ISSN 1021-4437, Russian Journal of Plant Physiology, 2017, Vol. 64, No. 6, pp. 861–868. © Pleiades Publishing, Ltd., 2017.

RESEARCH PAPERS

Salinity–Induced Modulations in the Protective Defense System and Programmed Cell Death in Nostoc muscorum1 A. Shamimb, A. Farooquia, *, M. H. Siddiquia, S. Mahfoozb, and J. Arifb aDepartment bDepartment

of Bioengineering, Faculty of Engineering, Integral University, Lucknow, 226026 India of Biosciences, Faculty of Applied Sciences, Integral University, Lucknow, 226026 India *e-mail: [email protected], [email protected] Received October 21, 2016

Abstract⎯To study the biochemical adaptive responses of the blue green algae Nostoc muscorum to the salinity-induced stress they were exposed to various concentrations (5, 10, 15, 20 or 200 mM) of sodium chloride (NaCl). A dose-dependent inhibition of total protein content showed an adverse effect of NaCl on the growth of N. muscorum. Four-day treatment of NaCl (5–20 mM) progressively increased the content of the total peroxide with subsequent increase of the superoxide dismutase (SOD) activity, proline and total phenol content only up to 10 mM NaCl. Higher concentrations of NaCl caused significant decrease in both the enzymatic and non-enzymatic antioxidants. Induction of two polypeptides of ~29.10 and 40.15 kD as well as upregulation of many polypeptides as compared to control indicates the induction of SOD and dehydrin-like proteins, which supports the theory of adaptation against the salt stress. Furthermore, adaptation of N. muscorum to lower concentrations (5–20 mM) of NaCl was also confirmed by no fragmentation of DNA while DNA fragmentation indicating programmed cell death (PCD) could only be seen at 200 mM NaCl for 12 hours. We hypothesized that proline may confer a positive role to combat salinity stress and the same was confirmed by treatment of the test blue green algae with exogenous proline (1 and 10 μM). The results exhibited 16% reduction in the level of total peroxides, which is a well known oxidative stress marker in the 10 μM proline-treated NaCl group as compared to direct exposure to NaCl. Keywords: Nostoc muscorum, sodium chloride stress, total peroxide, programmed cell death, adaptive response, dehydrin DOI: 10.1134/S1021443717060097

INTRODUCTION The major abiotic stresses that rank the most detrimental causing massive loss of crop productivity all over the world are soil salinity, drought and heat [1]. Soil salinity resulting from natural processes or from crop irrigation with saline water affects approximately 7% of the world’s total land area and nearly 40% of the world land surface can be categorized as from acute salinity problems. In warm and dry areas salt concentration increases in the upper soil layer due to high water losses which exceed precipitation. When a plant is exposed to high salinity, its major processes such as photosynthesis, protein synthesis, energy and lipid metabolism are affected. Crucial changes in ion and water homeostasis caused by high concentrations of salts lead to damage at the molecular level, arrested growth, and even death [2]. In addition, high salt, in the soil or in case of aquatic organisms in the sur1 The article is published in the original.

Abbreviations: chl⎯chlorophyll; DHN⎯dehydrin; MDA⎯ malondialdehyde; PCD⎯programmed cell death; ROS⎯reactive oxygen species; SDS-PAGE⎯sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SOD⎯superoxide dismutase.

rounding medium, lowers the osmotic potential and therefore leads to a restricted uptake of water. In the last two decades, there has been remarkable progress in understanding the biology of stress and adaptive responses to salinity in several bacteria and plants. Plant systems are rather difficult to study as compared to microbes as they grow very slowly and are less amenable to biochemical analysis and genetic manipulations. Although microbial models such as E. coli, Saccharomyces cerevisiae have indeed proved very useful in providing directions in which plant researches proceeded with beneficial consequences but as they do not have much in common to plants they can barely survive in soils. Plants exhibit much higher sensitivity to environmental stressors as compared to microbes [3]. In this context, cyanobacteria have emerged as a suitable system for studying plant responses to environmental stressors. Only recently it has been shown, that high salinity leads to programmed cell death (PCD) in higher plants which could be regarded as a salt adaptation mechanism [4]. Although PCD is a crucial event during normal plant growth and development, it is also induced by various biotic or abiotic stresses [5] in

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algae and higher plants such as hypersensitive response during pathogen attack, heat, UV-C irradiation or upon H2O2 induction. Srivastava et al. [6] provided a deep insight into the salinity-induced changes in the antioxidative defense system of Anabaena doliolum. Unfortunately, no report is available on the holistic view of salinity-induced changes in the antioxidative defense system, protein profile and DNA damage of N. muscorum, which is a dominant rice field blue green algae and exhibits salt tolerance comparable to Aulosira strains which was found to be widely distributed over a range of salinities [7]. It is opinioned that the enzymatic antioxidants may play a major role in detoxification of salinity-induced reactive oxygen species [8]. It is further hypothesized that, under severe salt stress condition, non-enzymatic antioxidants like proline will dominate over enzymatic antioxidants, owing to massive change in cellular homeostasis. Exogenous proline has been reported to improve the growth of salt stressed plants and the improvement was attributed to its role as an osmoprotectant for enzymes and membranes against salt inhibition rather than as a compatible solute [23]. The present study aims to explore the biochemical adaptive responses and the role of exogenous proline in conferring protection against the salt stress in the agriculturally important blue green algae, N. muscorum. MATERIALS AND METHODS Organism and growth conditions. Cyanobacterium Nostoc muscorum used in the present study as a test organism was obtained through the courtesy of Dr. S.M. Prasad, Department of Botany, University of Allahabad. The strain was originally isolated from rice fields near Allahabad, India. Axenic culture of N. muscorum was maintained in the culture room at the temperature 27 ± 2°C. For regular experiments, cultures were grown in BG-11medium (pH 7.0) [9] with nitrogen source under photosynthetic photon flux density of 75 μmol/(m2 s) and 14 hrs photoperiod. The cultures were manually shaken two to four times daily. Preparation of salt and proline solution for treatment. Stock solution of NaCl (1000 mM) was prepared by dissolving the required amount of sodium chloride in BG-11 media and from this stock solution working salt solutions having concentrations of 5, 10, 15, 20 and 200 mM was prepared by diluting the stock. All experiments were done using exponentially grown cultures (OD750-0.3) and conducted in triplicate as well as repeated at least twice to confirm the reproducibility of the results except for the crude protein extract preparation for which culture of cell density corresponding to (OD750) 0.5 was taken. 1000 μM stock solution of proline was freshly prepared and the solution was further sterilized by passing through Millipore membrane filter (0.22 μm). From

the stock solution, required concentrations of 1 and 10 μM of proline were freshly prepared in the BG-11 (positive) medium. To study the role of exogenous proline in ameliorating the effect of salt, logarithmic phase culture of N. muscorum was pre-incubated with various concentrations (1 and 10 μM) of proline for 24 hrs followed by removal of media. The proline pretreated N. muscorum was exposed to 10 mM NaCl for 4 days for different assays. The intracellular proline content of each sample was also determined. Growth experiments were performed in liquid medium and protein content was determined [10] after regular intervals for a period of 10 days. Photosynthetic pigment extraction and estimation. Chl a was extracted in 90% methanol and its concentration was determined from absorbance at 663 nm using the method of Mackinney [11]. Superoxide dismutase activity was measured spectrophotometrically [12]. Cyanobacterial cells after four days of treatment were collected by centrifugation and washed twice with 100 mM EDTA-phosphate buffer, pH 7.8. The cellular pellet was ground in an ice cold mortar and pestle with 100 mM EDTA-phosphate buffer, pH 7.8. The homogenate was centrifuged for 20 min at 8000 rpm. The supernatant fraction was used as the enzyme source. The 3 mL of reaction mixture contained 1.3 μM riboflavin, 13 mM L-methionine, 0.05 M Na2CO3, (pH 10.2), 63 μM p-nitroblue tetrazolium chloride and 0.1 mL of crude extract. Reaction was carried out in similar test tubes under illumination of 199 μmol/(m2 s) for 15 min from fluorescent lamp at 25°C. The initial rate of reaction as measured by the difference in increase in absorbance at 560 nm in the presence and absence of extract was proportional to the amount of enzyme. The unit of superoxide dismutase activity was defined as the amount of enzyme which caused a 50% inhibition of the reaction observed in the absence of enzyme. For the blank the reaction was run in darkness. For total peroxide estimation, 10 mL culture were extracted in 3.5 mL of 5% trichloroacetic acid and after centrifugation at 8000 rpm for 20 min level of total peroxide in the supernatant was measured by taking absorption at 480 nm [13]. The peroxide content was quantitatively estimated using the standard curve of hydrogen peroxide (H2O2) and results were expressed as μmol/g dry weight. Measurement of malondialdehyde. Lipid peroxidation in cyanobacterial sample under metal stress conditions was measured by estimating the end product malondialdehyde (MDA) in the metal treated and untreated cells of N. muscorum after each treatment as per the method of Heath and Packer [14]. Cells from different test samples were collected by centrifugation and washed twice in 5 mM phosphate buffer (pH 7.0). The cellular pellets were homogenised in 50 mM phosphate buffer. The resulting homogenate was centrifuged at 8000 rpm for 20 min. To 0.5 mL aliquot of

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4 6 Time, d

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Fig. 1. Growth characteristic of N. muscorum under different salt concentrations: 1—0 mM; 2—5 mM; 3—10 mM; 4—15 mM; 5—20 mM. Values are means ± SE (n = 3). The values are significant at (P < 0.01) compared to the control (Student’s t-test).

the supernatant, 2 mL of 20% trichloro acetic acid (TCA, w/v in TBA solution) containing 0.5% thiobarbituric acid (TBA, w/v in 0.2 N HCl) was added. The mixture was heated at 90°C for 20 min and then quickly cooled in ice bath. After centrifugation at 8000 rpm for 10 min, the absorbance of the supernatant was read at 532 nm. The value for nonspecific absorption of each sample at 600 nm was also recorded and substracted from the absorption recorded at 532 nm. The concentration of MDA, an end product of lipid peroxidation, was calculated from extinction coefficient 155 m mol–1 cm–1. Results are represented as nmol MDA/g dry weight. Estimation of proline. 20 mL of exponential phase culture was taken and centrifuged at 8000 rpm. The cells were homogenised in 10 mL of 3% sulfosalicylic acid were centrifuged at 8000 rpm for 10 min to remove cell debris. To the 2 mL of supernatant, 2 mL each of acid ninhydrin and glacial acetic acid was added and incubated at boiling temperature for 1 h. The mixture was extracted with toluene, and proline was quantified spectrophotometrically at 520 nm from the organic phase. Concentration of proline was estimated by referring to a standard curve of proline. The amount of proline in the sample was calculated in μg/g dry weight of samples. Estimation of total phenolics. 10 mL culture of cyanobacterium was centrifuged and cells were extracted in 2 mL of 80% of methanol at 4°C. After extraction, RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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the extracted sample was centrifuged at 8000 rpm. To 0.5 mL supernatant, 2 mL of sodium bicarbonate, 0.3 mL DDW, 0.2 mL Folin reagent was added and incubated in water bath until blue colour developed. The amount of phenol was calculated by measuring the optical density at 750 nm. The concentration of total phenol was calculated by standard curve of gallic acid and expressed as mg/g dry weight. Analysis of protein by SDS-PAGE. The total crude protein was prepared from cyanobacterial biomass by repeated freeze thawing and finally after cell disruption extraction was carried out in phosphate buffer saline (pH 7.4) [17]. Protein concentration was measured following the method of Lowry [10] using BSA as standard. The cell extract was used for SDS-PAGE analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out for comparative protein profile of untreated and NaCl treated N. muscorum [18]. Approximately 25 μL from treated and untreated extracts containing 25 μg of protein was loaded in each well. DNA extraction and analysis. A 50 mL of logarithmic phase culture of untreated and NaCl treated N. muscorum (OD750 0.5 at 750 nm) was taken and DNA was extracted by proteinase K digestion method [19]. Untreated and NaCl treated DNA were mixed with the sample buffer (0.125% bromophenol blue, 30% glycerol) in 3 : 1 ratio and loaded in the wells of 1% agarose gels. Electrophoresis was carried out for 2 hrs at 50 V and the gels were viewed under UV light. Statistical analysis was carried out using the SPSS software (SPSS, v. 7.0) to check the significant effects of the treatments. The number of independent variables for each experiment was three. RESULTS Effect on Growth Growth pattern of N. muscorum was monitored at regular intervals for 10 days in liquid medium by estimating the protein content after the supplementation of 0, 5, 10, 15 and 20 mM of NaCl (Fig. 1). NaCl at 5 mM did not cause marked effect even after 10 days of exposure as shown in the growth curve. Further, the effect became pronounced after 10 mM exposure. The lag phase continued for six days when culture was treated with 10 mM of NaCl while the high dose (15 and 20 mM) of NaCl did not show any growth even up to 10 days. Decline phase occurred at 15 and 20 mM concentrations after 10 days of the treatment. The inhibitory action of NaCl was more pronounced after 6 days of treatment, depicting a concentration and time dependent inhibition of growth (Fig. 1). Changes in Intracellular Peroxide After 4 days NaCl treatment induced the dosedependent formation of peroxide free radical (Fig. 2) No. 6

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Total perioxide, % control

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Fig. 2. Effect of different concentrations (0–20 mM) of NaCl on the peroxide radical level in N. muscorum. Total peroxide levels in untreated control was 610 ± 8 µmol/g dry weight × 102 Values are means ± SE (n = 3). The values are significant at (P < 0.01) compared to the control (Student’s t-test).

in N. muscorum. Low dose of NaCl (5 mM) increased the level of peroxide by 45% and the effect was doubled to 100% with 10 mM NaCl. Further 30% increase in total peroxide was also seen with 20 mM NaCl. Antioxidant Enzyme Data related to the SOD activity in N. muscorum under NaCl treatment are presented in Fig. 3. The activity of the SOD in untreated N. muscorum was 3.4 ± 0.23 Unit/mg protein. N. muscorum treated with NaCl at low concentrations (5 and 10 mM) stimulated the SOD activity by 27 and 38% respectively, whereas high concentrations (15 and 20 mM) showed moderate decrease in SOD enzyme activity by 13 and 25%, respectively, indicating the loss of enzymatic activity or synthesis due to cell death at high concentrations of NaCl. Non Enzymatic Antioxidants Similar to enzymatic antioxidants, non-enzymatic antioxidants also showed alterations following the exposure with NaCl. The data presented in Fig. 4a, represent changes in proline content in N. muscorum treated with various concentrations (5, 10, 15 and 20 mM) of NaCl. After 4 days of NaCl treatment free proline accumulated at all the concentrations except for a high dose of 20 mM. The increase in proline content was dose dependent only up to 10 mM NaCl. At 5 mM and 10 mM there was a significant increase of 20% and 45% in proline content, respectively, while the higher concentration (20 mM) reduced the level of proline by 15% as compared to control.

Superoxide dismutase activity, % control

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Fig. 3. Effect of different concentrations (0–20 mM) of NaCl on the superoxide dismutase activity (SOD) in N. muscorum. SOD activity in untreated control was 3.4 ± 0.23 Unit/mg protein. Values are means ± SE of three replicates. The values are significant at (P < 0.01) compared to the control (Student’s t-test).

Data related to accumulation of total phenols under salt stress is presented in Fig. 4b. Enhancement in total phenols in N. muscorum following the NaCl exposure (5 and 10 mM) was 45 and 55%, respectively. Interestingly, higher doses of 15 mM NaCl showed slight increase of 15% as compared to control whereas highest dose of 20 mM NaCl exhibited a decline of 10% in total phenols. Protein Profile The electrophoretic pattern of crude extract of protein isolated from the control and NaCl-treated cyanobacterial cells after four days of treatment are shown in Fig. 5. A moderately toxic dose of 10 mM was selected for visualisation of alterations at the protein level. The bands resolved were classified according to their molecular weight markers. The Lane 2 and Lane 3 showed the protein profiles in control and NaCltreated N. muscorum respectively. From the picture it is clear that a dose of 10 mM of NaCl led to the induction of polypeptide having molecular weight of around 29.10 kD. Another polypeptide of molecular weight about 40.15 kD was also induced. Two polypeptide bands of molecular weight around 36.55 and 63.22 kD showed remarkable upregulation. One polypeptide band of molecular weight 65.62 kD showed remarkable downregulation. DNA Profile The gel picture clearly shows the comparative DNA profile of salt treated and untreated samples of

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Fig. 4. Effect of different concentrations (0–20 mM) of NaCl on the proline content (a) and on the total phenols (b) of N. muscorum. Proline content in untreated control was 9.5 ± 0.6 µg/g dry weight. Total phenols in untreated control was 0.6 ± 0.03 mg/g dry weight. Values are means ± SE (n = 3). The values are significant at (P < 0.01)compared to the control (Student’s t-test).

N. muscorum (Fig. 6). Genomic DNA was detected in the form of a single sharp band on the upper portion of the gel in the untreated sample showing no fragmentation of DNA. Exposure of N. muscorum to 5–20 mM NaCl for 12 hrs did not show additional band or fragmentation of DNA. Further, higher dose of 200 mM NaCl also did not cause any fragmentation after 6 hrs of incubation, however, increased incubation of 12 hrs showed remarkable fragmentation.

37% at 1 and 10 µM proline, respectively. Further, the level of total peroxides and MDA was found to be lowered in the proline-treated NaCl group as compared to direct exposure to NaCl. NaCl treatment enhanced the peroxide content by over 2-fold after four days of exposure. Contrary to this, proline pre-treated 1

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Ameliorative Effect of Exogenous Proline on Chlorophyll a, Total Peroxides and MDA Content of N. muscorum Exposed to NaCl To verify whether exogenous proline modifies the internal amino acid content, the intracellular proline content of N. muscorum was estimated (Table 1). The level of intracellular proline increased by 1 and 25% at 1 and 10 µM proline supplementation as compared to control which indicates that proline was taken up by the cyanobacterium. Sodium chloride (10 mM) alone treatment increased intracellular proline by 22% compared to the control. While a decrease was seen in the intracellular proline content in N. muscorum exposed to higher concentration (20 mM) of NaCl (Fig. 5). This may suggest a role of proline in neutralizing the toxic effects. Further, the level of chlorophyll a (Chl a) increased by 6.7% and 12.7% respectively, as compared to control at 1 and 10 µM proline supplementation which indicates that proline is a growth promoter as Chl a content is growth linked (Table 1). NaCl treated N. muscorum exhibited 51% decrease in Chl a content, however, when proline-treated N. muscorum was exposed to NaCl, the decrease was reduced to 33 and RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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29 20 Fig. 5. Protein profile of N. muscorum treated with 10 mM NaCl (Lane 1—Marker; Lane 2—Control; Lane 3— 10 mM NaCl treated N. muscorum). The polypeptide band which is induced in response to NaCl is indicated by arrows. Molecular weights are expressed in kD. No. 6

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C

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Fig. 6. DNA fragmentation pattern in presence of 0– 200 mM NaCl. Panel A: Lane 1-DNA from untreated N. muscorum (C); Lanes 2-5-DNA from 5, 10, 15 and 20 mM NaCl-treated N. muscorum after 6 hrs incubation; Panel B: Lane 1—DNA from untreated N. muscorum (C); Lanes 2 and 3— DNA from 200 mM NaCl- treated N. muscorum after 6 and 12 hrs incubations, respectively.

N. muscorum showed a decrease of 9 and 16% in the level of total peroxides by 1 and 10 µM proline, respectively (Table 1). One of the very important stress markers, lipid peroxidation measured in terms of MDA content also showed a similar pattern of alteration. Proline pre-treatment at 1 and 10 µM proline reduced the MDA content by 5.8 and 10% in NaCl-treated group. These results reasonably indicate the ameliorative role of proline in protecting the cyanobacterial cells exposed to salt stress. DISCUSSION Salinity-induced disturbances in cellular homeostasis causes an increase in total peroxide production to a level much over their scavenging potential leading to oxidative stress as depicted by enhanced peroxide and subsequent lipid peroxidation. All these alterations finally led to arrested growth as evident from

Fig. 1. These results are in agreement with the findings of Srivastava [8]. Cyanobacteria have remarkable capacity to develop elaborate protective mechanisms and adapt to environmental changes [20]. One of the protective mechanisms is the induction in the activities of antioxidant enzymes by abiotic or biotic stresses in plants including algae and cyanobacteria to avoid damage caused by ROS [10]. SOD, which dismutates the superoxide radical, is known to be induced by different stresses in a variety of cyanobacteria. The increase in the activity of SOD of the N. muscorum observed in our study following NaCl treatment could be ascribed to the increased production of superoxide anion (O 2− ) in the cells of N. muscorum. Cells treated with NaCl at low concentration (5 and 10 mM) stimulated the SOD activity whereas loss of enzymatic activity or synthesis occurred at high concentrations due to arrested growth. Similar results have been reported by Tang et al. [21]. Not only the enzymatic but the non-enzymatic low molecular weight antioxidants such as ascorbate, proline, total phenols and glutathione, etc. also play an important role either alone or in association with enzymatic antioxidants. Many organisms belonging to bacteria, cyanobacteria, algae, fungi [22] intracellularly accumulate one or more low-molecular weight organic compounds called compatible solutes, so as to maintain osmotic balance of their cytoplasm against high osmolarity of the environment. Proline is the major amino acid associated with environmental stresses (salinity, extreme temperatures, UV radiation and heavy metals). Proline has been known as important indicator for stress tolerance and functions as stabilizer, a metal chelator, as an inhibitor of lipid peroxidation, a hydroxyl radical, and a singlet oxygen scavenger [23]. Not many reports are available on the role of proline in stress tolerance in cyanobacteria. Effect of salinity to proline content in Anabaena variabilis was reported previously [1]. In the present observation increased accumulation of proline following the treatment of NaCl could be due to an increase enzyme activity of glutamate kinase resulting in an increase in biosynthesis of proline.

Table 1. Ameliorative effect of exogenous proline on chlorophyll a, total peroxides, MDA and intracellular proline contents of N. muscorum exposed to NaCl Treatments

Chla

Total peroxide

Control NaCl (10 mM) Proline (1 µM) Proline (10 µM) Proline (1 µM) + NaCl (10 mM) Proline (10 µM) + NaCl (10 mM)

1.50 ± 0.02 0.74 ± 0.01 1.60 ± 0.03 1.69 ± 0.03 0.95 ± 0.02 1.00 ± 0.02

610 ± 8 1220 ± 16 614 ± 8ns 616 ± 7ns 1109 ± 14 1030 ± 11

MDA

Intracellular proline

661 ± 8 1310 ± 12 790 ± 8ns 784 ± 8ns 1234 ±12 1175 ± 11

9.5 ± 0.6 13.8 ± 1.0 14.7 ± 1.0ns 17.2 ± 1.0 18.0 ± 1.2 19.5 ± 1.5

The values represent means ± SE (n = 3). All the treatments are significantly different (P < 0.01) from control (Student’s t-test). ns – not significant. Units for Chla (µg/mL); total peroxide (μmol/102 g dry weight); MDA (nmol/g dry weight); proline (μg/g dry weight). RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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Phenolic acids are reported to have the potential to serve as antioxidants under stress conditions. Although positive correlations among the accumulation of polyphenolics, enhanced antioxidant activity and tolerance to stresses have been reported in plant species [24], only scarce reports are available on phenolic compounds-mediated salt-stress tolerance responses in cyanobacteria. We demonstrated induced accumulation of phenolic acids in N. muscorum under salt-stress conditions and correlated with enhanced antioxidant activity as a possible alternative mechanism to overcome stress-induced damages in the cells. Similar results have been reported by Tang et al. [21]. Earlier studies with two strains of Anabaena revealed that salinity-induced modification of protein synthesis occurs in cyanobacteria, like in plants, and that some proteins synthesized during salt stress may be essential for cyanobacterial osmotic adaptation [25]. Similarly, in our investigation exposure to salinity resulted in a qualitative and quantitative regulation of proteins in N. muscorum. Synthesis of a wide spectrum of proteins is either curtailed or enhanced, and in addition, synthesis of specific protein is induced de novo. The response to salt is very rapid, varies with duration of exposure to salinity and is dependent on the concentration of NaCl. The two polypeptides (29.10 and 40.15 kD) are induced which are likely to be that of SODs. Further, some of the protein bands also seem to be in range of dehydrin-like protein which is induced at 29–44 kD [26] indicating the up regulation of signaling pathways involved in salt/osmo regulation (Fig. 5). Ruibal et al. [27] demonstrated the expression of four DHNs and two DHN-like proteins in the predicted proteome of Physcomitrella patens which was induced by salt and osmotic stress. Programmed cell death is a key element in normal plant growth and development which may also be induced by various abiotic and biotic stress factors including salt stress. Increasing evidence has shown that PCD-like cell death can occur along the phylogenetic tree from unicellular amoebae and bacteria to multicellular ciliates and oomycete and to higher animals and plants [5]. The gel picture clearly shows the comparative DNA profile of NaCl treated and untreated samples of N. muscorum (Fig. 6). From the picture it is clear that a dose of 200 mM NaCl caused almost no fragmentation after 6 hrs incubation. However, prolonged salt stress (12 hrs) of similar dose led to the fragmentation of DNA of NaCl treated N. muscorum. This fragmentation may be due to degradation of organelles by autophagy, a special form of PCD, in NaCl treated cells [4]. Growth and pigment composition indicated an active metabolism which supports programmed rather than necrotic cell death in N. muscorum after salt stress. Thus, results suggest that like PCD in eukaryotes, PCD in N. muscorum may be an active RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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process towards adaptation to adverse environments [4]. Although in the past, most attention has been concerned with the role of proline as an osmoprotectant, its accumulation under heavy metals, high salinity and light-induced stress [23] nevertheless the underlying mechanism involved in stress tolerance have received far less attention. Further, exogenous proline has been reported to protect plants under stress, which improved tolerance of tobacco cell culture [28]. In our study, proline pretreatment of the blue green algae N. muscorum resulted in a decline of the parameters like total peroxides and MDA after exposure to NaCl. These results are in agreement to the findings of Chris et al. [29] who demonstrated the role of exogenous proline in detoxification of harmful ROS generated under UV stress. Khedr et al. [30] also demonstrated that proline induces the expression of salt-stress-responsive proteins and may improve the adaptation of Pancratium maritimum L. to salt-stress. It is concluded from this study that N. muscorum up to10 mM salt concentration has ability to with stand the mesosaline soil with strong antioxidant defense mechanism. Therefore, this blue green algae can be used as a model system for studies relating to stress induced biochemical and molecular alterations. ACKNOWLEDGMENTS We are grateful to the Vice Chancellor, Integral University, Lucknow, India, for providing financial assistance and necessary laboratory facilities. The manuscript has been approved by competent authority and the assigned communication number is IU/R&D/2016-MCN0004. RERFERENCES 1. Syiem, B.M. and Nongrum, A.N., Increase in intracellular proline content in Anabaena variabilis during stress conditions, J. Appl. Nat. Sci., 2011, vol. 3, pp. 119–123. 2. Demiral, T. and Türkan, I., Exogenous glycinebetaine affects growth and proline accumulation and retards senescence in two rice cultivars under NaCl stress, Environ. Exp. Bot., 2006, vol. 56, pp. 72–79. 3. Ma, P. and Liu, J., Leymus chinensis that enhances salt stress tolerance in Saccharomyces cerevisiae: isolation and characterization of a novel plasma membrane intrinsic protein gene, LcPIP1, Appl. Biochem. Biotechnol., 2012, vol. 166, pp. 479–485. 4. Affenzeller, M.J., Darehshouri, A., Andosch, A., Lutz, C., and Lutz, M.U., Salt stress-induced cell death in the unicellular green alga Micrasterias denticulate, J. Exp. Bot., 2009, vol. 60, pp. 939–954. 5. Unsal, N.P., Buyuktuncer, E.D., and Tufekci, M.A., Programmed cell death in plants, J. Cell Mol. Biol., 2005, vol. 4, pp. 9–23. No. 6

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RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

Vol. 64

No. 6

2017