) containing 4% riboflavin, 4% EDTA, and 20% NBT for 40 min in the dark at room temperature and then exposed to white light until white bands appeared in a violet background. For SOD isoform identification, assays were performed in the presence of selective inhibitors. KCN (3 mM) inhibits only Cu/Zn-SOD. H2O2 (5 mM) inhibited both Cu/Zn-SOD and Fe-SOD. Mn-SOD is not inhibited by KCN or H2O2 (Lee et al. 2001). Catalase (CAT; EC 1.11.1.6) activity was assayed by measuring the initial rate of disappearance of H2O2 according to Aebi (1984). The reaction mixture contains 50 mM phosphate buffer (pH 7.0), H 2 O 2 (3%), and 10 μL enzyme extract. The decrease in absorption was followed for 180 s and CAT activity was expressed as units per milligram of protein. Staining for CAT was performed using the method of Woodbury et al. (1971). The gel (10%) was soaked in 5 mM H2O2 for 10 min. The gels were washed with distilled water and CAT isoforms were detected by incubating the gels in 2% (w/v) ferric chloride and 2% (w/v) [K3Fe(CN)6] until yellow bands on the dark green background appeared. Peroxidase (POX; EC 1.11.1.7) activity was measured according to Abeles and Biles (1991). The assay mixture contains 4 mL of 200 mM acetate sodium buffer (pH 4.8), 400 μL H2O2 (3%), 200 μL of 20 mM benzidine, and 50 μL enzyme extract. The increase of absorbance was recorded at 530 nm. The POX activity was defined as units per milligram protein. Polyphenol oxidase (PPO; E.C. 1.14.18.1) was estimated following the method of Raymond et al. (1993) at 40 °C. The reaction mixture contained 2.5 mL of 200 mM potassium phosphate buffer (pH 7), 200 μL pyrogallol 20 mM, and 20 μL enzyme extract. The increase in absorbance was recorded at 430 nm. The PPO activity was defined as units per milligram protein.

Determination of chlorophyll, carotenoid, and PBP contents Chlorophyll content was measured according to Marker (1972) performing overnight extractions of cyanobacteria lysed cells using 90% aqueous methanol. Centrifuged extracts were analyzed at 665 nm and the extinction coefficient of Marker (1972) was used. The amount of total carotenoids was determined following Davis (1976). PBPs were extracted by the method of osmotic shock (Wyman and Fay 1986). One milliliter of microalga cell suspension was centrifuged and the pellets homogenized in 60–150 μL glycerol and incubated in the dark at 4 °C for 24 h. Water was then added to osmotically lyse the cells. The lysed cells were centrifuged and the PBPs were determined in the supernatant according to Bennett and Bogorad (1973). As a control, absorption spectra of the supernatants were measured (400–750 nm) to confirm the absence of chlorophyll contamination because the chlorophyll absorbance at 665 nm could interfere with the absorbance of APC at 650 nm. Finally, the absorbances were determined spectrophotometrically at 652, 615, 562, and 750 nm.

Statistical analysis Each experiment was repeated three times and the data were analyzed by using one-way analysis of variance (ANOVA) using SPSS (version 21) and means were compared by Duncan’s test at the 0.05 level of confidence.

Results Growth and contents of protein and proline The DW of both species was enhanced significantly under higher levels of salinity during various stages of growth compared to control (Fig. 1.) Nostoc ellipsosporum was more tolerant to NaCl stress than N. piscinale and N. ellipsosporum attained higher biomass under salinity stress. Both species showed significant increase in DW at 100 mM NaCl and more, compared with control, with about 500, 250, and 220% increase in N. ellipsosporum and 200, 100, and 37.5% increase in N. piscinale at 200 mM NaCl on days 5, 9, and 13, respectively. Protein content in N. ellipsosporum was higher than that of N. piscinale under control condition during various stages of growth. However, protein content in N. piscinale was more responsive to salt stress and significantly increased under different salinities in contrast to N. ellipsosporum as compared to control, and the increase level was higher on days 5 and 13 than the other times (Fig. 2a, b). Proline content in N. ellipsosporum remained relatively unchanged under NaCl stress on days 5 and 9, but increased steadily up to 150 mM NaCl on day 13,

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150 to 200 mM and more, significant accumulation of proline occurred in N. piscinale only on day 5. At 150 mM NaCl, the highest proline contents in cells of N. ellipsosporum and N. piscinale were 798 and 238% of the control on days 13 and 5, respectively (Fig. 2c, d).

Oxidative state in cyanobacteria under salt stress

Fig. 1 Effect of various NaCl concentrations on dry weight of N. ellipsosporum (a) and N. piscinale (b) during three harvest times (days 5, 9, and 13). Data are mean ± SE of three replicates. Bars for each harvest time followed by different letters show significant difference at P < 0.05 significance level

then decreased slightly at 200 and 250 mM NaCl. In contrast, when the level of NaCl was increased from

Lipid peroxidation was assessed as MDA content and for determination of ROS scavenging capacity the H2O2 content of the species under salt stress was estimated. MDA and H 2O 2 content in N. ellipsosporum under NaCl stress significantly increased on days 5 and 13, but decreased on day 9 compared to the control. When the level of NaCl was increased, in spite of increase in H2O2 content, significant reduction of MDA occurred in N. piscinale on days 5 and 9 (Fig. 3a, b). A significant increase in H2O 2 content was seen in N. piscinale under most levels of salinity as compared to the control (Fig. 3d). However, H 2O2 content in N. ellipsosporum on day 5 increased from 40 at 0 mM NaCl to 160 nmol g−1 FW at 200 mM NaCl (ca. a 250% increase), then decreased slightly at final concentration of salt. In contrast, N. piscinale on day 5 showed much more enhancement in the H2O2 content under salinity, with the H2O2 content at 150 mM NaCl being 400% higher than the control level.

Fig. 2 Effect of NaCl on contents of protein (a, b) and proline (c, d) of N. ellipsosporum and N. piscinale during three harvest times (days 5, 9, and 13). Data are mean ± SE of three replicates. Bars for each harvest time followed by different letters show significant difference at P < 0.05 significance level

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Antioxidant enzymes CAT activity in N. ellipsosporum under salinity was higher than that of N. piscinale. Salinity at 50 mM NaCl significantly increased activity of CAT in N. ellipsosporum on days 5 and 9 and the activity decreased gradually thereafter. Moreover, CAT activity under all salinity concentrations was higher than that of control (Fig. 4a, b). In contrast to CAT, POX activity in N. piscinale under salinity stress was higher than that of N. ellipsosporum. POX activity in both species on day 13 increased significantly under NaCl. At 200 mM NaCl, POX activity in N. ellipsosporum and N. piscinale amounted to 2811 and 1164% of the control value, respectively (Fig. 4c, d). Polyacrylamide gel electrophoresis (PAGE) analysis of CAT activity revealed two isoforms (CAT1 and CAT2) in N. ellipsosporum during various stages of growth Fig. 5. SOD and PPO activities in both species were induced under NaCl stress and the activities nearly at all NaCl concentrations during various stages of growth were higher than that of control. Moreover, maximum activities of these two enzymes in both species under salinity treatments were detected on day 13 of growth (Figs. 6). Activity staining of SOD separated by native PAGE revealed that cells of N. ellipsosporum had one Cu/Zn-SOD isoform, one Mn-SOD isoform, and five Fe-SOD isoforms, all of which were affected by salinity Fig. 7a. In N. piscinale, one Cu/Zn-SOD isoform, three Mn-SOD isoforms, and one Fe-SOD isoforms were detected (Fig. 7b).

Chlorophyll a, carotenoid, and phycobiliprotein contents Chlorophyll a (Chla) content increased at 50 and 100 mM NaCl only on the first stage of growth and decreased later (Fig. 9a, b). Chla content in N. ellipsosporum at 0 mM NaCl ranged from 33.76 μg g−1 FW on day 5 to 99.32 μg g−1 FW on day 9, while those of N. piscinale ranged from 204.65 μg g−1 FW on day 5 to 315.22 μg g−1 FW on day 13. Car content in N. ellipsosporum and N. piscinale at 0 mM NaCl ranged from 2.9 on day 13 to 8.4 μg g−1 FW on day 5, and from 6.4 on day 13 to 13.2 μg−1 g−1 FW on day 9, respectively. A significant decrease was detected in the Car content of both species under salinity on day 9 as compared to control (Fig. 8c, d). Contents of AP, PC, PE, and total PBP in N. ellipsosporum cells during growth stages under control condition varied from 38.53 to 89.62, 20.84 to 138.43, 5.52 to 15.83, and 65.45 to 243.88 μg g−1 FW, significantly higher than those in N. piscinale (1.77 to 4.61, 1.27 to 2.92, 0.92 to 2.4, and 3.82 to 9.94 μg g−1 FW), respectively (Figs. 9 and 10). Moreover, NaCl stress mostly at moderate concentrations significantly increased AP, PC, PE, and total PBP contents in both species on day 9 (at mid of log phase), except for PC in N. piscinale, compared to that of control. The variability of phycobilisome size [PE + PC/APC] was also examined. In contrast to N. piscinale, the size of phycobilisomes in N. ellipsosporum increased significantly at 100 mM NaCl and more on day 9 and 13 (Fig. 11a and b).

Fig. 3 Effect of NaCl on contents of MDA (a, b) and H2O2 (c, d) of N. ellipsosporum and N. piscinale during three harvest times (days 5, 9, and 13). Data are mean ± SE of three replicates. Bars for each harvest time followed by different letters show significant difference at P < 0.05 significance level

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Fig. 4 Changes in the activities of CAT (a, b) and POX (c, d) enzymes in N. ellipsosporum and N. piscinale under NaCl stress during three harvest times (days 5, 9, and 13). Data are mean ± SE of three replicates. Bars for

each harvest time followed by different letters show significant difference at P < 0.05 significance level

Discussion

stress was previously reported in Chlorella vulgaris and Chlorococcum humicola (Abdel-Rahman et al. 2005) and Botryococcus braunii (Ranga Rao et al. 2007). A different result was also reported in the cyanobacterium Anabaena cylindrica under NaCl stress (Bhadauriya et al. 2007). NaCl in trace

The present investigation indicates the halotolerant characteristics of the two Nostoc species as evidenced by the positive effect of NaCl on the growth (Fig. 1). Increase in growth under salt

Fig. 5 Changes in the isoform patterns of CAT enzyme in N. ellipsosporum subjected to various salt concentrations during three harvest times (days 5, 9, and 13)

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Fig. 6 Changes in the activities of SOD (a, b) and PPO (c, d) enzymes in N. ellipsosporum and N. piscinale under NaCl stress during three harvest times (days 5, 9, and 13). Data are mean ± SE of three replicates. Bars for

each harvest time followed by different letters show significant difference at P < 0.05 significance level

amounts appears essential for some of the metabolic functions in cyanobacteria (e.g., Garcia-Gonzalez et al. 1987), but elevated levels of NaCl might inhibit growth (Jeanjean et al. 1993). In response to stress, plants make new proteins that may provide a storage form of nitrogen which is reutilized when stress is over (Torabi and Niknam 2011). The increase in total protein in response to stress is in accordance with the recent findings for in vitro grown cyanobacterium Lyngbya aestuarii (Rath et al. 2014) and two Acanthophyllum species (Niknam et al. 2011). According to Rath et al. (2014), one major difference between the cyanobacterial response and that of plants is that the stress proteins of plants are predominantly of low molecular mass (Kimpel and Key 1985; Ramagopal 1987). Proline is a compatible solute reported in salt-stressed cells of heterotrophic bacteria and more frequently in plants (Hagemann 2011). Proline has been found and reported in salt-shocked cyanobacteria, for example, in cells of Synechococcus sp. PCC 7418 (Fulda et al. 1999) and Nostoc muscorum (Singh et al. 1996). Proline accumulation under salt in the two species of Nostoc is time dependent consistent with results obtained in higher plants (Aghaleh et al. 2009; Niknam et al. 2011; Torabi and Niknam2011) and the cyanobacterium Oscillatoria acuminata (Senthilkumar and Jeyachandran 2006). It is possible that proline accumulation under stress could be because of a stimulated synthesis or because of a lower rate of oxidation and a slow incorporation of proline into proteins. Proline contributes to cytoplasmic osmotic adjustment, stabilizing membranes, protein, and scavenging free radicals under stress conditions (Szabados and Savoure 2009).

Salt stress leads to increase of ROS and oxidative stress. ROS are highly reactive and might cause oxidative damage to lipids, protein, and nucleic acids. In cyanobacteria, lipids present in thylakoids have a high percentage of polyunsaturated fatty acid (PUFA) residues and are susceptible to peroxidation (Tang et al. 2007). When the level of NaCl was increased, in spite of increase in H2O2 content, a significant reduction of MDA occurred in N. piscinale on days 5 and 9 (Fig. 3a, b). Reduction of MDA content in N. piscinale could be due to increased activities of antioxidant enzymes or other compounds. Moreover, in contrast to H 2 O 2 , MDA content was always lower in N. ellipsosporum cells than that in N. piscinale throughout the experiments performed here (Fig. 3a, b). This indicates that N. ellipsosporum cells are able to tolerate salinity-induced oxidative damage better than those of N. piscinale. Lower content of MDA in N. ellipsosporum cells could be due to the efficient antioxidative systems (mainly phycobiliproteins) compared to that of N. piscinale. This result is also in accordance with our findings on biomass of both species (Fig. 1a, b). Changes in lipid peroxidation serve as an indicator of the extent of oxidative damage under stress, with a lower level of MDA seeming to be a characteristic of tolerant species coping with elevated salinity (e.g., Niknam et al. 2011). SOD catalyzes the dismutation of superoxide radical to molecular oxygen and H2O2 and hence decreases the risk of hydroxyl radical formation from superoxide via the Haber–Weiss type reaction. PPO is the major enzyme responsible for oxidation of phenolic compounds (Torabi and Niknam 2011). Out of

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Fig. 7 Changes in the isoform patterns of SOD in ellipsosporum (a) and N. piscinale (b) subjected to various NaCl concentrations on day 9 of harvest time

the SOD isoforms, expression of the Mn-SOD isoforms was prominent in both species (Fig. 7a, b). According to Ismaiel et al. (2014), five SOD isoforms including Cu/ZnSOD, Mn-SOD, Fe-SOD, Ni-SOD, and Fe/ZnSOD have been identified in Spirulina (Arthrospira) platensis. Based on Li et al. (2002), most cyanobacteria have the Fe-SOD and Mn-SOD isoforms varying in their metallic cofactors, but the marine cyanobacterium Synechococcus sp. has both Fe-SOD and Cu/ZnSOD (Chadd et al. 1996), whereas Synechocystis sp. contains only the Fe-SOD (Tichy and Vermaas 1999). The main point regarding the analysis of SOD and its isoforms is that for the survival of cyanobacteria with oxygenic photosynthesis, the selection pressure led to the evolution of SODs as the first line of antioxidative systems against ROS. Studies on cyanobacterial SODs would serve as a window into the past and present evolutionary events of these primitive photosynthetic organisms. The SOD is the first line of defense against ROS and it catalyzes the disproportionation of highly damaging O2•– into comparatively less damaging H2O2. The balance between the activity of SOD and that of the H2O2-scavenging enzymes such as CAT and POX in cells plays an important role in providing a defense mechanism against oxidative damage

(Badawi et al. 2004). As the salt tolerance is often correlated with a more efficient antioxidative system (e.g., Jahnke and White 2003; Lu et al. 2006), then constitutive and/or induced activities of CAT and SOD suggest better tolerance to salt stress in N. ellipsosporum in comparison with N. piscinale. Because NaCl stress is supposed to inactivate the photosynthetic machinery, an attempt has also been made to study the changes of important pigments of photosynthesis (i.e., Chl, Car, AP, PC, PE, and PBP) in response to NaCl stress. Moreover, cyanobacteria are valuable resource for natural non-enzymatic antioxidants such as phycobiliproteins and carotenoids. Consequently, investigations were carried out with the aims of assessment and production of these antioxidants under salinity stress. Chla and Car contents in N. piscinale were higher than those of N. ellipsosporum. Conversely, phycobiliprotein accumulation was observed in N. ellipsosporum cells. The decrease in Chla content with increasing salinity which occurred under salt treatment has also been recorded in Spirulina sp. (Deniz et al. 2011). Decrease in Chla content could be a symptom of oxidative stress and may be due to an increase in pigment degradation or decrease in its synthesis. Reductions in Chla may also be explained by decrease in rubisco activity under stress (Lawlor 1995). Saltinduced decreases in Chla have also been reported previously in various organisms including cyanobacteria (e.g., Aghaleh et al. 2009; Deniz et al. 2011; Torabi and Niknam 2011; Rath et al. 2014; Sharma et al. 2014). Conversely and in contrast to higher plants, the accumulation of various kinds of carotenoids under stress has been reported in cyanobacteria and green algae (Masojídek et al. 2000). Carotenoids are essential for photosynthesis and serve as precursors for signaling molecules such as abscisic acid which plays an important role in plant development as well as stress responses (Li et al. 2002). They are also recognized as efficient antioxidants against oxidative damage and could quench singlet oxygen, resulting in the suppression of lipid peroxidation (Burton and Ingold 1984). Finally, N. piscinale in spite of lower salinity tolerance could be considered as a potential biosource of carotenoids. Contents of AP, PC, PE, and total PBP in N. ellipsosporum during various stages of growth both under control and nearly all levels of NaCl stress were higher than those of N. piscinale Figs. 9 and 10. NaCl stress mostly at moderate concentrations significantly increased AP, PC, and total PBP contents in both species compared to those of control. Similar changes in the pigment content in the green alga Chlorococcum sp. have also been reported under stress conditions (Masojídek et al. 2000). Thus, an increase in phycobiliproteins and a subsequent decrease in Chla and Car measured in NaCl-treated cells of N. ellipsosporum and N. piscinale might be considered as a regulatory mechanism for the maintenance of the biosynthetic machinery under salt stress. To mitigate oxidative stress, organisms develop an efficient antioxidant system to scavenge ROS. Among non-enzymatic

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Fig. 8 Chlorophyll a (a, b) and carotenoid (c, d) contents in N. ellipsosporum and N. piscinale subjected to various salt concentrations during three harvest times (days 5, 9, and 13). Data are mean ± SE of three

replicates. Bars for each harvest time followed by different letters show significant difference at P < 0.05 significance level

antioxidants, phycobiliproteins accumulate strongly in several cyanobacteria under various abiotic stresses such as salinity. Our results showed that the content of these biopigments increased under salt treatments (Figs. 9 and 10). It was also earlier demonstrated that microalgae exposed to salinity (Hifney et al. 2013) and microwave radiation (Asadi et al.

2013) accumulate phycobiliproteins to protect the cells against stress damage. According to this research, the induced effect of NaCl stress on these pigments content was found to be concentration dependent. Because of high contents of AP, PC, PE, and PBP in N. ellipsosporum, this species could be considered as a potential biological resource for

Fig. 9 AP (a, b) and PC (c, d) contents in N. ellipsosporum and N. piscinale subjected to various salt concentrations during three harvest times (days 5, 9, and 13). Data are mean ± SE of three replicates. Bars

for each harvest time followed by different letters show significant difference at P < 0.05 significance level

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Fig. 10 PE (a, b) and total PBP (c, d) contents in N. ellipsosporum and N. piscinale subjected to various salt concentrations during three harvest times (5, 9 and 13 days). Data are mean ± SE of three replicates. Bars

for each harvest time followed by different letters show significant difference at P < 0.05 significance level

phycobiliproteins. Moreover, antioxidative and ROS scavenging properties of phycobiliproteins also have been reported (e.g., Bhat and Madyastha 2000; Reddy et al. 2000; Estrada

et al. 2001; Benedetti et al. 2004; Huang et al. 2007). Therefore, accumulation of phycobiliproteins under salinity stress along with some antioxidant enzymes could contribute to protection against oxidative damages and/or stabilization. Moreover, the variability of phycobilisome size was also examined. The size of phycobilisomes can be usually represented by the ratio [PE + PC/APC] (Asadi et al. 2013). The size of phycobilisomes in N. ellipsosporum increased significantly under higher levels of NaCl compared to that of control. Phycobilisomes can exhibit a high sensitivity to external factors. Transfer of energy within these additional pigments follows the path from phycoerythrin to phycocyanin to allophycocyanin to the longwavelength pigment (Mimuro et al. 1986). PBSs are typically associated with PS II, and most of the light energy absorbed by phycobiliproteins is usually delivered to PS II reaction centers (Wang et al. 1977) and rarely to PS I (Mullineaux 1992). Increasing the size of phycobilisomes under salinity stress in N. ellipsosporum (Fig. 11a) could be considered as an adaptive mechanism to promote the efficiency of light energy transfer from antenna pigments to photosystem reaction centers.

Fig. 11 PE + PC/AP ratio in N. ellipsosporum (a) and N. piscinale (b) subjected to various salt concentrations during three harvest times (days 5, 9, and 13). Data are mean ± SE of three replicates. Bars for each harvest time followed by different letters show significant difference at P < 0.05 significance level

Conclusion The present study reveals substantial differences in the cellular responses between N. ellipsosporum and N. piscinale in response to salinity. The lower content of MDA combined with

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a higher content of phycobiliproteins and higher capacity for oxygen radical scavenging could possibly explain the ability of N. ellipsosporum to grow at higher NaCl concentrations than N. piscinale which appears to be less salt tolerant. According to this study, the content of phycobiliproteins increased under salt treatments in N. ellipsosporum and this species could be considered as a source of these valuable biopigments. Finally, the present study provides a better understanding of salinity effects on N. ellipsosporum and N. piscinale that can be used to enhance the yield and quality of Nostoc species in industrial production. Acknowledgments We thank the editor and two reviewers for constructive comments on the earlier version of this paper. Author contribution Maryam Rezayian has contributed in the major bench experiments. Dr. Vahid Niknam and Dr. Mohammad Ali Faramarzi equally designed the experiments, supervised the entire work, and framed the manuscript. All the authors read and approved the manuscript. Funding information The financial support of this research was equally provided by College of Science, University of Tehran and Tehran University of Medical Sciences.

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