Cm 2007a Physiological And Biochemical

Curr Microbiol (2007) 55:334–338 DOI 10.1007/s00284-007-0191-1

Physiological and Biochemical Alterations in a Diazotrophic Cyanobacterium Anabaena cylindrica Under NaCl Stress Pratiksha Bhadauriya Æ Radha Gupta Æ Surendra Singh Æ Prakash Singh Bisen

Received: 23 March 2007 / Accepted: 28 April 2007 Ó Springer Science+Business Media, LLC 2007

Abstract Growth, morphological variation, and liquid chromatography–photodiode array detection–mass spectrometric analysis of pigments have been studied in a diazotrophic cyanobacterium Anabaena cylindrica in response to NaCl stress. The chlorophyll and cellular protein contents increased initially in response to 50 mM NaCl. Further increment in NaCl concentration, however, resulted in a significant decrease in both chlorophyll and cellular protein. A. cylindrica cells subjected to NaCl stress also showed morphological variations by having alteration in their size and volume. A. cylindrica cells subjected to NaCl stress also exhibited altered plastoquinone and chlorophyll-a (chl a) levels in comparison to its NaCl-untreated counterpart. Furthermore, a relative increase in plastoquinone level and a subsequent decrease in chl a level were recorded in NaCl adapted cells of A. cylindrica in response to NaCl stress. These results suggest that owing to adaptation various morphological, physiological, and biochemical changes occur in the cyanobacterium A. cylindrica in response to NaCl stress. Keywords Anabaena
Cyanobacterium
Stress biology
Physiology
Environmental stress response

P. Bhadauriya
R. Gupta
P. S. Bisen (&) Department of Biotechnology, Madhav Institute of Technology & Science, Race Course Road, 474001 Gwalior, India e-mail: [email protected] S. Singh School of Studies in Microbiology, Jiwaji University, 474011 Gwalior, India

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Introduction The environment is fast becoming increasingly stressful. Studies on the response of microbes and plants to environmental stressors, such as salinity, drought, heat, and so forth, form a thrust area of contemporary biological research. Cyanobacteria exhibit a close phylogenetic relationship with plant chloroplast and therefore, are regarded as most appropriate model system for studying plant responses to various stresses [1, 13]. Cyanobacteria are also able to adapt to a wide range of environmental conditions [23]. Among them, salinity is one of the most important factors that limits the growth and productivity of plants, eukaryotic microorganisms, and bacteria [11]. Salinity has a considerable effect on agriculture, affecting almost half of the world’s irrigated areas of land to a moderate or high degree. Biological reclamation of such soil with cyanobacterial species was first advocated by Singh [22]. To NaCl in trace amounts appears essential for some of the metabolic functions in cyanobacteria [2, 7, 20, 24], but elevated levels might inhibit growth [4, 12]. Survival and growth of cyanobacteria in habitats with high or fluctuating salinities require the adjustment of their cytoplasmic water potential by active extrusion of excess inorganic ions from the cell. Accumulation of compatible solutes [10] and expression of salinity stress proteins [13] are prerequisites for adaptation to a high salt concentration. This article deals with the growth, morphological variation, and liquid chromatography–photodiode array detection–mass spectrometric (LCMS) analysis of pigments in a diazotrophic cyanobacterium Anabaena cylindrica in response to NaCl stress.

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Materials and Methods

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Anabaena cylindrica was axenically grown in BG-110 medium [19] devoid of any nitrogen source. The cultures were incubated in a culture room at 25 ± 1°C and illuminated with day-light fluorescent tubes having the photon fluence rate of 50 lmol/m2/s at the surface of the culture vessels. All experiments were performed with log-phase cultures having a cell density of 400 lg protein/mL. Exponentially growing cells of A. cylindrica grown in BG-110 medium containing 200 mM NaCl for 6 days were designated as NaCl-adapted cells. Scanning Electron Microscopy Anabaena cylindrica cells were harvested by centrifugation and prefixed in culture medium by the addition of an equal volume of 1% glutraldehyde in phosphate buffer. Cells were allowed to stand for 30 min on ice, pelleted, suspended in phosphate buffer with 2% glutraldehyde, and incubated for 1 h at room temperature. The samples were washed with phosphate buffer, postfixed in 1% osmium tetraoxide in the same buffer and washed once in distilled water. The samples were then kept on carbon stubs and gold coatings were done with a fine-coat ion sputter JFC 1100. The samples were observed under scanning electron microscope (JEOL JSM-840).

Growth (µg chl a ml -1)

Organism and Culture Conditions

12 10 8 6 4 2 0 2

4

8

12

16

Days 0mM

50mM

100mM

200mM

Fig. 1 Growth (in terms of chl a) of Anabaena cylindrica in response to various concentrations of NaCl

To Octadecyl silane (ODS) column, utilizing methanol/ propanol as mobile phase by using atmospheric pressure chemical ionization (APCI) method for comparative analysis of chl a and plastoquinone, was used. Total ion chromatogram was taken and photodiode array (PDA) detector was used for level of absorption of specific wavelength.

Results and Discussion Analytical Methods Whereas chlorophyll-a (chl a) was assayed following the method of Mackinney [16], and cellular protein was estimated by the method of Lowry et al. [15] using bovine serum albumin as the standard. Pigment Sample Preparation Samples for photopigment analysis were obtained by vacuum filtration onto a glass fiber filters (Whatman GF/ F). Filters were placed in microcentrifuge tubes (2 mL); a known volume (0.5–1.5 mL) of 100% acetone was added and the mixture was sonicated for 30–60 s in an ice slurry to reduce heating. Tubes were wrapped in aluminum foil, placed in freezer (–20°C) and extracted overnight (12 h). After extraction, the supernatant was filtered through a 0.45-lm polytetrafluoroethylene (PTFE) filter and a known volume of the extract was dispensed into a fresh vial. Ammonium formate (1 M) was added to the vial in a ratio of three parts extract to one part of ammonium formate just prior to run an ionpairing (IP) solution.

Because, the growth of most of the plants are adversely affected beyond 50 mM NaCl concentration [3, 21], an attempt was made to evaluate the effect of 50–200 mM NaCl on the growth of A. cylindrica, which also shows plantlike photosynthesis. Figures 1 and 2 show the effect of various concentrations of NaCl on the growth of A. cylindrica in terms of chl a and protein, respectively. It is evident that growth of A. cylindrica (both in terms of chl a and protein) was slightly increased at 50 mM NaCl up to day 4. A further increment in NaCl concentration, however, resulted in a reduction of growth. This indicated that whereas NaCl at 50 mM is required for the growth of A. cylindrica, its higher concentrations are found to be cytotoxic. The inhibition in growth in response to a high concentration of NaCl might be due to NaCl-induced inhibition of photosynthetic and respiratory systems. It has also been reported that cyanobacterium cultured with a higher salt concentration had lower chlorophyll and protein contents [25]. Thus, NaCl stress adversely limits the growth and protein synthesis in A. cylindrica because of either its osmotic or ionic impact on the cell metabolism and consequently causing other physi-

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Growth (µg protein ml-1)

350 300 250 200 150 100 50 0 2

4

0mM

8 Days

50mM

100mM

12

16

200mM

Fig. 2 Growth (in terms of protein) of Anabaena cylindrica in response to various concentrations of NaCl

Fig. 4 Part of LC-MS total ion chromatogram of pigments Chl a and Plastoquinone of NaCl-untreated (a) and NaCl (200 mM) adapted cells (b) of Anabaena cylindrica

Fig. 5 Mass spectra of chlorophyll a (a) and plastoquinone (b) of Anabaena cylindrica Fig. 3 Scanning electron photomicrograph (magnification at 5000x) of NaCl-untreated (a) and NaCl (200 mM)-adapted cells (b) of Anabaena cylindrica

ological and biochemical imbalances. These results are also supported by the earlier findings [5, 8, 18]. In order to see whether A. cylindrica cells could produce any morphological change in response to NaCl stress, the

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scanning electron microscopy of the NaCl-untreated and NaCl-adapted cells of A. cylindrica was done (Figs 3a and 3b). It is evident from the scanning electron photomicrographs that A. cylindrica cells subjected to NaCl stress showed morphological changes by having alteration in their size and volume (Fig. 3b). The cell wall of A. cylindrica

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chlorophyll was altered to 915 while the m/z ratio of plastoquinone remained at 750. Chl a and plastoquinone had a specific absorbance peak at 660 and 270 nm, respectively (Fig. 6). In cyanobacteria, photosystem I (PSI) functions as a biosolar energy converter, catalyzing one of the first steps of oxygenic photosynthesis. Chl a is one of the most important components of PSI. It captures the solar light by means of large antenna systems and transfers energy to the center of the complex, driving the transmembrane electron transfer from plastoquinone to ferredoxin [9]. Thus, the observed changes in pigments in response to NaCl stress might be correlated with the accumulation of intracellular sodium ions as salt stress changes the ratio K : Na, which seems to affect the bioenergetic processes of photosynthesis. Similar changes in the pigment composition in the green alga Chlorococcum sp. have also been reported under stress conditions [17]. Thus, an increase in plastoquinone and a subsequent decrease in chl a recorded in NaCladapted cells of A. cylindrica might be considered one of the important unknown regulatory mechanism for the maintenance of the biosynthetic machinery with an important role in salinity stress. Fig. 6 Absorption spectra (PDA detector) of chlorophyll a (a) and plastoquinone (b) at 660

also seemed to be expanded and stretched at the junction of the septum and nascent pole in response to tension created by osmotically derived hydrostatic pressure. However, the shape of the cell was found to be generally preserved and deformations of the cell surface were found to be almost absent. Similar stress-induced morphological changes have also been reported in microorganisms [6, 14]. Because NaCl stress is supposed to inactivate the photosynthetic machinery in A. cylindrica, an attempt has also been made to see the changes of two important pigments of photosynthesis (i.e., chl a and plastoquinone) in response to NaCl stress by using the LCMS technique. Chl a and plastoquinone of the NaCl-adapted and NaCl-untreated cells of A. cylindrica were separated and purified from other pigments on a ODS column and subjected to a PDA detector. It is evident from the data of the LCMS analysis of pigments of A. cylindrica that pigments of NaCl-adapted cells of A. cylindrica exhibited altered plastoquinone and chl a levels compared to its NaCl-untreated counterpart (Figs. 4a and 4b). An increase in plastoquinone and a subsequent decrease in chl a were recorded in NaCladapted cells of A. cylindrica (Fig. 4b). The mass spectra of chl a and plastoquinone of A. cylindrica are shown in Figs. 5a and 5b, respectively. Chl a is a porphyrin derivative having molecular weight 893.49. Due to Na adduction of the molecular ion (M+Na+), the m/z ratio of

Acknowledgments We thank Defence Research Development and Establishment, Gwalior, M.P. India for providing Instrumental Facility and Professor. N. K. Sah, Head, Department of Biotechnology, MITS Gwalior M.P. India, for his support.

References 1. Apte SK (2001) Coping with salinity/water stress: Cyanobacteria show the way. Proc Indian Nat Sci Acad B 67:285–310 2. Apte SK, Thomas J (1984) Effect of sodium on nitrogen fixation in Anabaena torulosa and Plectonema boryanum. J Gen Microbiol 130:1161–1168 3. Apte SK, Thomas J (1997) Possible amelioration of coastal soil salinity using halotolerant nitrogen-fixing cyanobacteria. Plant Soil 189:205–211 4. Apte SK, Reddy BR, Thomas J (1987) Relationship between sodium influx and salt tolerance of nitrogen-fixing cyanobacteria. Appl Environm Microbiol 53:1934–1939 5. Blumwald E, Wolosin JM, Packer L (1984) Na+/H+ exchange in cyanobacterium Synechococcus 6311. Biochem Biophys Res Commun 122:452–459 6. Cefalı` E, Patane` S, Arena A, Saitta G, Guglielmino S, Cappello S, Allegrini M, Nicolo` M (2002) Morphologic variations in bacteria under stress conditions: Near-Field Opt Studies Scan 24:274–283 7. Garcia-Gonzalez M, Sanchez-Maeso E, Quesada A, Fernandez E (1987) Sodium requirement for photosynthesis and nitrate assimilation in a mutant of Nostoc muscorum. J Plant Physiol 127:423–429 8. Gorham J, Wyn-Joes RG, MCDonnel E (1985) Some mechanism of salt tolerance in crop plants. Plant Soil 89:15–40 9. Grotjohann I, Fromme P (2005) Structure of cyanobacterial photosystem I. Photosynth Res 85(1):51–72 10. Hangemann M, Erdmann N (1997) Environmental stresses. Pages 156–221 in Rai AK (ed) Cyanobacterial nitrogen metab-

123

338

11.

12.

13.

14.

15.

16. 17.

P. Bhadauriya et al.: Alterations in Anabaena cylindrica olism and environmental biotechnology, Springer, Heidelberg/ Narosa Publishing, New Delhi Inabha M, Sakamato A, Murata N (2001) Functional expression in E.coli of low-affinity and high-affinity Na+ (Li)+/H+ antiporters of Synechocystis. J Bacteriol 183:1376–1384 Jeanjean R, Matthijs HCP, Onana B, Havaux M, Joset F (1993) Exposure of cyanobacterium Synechocystis PCC 6803 to salt stress induces concerted changes in respiration and photosynthesis. Plant Cell Physiol 43:1073–1079 Joset F, Jeanjean R, Hagemann M (1996) Dynamics of response of cyanobacteria to salt stress: deciphering the molecular events. Physiol Plant 96:738–744 Koch AL, Higgins ML, Doyle RJ (1982) The role of surface stress in the morphology of microbes. J Gen Microbiol 128(5):927–945 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with folin phenol reagent. J Biol Chem 193:265– 275 Mackinney G (1941) Absorption of light by chlorophyll solutions. J Biol Chem 140:315–322 Masojı´dek J, Torzillo G, Kopecky´ J, et al. (2000) Changes in chlorophyll fluorescence quenching and pigment composition the green alga Chlorococcum sp. grown under nitrogen deficiency and salinity stress. J App Phycol 12:417–426

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18. Moisander PH, McClinton E, Paeral HW (2002) Salinity effects on growth, photosynthetic parameters, and nitrogenase activity in eustarine planktonic cyanobacteria. Microbial Ecol 43(4):432–434 19. Rippka R, Deruelles J, Waterbury JB, Herdmann M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1–61 20. Sanchez-Maeso E, Fernandez-pinas F, Garcia-Gonzalez M, Fernandez-Valiente E (1987) Sodium requirement for photosynthesis and its relationship with dinitrogen fixation and external CO2 concentration in cyanobacteria. Plant Physiol 85:585–587 21. Serrano R, Gaxiolam R (1994) Microbial models and salt stress tolerance in plants. Crit Rev Plant Sci 13:121–138 22. Singh RN (1961) Role of blue-green algae in nitrogen economy of Indian agriculture. Indian Council of Agricultural Research, New Delhi 23. Tandeau de Marsac N, Houmard J (1993) Adaptation of cyanobacteria to environmental stimuli: new steps towards molecular mechanisms. FEMS Microbiol Rev 104:119–190 24. Thomas J, Apte SK (1984) Sodium requirement and metabolism in nitrogen-fixing cyanobacteria. J Biosci 6:771–794 25. Vonshak A, Aksa N, Bunnag B, Tanticharoen M (1996) Role of light and photosynthesis on the acclimation process of the cyanobacterium Spirulina platensis to salinity stress. J Appl Phycol 8:119–124