Physiological characterization and stress-induced metabolic responses of Dunaliellasalina isolated from salt pan

A Dunaliella strain was isolated from salt crystals obtained from experimental salt farm of the institute (latitude 21.46 N, longitude 72.11°E). The comparative homology study of amplified molecular signature 18S rRNA, proves the isolated strain as D. salina. The growth pattern and metabolic responses such as proline, glycine betaine, glycerol, total protein and total sugar content to different salinity (from 0.5 to 5.5 M NaCl) were studied. The optimum growth was observed at 1.0 M NaCl and thereafter it started to decline. Maximum growth was obtained on 17th day of inoculation in all salt concentrations except 0.5 M NaCl, whereas maximum growth was observed on 13th day. There were no significant differences (P < 0.01) in chlorophyll a/b contents (1.0–1.16 ± 0.05 μg chl. a and 0.2–0.29 ± 0.01 μg chl. b per 106 cells) up to 2.0 M NaCl, however at 3.0 M NaCl a significant increase (2.5 ± 0.12 μg chl. a and 0.84 ± 0.4 μg chl. b per 106 cells) was observed which declined again at 5.5 M NaCl concentration (2.0 ± 0.1 μg chl. a and 0.52 ± 0.03 μg chl. b per 106 cells). Stress metabolites such as proline, glycine betaine, glycerol and total sugar content increased concomitantly with salt concentration. Maximum increase in proline (1.4 ± 0.07 μg), glycine betaine (5.7 ± 0.28 μg), glycerol (3.7 ± 0.18 ml) and total sugar (250 ± 12.5 μg) per 105 cells was observed in 5.5 M NaCl. A decrease in total protein with reference to 0.5 M NaCl was observed up to 3.0 M NaCl, however, a significant increase (P < 0.01) was observed at 5.5 M NaCl (0.19 ± 0.01 μg per 105 cells). Inductive coupled plasma (ICP) analysis shows that intracellular Na+ remained unchanged up to 2.0 M NaCl concentration and thereafter a significant increase was observed. No relevant increase in the intracellular level of K+ and Mg++ was observed with increasing salt concentration. Evaluation of physiological and metabolic attributes of Dunaliellasalina can be used to explore its biotechnological and industrial potential.

[1]  C. Hirsch,et al.  Method for Isolation and Purification of Cyanobacteria , 1991, Applied and environmental microbiology.

[2]  M. E. Clark,et al.  Living with water stress: evolution of osmolyte systems. , 1982, Science.

[3]  G. F. Humphrey,et al.  New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton , 1975 .

[4]  Peng Wang,et al.  Stable expression of hepatitis B surface antigen gene in Dunaliella salina (Chlorophyta) , 2003, Journal of Applied Phycology.

[5]  A. Kaplan,et al.  Salt-Induced Metabolic Changes in Dunaliella salina. , 1980, Plant physiology.

[6]  J. Cousin,et al.  Ionic regulation of the unicellular green algaDunaliella tertiolecta: Response to hypertonic shock , 1984, The Journal of Membrane Biology.

[7]  A. Wrona,et al.  Saline Culture of Crops: A Genetic Approach , 1980, Science.

[8]  F. James Rohlf,et al.  Biometry: The Principles and Practice of Statistics in Biological Research , 1969 .

[9]  S. Goodison,et al.  16S ribosomal DNA amplification for phylogenetic study , 1991, Journal of bacteriology.

[10]  J. González,et al.  Effect of NaCl on germination, growth, and soluble sugar content in Chenopodium quinoa Willd. seeds , 2000 .

[11]  A. Cowan,et al.  Dunaliella salina: A model System for Studying the Response of Plant Cells to Stress , 1992 .

[12]  F. Smith,et al.  COLORIMETRIC METHOD FOR DETER-MINATION OF SUGAR AND RELATED SUBSTANCE , 1956 .

[13]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[14]  A. Oren,et al.  Interrelationships between Dunaliella and halophilic prokaryotes in saltern crystallizer ponds , 2006, Extremophiles.

[15]  P. Singh,et al.  Isolation and characterization of an indigenous isolate of Dunaliella sp. for β‐carotene and glycerol production from a hypersaline lake in India , 2003, Journal of basic microbiology.

[16]  R. Contreras,et al.  Molecular identification of Dunaliella sp. utilizing the 18S rDNA gene , 2000, Letters in applied microbiology.

[17]  R. Raja,et al.  PCR-identification of Dunaliella salina (Volvocales, Chlorophyta) and its growth characteristics. , 2007, Microbiological research.

[18]  M. Vaara,et al.  Two Improved Methods for Obtaining Axenic Cultures of Cyanobacteria , 1979, Applied and environmental microbiology.

[19]  M. Ginzburg Measurements of Ion Concentrations in Dunaliella parva Subjected to Hypertonic Shock , 1981 .

[20]  A. Shevchenko,et al.  Enhanced Photosynthesis and Redox Energy Production Contribute to Salinity Tolerance in Dunaliella as Revealed by Homology-Based Proteomics1 , 2004, Plant Physiology.

[21]  A. Sharma,et al.  EFFECT OF VARIOUS ABIOTIC STRESSES ON THE GROWTH, SOLUBLE SUGARS AND WATER RELATIONS OF SORGHUM SEEDLINGS GROWN IN LIGHT AND DARKNESS. , 2001 .

[22]  J. Cousin,et al.  Ionic regulation of the unicellular green algadunaliella tertiolecta , 1982, The Journal of Membrane Biology.

[23]  A. Ben‐Amotz,et al.  The Role of Glycerol in the Osmotic Regulation of the Halophilic Alga Dunaliella parva. , 1973, Plant physiology.

[24]  K. Strange,et al.  Cellular and Molecular Physiology of Cell Volume Regulation , 1993 .

[25]  H. U. Riisgård,et al.  Further studies on volume regulation and effects of copper in relation to pH and EDTA in the naked marine flagellate Dunaliella marina , 1980 .

[26]  M. Borowitzka,et al.  The taxonomy of the genus Dunaliella (Chlorophyta, Dunaliellales) with emphasis on the marine and halophilic species , 2007, Journal of Applied Phycology.

[27]  Zhengkai Xu,et al.  Isolation and characterization of a sodium-dependent phosphate transporter gene in Dunaliella viridis. , 2006, Biochemical and biophysical research communications.

[28]  Md. Anamul Hoque,et al.  Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities. , 2007, Journal of plant physiology.

[29]  H. Bohnert,et al.  Adaptations to Environmental Stresses. , 1995, The Plant cell.

[30]  Mariela A. González,et al.  The effect of temperature and irradiance on the growth and carotenogenic capacity of seven strains of Dunaliella salina (Chlorophyta) cultivated under laboratory conditions. , 2005, Biological research.

[31]  Mariela A. González,et al.  REAPPRAISAL OF PHYSIOLOGICAL ATTRIBUTES OF NINE STRAINS OF DUNALIELLA (CHLOROPHYCEAE): GROWTH AND PIGMENT CONTENT ACROSS A SALINITY GRADIENT , 2001 .

[32]  R. Guillard Purification Methods for Microalgae , 2005 .

[33]  Md. Anamul Hoque,et al.  Exogenous proline and glycinebetaine increase NaCl-induced ascorbate-glutathione cycle enzyme activities, and proline improves salt tolerance more than glycinebetaine in tobacco Bright Yellow-2 suspension-cultured cells. , 2007, Journal of plant physiology.

[34]  H. Degani,et al.  In Vivo pH Regulation by a Na/H Antiporter in the Halotolerant Alga Dunaliella salina. , 1991, Plant physiology.

[35]  A. Oren A hundred years of Dunaliella research: 1905–2005 , 2005, Saline systems.

[36]  H. Archbold FRUCTOSANS IN THE MONOCOTYLEDONS. A REVIEW , 1940 .

[37]  P. Singh,et al.  Effect of nutrient depletion on beta-carotene and glycerol accumulation in two strains of Dunaliella sp. , 2003, Bioresource technology.

[38]  J. Harborne Encyclopedia of plant physiology, New series , 1978 .

[39]  J. Staden,et al.  Dissecting the roles of osmolyte accumulation during stress , 1998 .

[40]  P. Yancey Compatible and Counteracting Solutes: Protecting Cells from the Dead Sea to the Deep Sea , 2004, Science progress.

[41]  I. D. Teare,et al.  Rapid determination of free proline for water-stress studies , 1973, Plant and Soil.

[42]  S. Mudge,et al.  Effects of ionic strength on the production of short chain volatile hydrocarbons by Dunaliella salina (Teodoresco). , 2004, Chemosphere.

[43]  D. Galbraith,et al.  Monitoring large-scale changes in transcript abundance in drought- and salt-stressed barley , 2004, Plant Molecular Biology.

[44]  R. Barg,et al.  Developmental and organ-specific expression of an ABA- and stress-induced protein in barley , 1992, Plant Molecular Biology.

[45]  A. Ben‐Amotz,et al.  The biotechnology of cultivating the halotolerant algaDunaliella , 1990 .

[46]  H. Gimmler,et al.  Cation Permeability of the Plasmalemma of the Halotolerant alga Dunaliella parva , 1978 .

[47]  H. Bohnert,et al.  PLANT CELLULAR AND MOLECULAR RESPONSES TO HIGH SALINITY. , 2000, Annual review of plant physiology and plant molecular biology.

[48]  D. Robledo,et al.  Physiological characterization of Dunaliella sp. (Chlorophyta, Volvocales) from Yucatan, Mexico. , 2007, Bioresource technology.

[49]  Karseno,et al.  Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells. , 2006, Journal of bioscience and bioengineering.

[50]  Hong Wang,et al.  Gene Expression Profiles during the Initial Phase of Salt Stress in Rice , 2001, Plant Cell.

[51]  A. Ben‐Amotz,et al.  PRODUCTION AND SELECTION OF HIGH β‐CAROTENE MUTANTS OF DUNALIELLA BARDAWIL (CHLOROPHYTA) 1 , 1991 .

[52]  S. Grattan,et al.  Rapid assay for determination of water soluble quaternary ammonium compounds , 1983, Plant and Soil.

[53]  R. Andersen,et al.  Algal culturing techniques , 2005 .

[54]  A. Coleman,et al.  PHYLOGENETIC RELATIONSHIP AMONG VARIOUS STRAINS OF DUNALIELLA (CHLOROPHYCEAE) BASED ON NUCLEAR ITS rDNA SEQUENCES , 2001 .

[55]  A. Young,et al.  LOW‐TEMPERATURE‐INDUCED SYNTHESIS OF α‐CAROTENE IN THE MICROALGA DUNALIELLA SALINA (CHLOROPHYTA) , 1999 .

[56]  A. Neish,et al.  RAPID METHOD FOR ESTIMATION OF GLYCEROL IN FERMENTATION SOLUTIONS , 1950 .

[57]  A. Katz,et al.  Determination of intracellular osmotic volume and sodium concentration in dunaliella. , 1985, Plant physiology.

[58]  U. Pick,et al.  Regulation of glycerol synthesis in response to osmotic changes in dunaliella. , 1991, Plant physiology.

[59]  J. Reid,et al.  Reserve Polysaccharides Other Than Starch in Higher Plants , 1982 .

[60]  G. Abogadallah,et al.  Proline induces the expression of salt-stress-responsive proteins and may improve the adaptation of Pancratium maritimum L. to salt-stress. , 2003, Journal of experimental botany.

[61]  M. Avron The osmotic components of halotolerant algae , 1986 .

[62]  Short Communication Effects of salinity on b-carotene production by Dunaliella tertiolecta DCCBC26 isolated from the Urmia salt lake, north of Iran , 2006 .

[63]  K. Akiyama,et al.  Monitoring the expression pattern of around 7,000 Arabidopsis genes under ABA treatments using a full-length cDNA microarray , 2002 .

[64]  James B. Hicks,et al.  A plant DNA minipreparation: Version II , 1983, Plant Molecular Biology Reporter.

[65]  G. L. Floyd,et al.  Group I introns within the nuclear-encoded small-subunit rRNA gene of three green algae. , 1992, Molecular biology and evolution.