Characterization of the glucose-6-phosphate isomerase (GPI) gene from the halotolerant alga <Emphasis Type="Italic">Dunaliella salina</Emphasis>

Although glucose-6-phosphate isomerase (GPI) plays an important role in glycolysis of both the prokaryotes and eukaryotes, studies on the GPI have not been involved in the halotolerant, unicellular green alga Dunaliella salina (D. salina). In this study, a 2,338 bp of full-length cDNA cloned using rapid amplification of cDNA end (RACE) technique contained an open reading frame (ORF) of 1,980 bp encoding 660 amino acids, which has a predicted molecular weight of 73.3 kD and pI of 6.22 and shares high homology with other organisms. The cloned full-length cDNA was heterologously expressed in Escherichia coli and the recombinant GPI proteins purified using Ni-NTA His Bind column were consistent with the anticipated size of ~75 kD. Predicted 2D and 3D structures of GPI proteins possessed potential active motifs including “GEPGTNGQHSFYQLIHQG” and “VQGFIWGINSFDQWGVELGK”, and critical active site residues, such as Ser 241, Ser 296, Thr 298, Thr 301, Arg 358, Glu 444, His 475 and Lys 600. Real time quantitative RT-PCR demonstrated that the expression level of the GPI gene from D. salina (DsGPI) was induced by 3.5 M NaCl with 14-fold higher than that by 1.5 M NaCl (P < 0.01), but inhibited by the light with 4-fold lower than that in the dark (P < 0.05). It is concluded that the cloned GPI gene is indeed from D. salina and may respond to salt and light.     

Protein profile analysis of salt-responsive proteins in leaves and roots in two cultivars of creeping bentgrass differing in salinity tolerance

Knowledge of stress-responsive proteins is critical for further understanding the molecular mechanisms of stress tolerance. The objectives of this study were to establish a proteomic map for a perennial grass species, creeping bentgrass (A. stolonifera L.), and to identify differentially expressed, salt-responsive proteins in two cultivars differing in salinity tolerance. Plants of two cultivars (‘Penncross’ and ‘Penn-A4’) were irrigated daily with water (control) or NaCl solution to induce salinity stress in a growth chamber. Salinity stress was obtained by adding NaCl solution of 2, 4, 6, and 8 dS m⁻¹ in the soil daily for 2-day intervals at each concentration, and then by watering soil with 10 dS m⁻¹ solution daily for 28 days. For proteomic map, using two-dimensional electrophoresis (2-DE), approximately 420 and 300 protein spots were detected in leaves and roots, respectively. A total of 148 leaf protein spots and 40 root protein spots were excised from the 2-DE gels and subjected to mass spectrometry analysis. In total, 106 leaf protein spots and 24 root protein spots were successfully identified. Leaves had more salt-responsive proteins than roots in both cultivars. The superior salt tolerance in ‘Penn-A4’, indicated by shoot extension rate, relative water content, and cell membrane stability during the 28-day salinity stress could be mainly associated with its higher level of vacuolar H⁺-ATPase in roots and UDP-sulfoquinovose synthase, methionine synthase, and glucan exohydrolase in leaves, as well as increased accumulation of catalase and glutathione S-transferase in leaves. Our results suggest that salinity tolerance in creeping bentgrass could be in part controlled by an alteration of ion transport through vacuolar H⁺-ATPase in roots, maintenance of the functionality and integrity of thylakoid membranes, sustained polyamine biosynthesis, and by the activation of cell wall loosening proteins and antioxidant defense mechanisms.     

OPTIMIZATION OF SALT CONCENTRATIONS FOR A HIGHER CAROTENOID PRODUCTION IN DUNALIELLA SALINA (CHLOROPHYCEAE)1

Dunaliella salina (Dunal) Teodor, when treated over 25 d with a wide range of NaCl salinities (0.6–4.5 M), showed its maximal growth potentialities at 1.5–3.0 M NaCl and was able to survive even at 4.5 M NaCl. Sodium concentrations increased significantly at the supraoptimal salinities, reaching up to 5 mmol · g^?1 dry weight (dwt) at 4.5 M NaCl. Interestingly, ability of D. salina to take up essential mineral nutrients was not impaired by increased salinity. As for growth, chl concentrations were maximal in the 1.5–3.0 M NaCl range. Interestingly, carotenoid concentrations increased with the increasing salinity. The highest values of total antioxidant activity (5.2–6.9 mg gallic acid equivalents [GAE] · g^?1 dwt), antiradical activity, and reducing power were measured at 1.5–3.0 M NaCl. As a whole, these results showed that at 1.5–3.0 M NaCl, D. salina produce appreciable antioxidant level. But, once it reaches its growth maximum, a salt addition up to 4.5 M could enhance its carotenoid yield.     

Biochemical and physiological characterization of a halotolerant Dunaliella salina isolated from hypersaline Sambhar Lake,India

The objective of the present study was to characterize intrinsic physiological and biochemical properties of the wall‐less unicellular cholorophyte Dunaliella salina isolated from a hypersaline Sambhar Lake. The strain grew optimally at 0.5 M NaCl and 16:8 h L:D photoperiod along with maintaining low level of intracellular Na⁺ even at higher salinity, emphasizing special features of its cell membranes. It was observed that the cells experienced stress beyond 2 M NaCl as evidenced by increased intracellular reactive oxygen species and antioxidative enzymes, nevertheless proline and malondialdehyde content declined sharply accompanied by higher neutral lipid accumulation. Salinity exceeding 2 M resulted decrease in photosynthetic quantum yield (Fv/Fm) and enhanced glycerol synthesis accompanied by leakage. Super oxide dismutase seemed to play a pivotal role in antioxidative defense as eight isoforms were expressed differentially while catalase and glutathione peroxidase showing no significant change in their expression at higher salinity. The ability of D. salina to grow in range of salinities by sustaining healthy photosynthetic apparatus along with accumulation of valuable products made this alga an ideal organism that can be exploited as resource for biofuel and commercial products.     

Anatomical changes induced by increasing NaCl salinity in three fodder shrubs, <Emphasis Type="Italic">Nitraria retusa</Emphasis>, <Emphasis Type="Italic">Atriplex halimus</Emphasis> and <Emphasis Type="Italic">Medicago arborea</Emphasis>

Nitraria retusa and Atriplex halimus (xero-halophytes) plants were grown in the range 0–800 mM NaCl while Medicago arborea (glycophyte) in 0–300 mM NaCl. Plants were harvested after 120 days of salt-treatment. The present study was designed to study the effect of salinity on root, stem and leaf anatomy, water relationship, and plant growth in greenhouse conditions. Salinity induced anatomical changes in the roots, stems and leaves. The cuticle and epidermis of N. retusa and A. halimus stems were unaffected by salinity. However, root anatomical parameters (root cross section area, cortex thickness and stele to root area ratio), and stem anatomical parameters (stem cross section area and cortex area) were promoted at 100–200 mM NaCl. Indicating that low to moderate salinity had a stimulating effect on root and stem growth of these xero-halophytic species. At higher salinities, root and stem structures were altered significantly, and their percentages of reduction were higher in A. halimus than in N. retusa whereas, in M. arborea, they were strongly altered as salinity rose. NaCl (100–300 mM) reduced leaf water content by 21.2–56.2% and specific leaf area by 51–88.1%, while increased leaf anatomical parameters in M. arborea (e.g. increased thickness of upper and lower epidermis, palisade and spongy mesophyll, entire lamina, and increased palisade to spongy mesophyll ratio). Similar results were evidenced in A. halimus leaves with salinity exceeding 100 mM NaCl. Leaves of N. retusa were thinner in salt-stressed plants while epidermis thickness and water content was unaffected by salinity. The size of xylem vessel was unchanged under salinity in the leaf’s main vein of the three species while we have increased number in M. arborea leaf main vein in the range of 200–300 mM NaCl. A longer distance between leaf vascular bundle, a reduced size and increased number of xylem vessel especially in stem than in root vascular system was evidenced in M. arborea treated plants and only at (400–800 mM) in the xero-halophytic species. The effects of NaCl toxicity on leaf, stem and root ultrastructure are discussed in relation to the degree of salt resistance of these three species. Our results suggest that both N. retusa and A. halimus show high tolerance to salinity while M. arborea was considered as a salt tolerant species.     

Carrageenan of <Emphasis Type="Italic">Eucheuma isiforme</Emphasis> (Solieriaceae,Rhodophyta) from Nicaragua

The yield and physicochemical properties of native and alkali treated carrageenan from Eucheuma isiforme harvested from the Nicaraguan coast were investigated. The native carrageenan yield was 57.2% of dry weight and decreased to 43.5% when the alga was alkali treated. Native carrageenan viscosities showed significant differences between native (144.6 ± 3.3 cPs) and treated carrageenan (113.9 ± 2.6 cPs) (p < 0.01). Alkali treatment reduced carrageenan sulphate content by 19.3% and increased 3,6 AG content by 13%. Alkali-treated carrageenan formed very weak gels in 1.5% solutions (<50 g cm⁻²). Chemical analysis and FTIR spectra revealed that Eucheuma isiforme from Nicaragua is a good source of relatively pure iota-carrageenan with sufficient quality to serve as a substitute for traditional iota-carrageenan sources.     

Subcellular Adaptation to Salinity and Irradiance in Dunaliella salina

Dunaliella salina V-63 was cultivated in different concentrations of NaCl (0.5, 1.0, 2.5, 3.0, or 4.0 M) and at two irradiances (170 or 220 μmol m⁻²s⁻¹). Concentration-dependent suppression of growth was observed above 1 M NaCl, and elevated salinity induced formation of salt-containing vacuoles. However, the changes in the chloroplast ultrastructure following changes in salinity and irradiance (increase of invaginations and protuberances, numerous grana with low number of thylakoids, less number of starch grains, etc.) appeared to be of primary importance. This revised version was published online in July 2006 with corrections to the Cover Date.     

Regeneration of plants from <Emphasis Type="Italic">Fraxinus americana</Emphasis> hypocotyls and cotyledons

A plant regeneration protocol was developed for white ash (Fraxinus americana L.). Hypocotyls and cotyledons excised from embryos were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) plus thidiazuron (TDZ), and compared for organogenic potential. Sixty-six percent of hypocotyl segments and 10.4% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 3.5 ± 0.9 and 2.5 ± 1.5, respectively. The best regeneration medium (52% shoot formation; 47% shoot elongation) for hypocotyls was MS basal medium containing 22.2 μM BA plus 0.5 μM TDZ, producing a mean of 3.9 ± 0.4 adventitious shoots. Adventitious shoots were established as proliferating shoot cultures following transfer to MS medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. For in vitro rooting, woody plant medium with indole-3-acetic acid (IAA) at 0, 2.9, 5.7, or 8.6 μM in combination with 4.9 μM indole-3-butyric acid (IBA) was tested for a 5- or 10-d dark culture period, followed by culture under a 16-h photoperiod. The best rooting (78% to 81%) of in vitro shoots was obtained with a 5 d dark culture treatment on medium containing 2.9 or 5.7 μM IAA plus 4.9 μM IBA, with an average of 2.6 ± 0.4 roots per shoot. Rooted plants were successfully acclimatized to the greenhouse. This adventitious shoot regeneration and rooting protocol will be used as the basis for experimental studies to produce transgenic white ash with resistance to the emerald ash borer.     

Identification of the proteins related to cytochrome P450 induced by fenvalerate in a <Emphasis Type="Italic">Trichoplusia ni</Emphasis> cell line

In order to reveal the metabolic reaction to the presence of fenvalerate mediated by P450 in insects, we used the trypan blue exclusion technique and 3-(4,5-dimethylthiazol)-2,5-diphenyltrazolium bromide (MTT) reduction assay to assess the vitality of Trichoplusia ni (Tn) cells treated with fenvalerate, and observed dose- and time-dependent changes in total cellular P450s. In addition, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify the proteins involved in the fenvalerate reaction process. Finally, the cDNA of P450 fragments was cloned and real-time RT-PCR was performed. Our data showed that at the 0–15 μmol/L challenge concentration of fenvalerate, at which the vitality of Tn cells was not affected (p > 0.05), there was a tendency toward a dose- and time-response of total cellular P450s, which peaked at the 9 h (p < 0.05) and 12 h (p < 0.01) time points following 12.5 μmol/L stimulation with fenvalerate. The 2-DE assay detected more than 1300 protein spots in each two-dimensional gel, of which 33 spots displayed significant differences. Among the changed spots, three isoforms of P450 were identified. One of the three P450 cDNA fragments (CYP4L4) was cloned and sequenced, and its expression in treated Tn cells increased significantly (p < 0.01). It was found that fenvalerate induced the expression of P450s in insect cells. This suggests that fenvalerate could be metabolized by CYP4L4 through a hydroxylation reaction in insect cells.     

Cloning and characterization of a thermostable and halo-tolerant endoglucanase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis> MB4

A β-1,4-endoglucanase (Cel5A) was cloned from the genomic DNA of saccharolytic thermophilic eubacterium Thermoanaerobacter tengcongensis MB4 and functionally expressed in Escherichia coli. Substrate specificity analysis revealed that Cel5A cleaves specifically the β-1,4-glycosidic linkage in cellulose with high activity (294 U mg⁻¹; carboxymethyl cellulose sodium (CMC)). On CMC, kinetics of Cel5A was determined (K _m 1.39 ± 0.12 g l⁻¹; k _cat/K _m 1.41 ± 0.13 g⁻¹ s⁻¹). Cel5A displays an activity optimum between 75 and 80 °C. Residues Glu187 and Glu289 were identified as key catalytic amino acids by sequence alignment. Interestingly, derived from a non-halophilic bacterium, Cel5A exhibits high residual activities in molar concentration of NaCl (3 M, 49.3%) and KCl (4 M, 48.6%). In 1 M NaCl, 82% of Cel5A activity is retained after 24 h incubation. Molecular Dynamics studies performed at 0 and 3 M NaCl, correlate the Cel5A stability to the formation of R-COO⁻···Na⁺ ···⁻OOC-R salt bridges within the Cel5A tertiary structure, while activity possibly relates to the number of Na⁺ ions trapped into the negatively charged active site, involving a competition mechanism between substrate and Na⁺. Additionally, Cel5A is remarkably resistant in ionic liquids 1-butyl-3-methyllimidazolium chloride (1 M, 54.4%) and 1-allyl-3-methylimidazolium chloride (1 M, 65.1%) which are promising solvents for cellulose degradation and making Cel5A an attractive candidate for industrial applications.     

Photosystem II photochemistry and physiological parameters of three fodder shrubs, <Emphasis Type="Italic">Nitraria retusa</Emphasis>, <Emphasis Type="Italic">Atriplex halimus</Emphasis> and <Emphasis Type="Italic">Medicago arborea</Emphasis> under salt stress

Nitraria retusa and Atriplex halimus (xero-halophytes) plants were grown in the range 0–800 mM NaCl while Medicago arborea (glycophyte) in 0–300 mM NaCl. Salt stress caused a marked decrease in osmotic potential and a significant accumulation of Na⁺ and Cl⁻ in leaves of both species. Moderate salinity had a stimulating effect on growth rate, net CO₂ assimilation, transpiration and stomatal conductance for the xero-halophytic species. At higher salinities, these physiological parameters decreased significantly, and their percentages of reduction were higher in A. halimus than in N. retusa whereas, in M. arborea they decreased linearly with salinity. Nitraria retusa PSII photochemistry and carotenoid content were unaffected by salinity, but a reduction in chlorophyll content was observed at 800 mM NaCl. Similar results were found in A. halimus, but with a decrease in the efficiency of PSII (F′v/F′m) occurred at 800 mM. Conversely, in M. arborea plants we observed a significant reduction in pigment concentrations and chlorophyll fluorescence parameters. The marked toxic effect of Na⁺ and/or Cl⁻ observed in M. arborea indicates that salt damage effect could be attributed to ions’ toxicity, and that the reduction in photosynthesis is most probably due to damages in the photosynthetic apparatus rather than factors affecting stomatal closure. For the two halophyte species, it appears that there is occurrence of co-limitation of photosynthesis by stomatal and non-stomatal factors. Our results suggest that both N. retusa and A. halimus show high tolerance to both high salinity and photoinhibition while M. arborea was considered as a slightly salt tolerant species.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange activity in the alkaliphile halotolerant cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

The activity of Na⁺/H⁺ exchanger to remove toxic Na⁺ is important for growth of organisms under high salinity. In this study, the halotolerant cyanobacterium Aphanothece halophytica was shown to possess Na⁺/H⁺ exchange activity since exogenously added Na⁺ could dissipate a pre-formed pH gradient, and decrease extracellular pH. Kinetic analysis yielded apparent K _m (Na⁺) and V _max of 20.7 ± 3.1 mM and 3,333 ± 370 nmol H⁺ min⁻¹ mg⁻¹, respectively. For cells grown under salt-stress condition, the apparent K _m (Na⁺) and V _max was 18.3 ± 3.5 mM and 3,703 ± 350 nmol H⁺ min⁻¹ mg⁻¹, respectively. Three cations with decreasing efficiency namely Li⁺, Ca²⁺, and K⁺ were also able to dissipate pH gradient. Only marginal exchange activity was observed for Mg²⁺. The exchange activity was strongly inhibited by Na⁺-gradient dissipators, monensin, and sodium ionophore as well as by CCCP, a protonophore. A. halophytica showed high Na⁺/H⁺ exchange activity at neutral and alkaline pH up to pH 10. Cells grown at pH 7.6 under high salinity exhibited higher Na⁺/H⁺ exchange activity than those grown under low salinity during 15 days of growth suggesting a role of Na⁺/H⁺ exchanger for salt tolerance in A. halophytica. Cells grown at alkaline pH of 9.0 also exhibited a progressive increase of Na⁺/H⁺ exchange activity during 15 days of growth.     

Efficient production of ectoine using ectoine-excreting strain

Halophilic bacteria strain Halomonas salina DSM 5928 was found to excrete ectoine, suggesting its potential in the development of a new method of ectoine production. We performed HPLC and LC–MS analyses that showed that Halomonas salina DSM 5928 excreted ectoine under constant extracellular osmolarity. Medium adopting monosodium glutamate as a sole source of carbon and nitrogen was beneficial for ectoine synthesis. The total concentration of ectoine was not affected by NaCl concentration in the range 0.5–2 mol l⁻¹. The total concentration of ectoine and productivity in a 10-l fermentor with 0.5 mol l⁻¹ NaCl were 6.9 g l⁻¹ and 7.9 g l⁻¹ d⁻¹, respectively. These findings show that Halomonas salina DSM 5928 efficiently produces ectoine at relatively low NaCl concentration. This research also indicates the potential application of free or immobilized cells for continuous culture to produce ectoine.     

Microalgal biotechnology: Carotenoid and glycerol production by the green algae <Emphasis Type="Italic">Dunaliella</Emphasis> isolated from the Gave-Khooni salt marsh,Iran

In this study, carotenoid and glycerol production in two unicellular green algae (Dunaliella salina and D. viridis) isolated from the Gave-Khooni salt marsh grown in media containing five different salt concentrations (0.17, 1, 2, 3, and 4 M NaCl) were evaluated under sterile conditions. Algae growth decreased as the medium salinity increased. Optimum growth of D. salina and D. viridis were obtained at 2 and 1 M NaCl, respectively. As salinity increased, glycerol and carotenoid production were increased in D. salina, whereas lower values for these products were produced in D. viridis under the same conditions. Furthermore, the cell color of D. salina changed from green to orange-red following accumulation of carotenoid, but the color of D. viridis was not changed. Thereby, it seems that the Iranian D. salina may be suitable for carotenoid production (betacarotene) on a large scale. In addition, since carotenoid compounds enhance the efficiency of photosynthesis and glycerol synthesis, it appears that the pathway for glycerol production and mechanisms of salt tolerance in D. viridis are unique from those of D. salina.     

Chromotoxic effects of exogenous hydrogen peroxide (H<Subscript>2</Subscript>O<Subscript>2</Subscript>) in barley seeds exposed to salt stress

The effect of exogenous hydrogen peroxide (H₂O₂) on mitotic activity and chromosomal aberrations in root tip meristems of barley (Hordeum vulgare L. var. Tokak 157/37) germinated under salinity was analyzed. The inhibitory effect of salinity on mitotic index and the frequency of chromosomal aberrations increased with increasing salt concentration (0.00 control, 0.35, 0.40, 0.45 M, molal NaCl). The frequency of chromosomal aberrations of seeds germinated in medium with 0.40 M NaCl after pretreatment with H₂O₂ (30 μM, micromolal) was significantly higher than the control group. The highest concentration of NaCl (0.45 M) together with H₂O₂ caused total inhibition of germination. In this study, the intention was to determine the performance of H₂O₂ in alleviating detrimental effect of salt stress on mitotic activity and chromosomal aberrations. However, H₂O₂ did not reduce the detrimental effect of NaCl on these parameters. Also, it caused higher chromotoxic effect compared to those of control groups.     

Cloning and heterologous expression of nitrate reductase genes from <Emphasis Type="Italic">Dunaliella salina</Emphasis>

A cDNA corresponding to the nitrate reductase (NR) gene from Dunaliella salina was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3,694 bp contained an open reading frame of 2,703 bp encoding 900 amino acids, a 5′-untranslated region of 151 bp and a 3′-untranslated sequence of 840 bp with a poly (A) tail. The putative gene product exhibited 78%, 65%, 59% and 50% identity in amino acid sequence to the corresponding genes of Dunaliella tertiolecta, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. Phylogenetic analysis showed that D. salina NR clusters together with known NR proteins of the green algae. The molecular mass of the encoded protein was predicted to be 99.5 kDa, with an isoelectric point of 8.31. This protein shares common structural features with NRs from higher plants and green algae. The full-length cDNA was heterologously expressed in Escherichia coli as a fusion protein, and accumulated to up to 21% of total bacteria protein. Recombinant NR protein was active in an enzyme assay, confirming that the cloned gene from D. salina is indeed NR.     

The effects of salinity on photosynthesis and growth of the single-cell C<Subscript>4</Subscript> species <Emphasis Type="Italic">Bienertia sinuspersici</Emphasis> (Chenopodiaceae)

Recent research on the photosynthetic mechanisms of plant species in the Chenopodiaceae family revealed that three species, including Bienertia sinuspersici, can carry out C₄ photosynthesis within individual photosynthetic cells, through the development of two cytoplasmic domains having dimorphic chloroplasts. These unusual single-cell C₄ species grow in semi-arid saline conditions and have semi-terete succulent leaves. The effects of salinity on growth and photosynthesis of B. sinuspersici were studied. The results show that NaCl is not required for development of the single-cell C₄ system. There is a large enhancement of growth in culture with 50–200 mM NaCl, while there is severe inhibition at 400 mM NaCl. With increasing salinity, the carbon isotope values (δ¹³C) of leaves increased from −17.3^o/_oo (C₄-like) without NaCl to −14.6^o/_oo (C₄) with 200 mM NaCl, possibly due to increased capture of CO₂ from the C₄ cycle by Rubisco and reduced leakiness. Compared to growth without NaCl, leaves of plants grown under saline conditions were much larger (~2 fold) and more succulent, and the leaf solute levels increased up to ~2000 mmol kg solvent⁻¹. Photosynthesis on an incident leaf area basis (CO₂ saturated rates, and carboxylation efficiency under limiting CO₂) and stomatal conductance declined with increasing salinity. On a leaf area basis, there was some decline in Rubisco content with increasing salinity up to 200 mM NaCl, but there was a marked increase in the levels of pyruvate, Pi dikinase, and phosphoenolpyruvate carboxylase (possibly in response to sensitivity of these enzymes and C₄ cycle function to increasing salinity). The decline in photosynthesis on a leaf area basis was compensated for on a per leaf basis, up to 200 mM NaCl, by the increase in leaf size. The influence of salinity on plant development and the C₄ system in Bienertia is discussed.     

Over-Expression in <Emphasis Type="Italic">E. coli</Emphasis> and Purification of the Human OCTN2 Transport Protein

The OCTN2 cDNA amplified from human skin fibroblast was cloned in pET-41a(+) carrying the glutathione S-transferase (GST) gene. The construct pET-41a(+)–hOCTN2 was used to express the GST–hOCTN2 fusion protein in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained after 6 h of induction with IPTG at 28°C. The GST–hOCTN2 polypeptide was collected in the inclusion bodies and showed an apparent molecular mass on SDS-PAGE of 85 kDa. After solubilization with a buffer containing 0.8% sarkosyl and 3 M urea, the fusion protein was applied onto a Ni²⁺-chelating chromatography column. The purified GST–hOCTN2 was treated with thrombin, and the hOCTN2 was separated from the GST by size exclusion chromatography. After the whole procedure, a yield of about 0.2 mg purified protein per liter of cell culture was obtained. To improve the protein yield, hOCTN2 cDNA was subjected to codon bias. The second codon CGG was substituted with AAA; the substitution led to the mutation R2K in the hOCTN2 protein. hOCTN2(R2K) cDNA was cloned in pET-21a(+) carrying a C-terminal 6His tag. The resulting protein was expressed in E. coli Rosetta(DE3)pLysS and purified by Ni²⁺-chelating chromatography. A yield of about 3.5 mg purified protein per liter of cell culture was obtained with this procedure.     

A METHOD FOR THE CRYOCONSERVATION OF DUNALIELLA SALINA (CHLOROPHYCEAE): EFFECT OF GLYCEROL AND COLD ADAPTATION1

The unicellular green alga Dunaliella salina Teod. was frozen according to the following procedure: 3 days cold adaptation at 4°C, addition of 3.5 M glycerol as a cryoprotectant, slow cooling to –40°C, immersion in liquid nitrogen, and rapid thawing. The survival rate was higher when cells were grown, before freezing, in the presence of 2 M NaCl instead of 1 M NaCl (78 and 48% survival, respectively). This difference is probably due to the intracellular amount of glycerol, which increases with external NaCl concentration and, therefore, may enhance cell protection. Although cells grown in 4 M NaCl accumulated a large amount of glycerol in response to osmotic stress, they did not withstand freezing. The use of cryoprotectant was absolutely necessary for the cells to recover from storage at –196°C. Glycerol was used because it is naturally produced by Dunaliella salina and therefore is not toxic. Provided it was added slowly to avoid osmotic shock, 3.5 M glycerol gave better results than 1M glycerol (48 and 18% survival, respectively). Cold adaptation in the dark increased postthaw viability. Cells grown in 1 M or 2 M NaCl had a survival rate of 48 and 78%, respectively, when cold-adapted, against 10 and 42% when not cold-adapted. This adaptation could be due to the synthesis, at low temperature, of specific proteins because two bands (28–29 kDa) appeared when electrophoretically separated proteins from cold-adapted cells and control cells were compared. Also, it could be due to the degradation of starch that occurs in the dark and leads to glycerol accumulation. Our procedure has never been used to cryopreserve microalgae and could enhance reported survival rates.