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Received for publication May 9, 1988 and in revised form September 8, 1988 Plant Physiol. (1989) 89, 970-976 0032-0889/89/89/0970/07/$01 .00/0 Lipid Characterization of an Enriched Plasma Membrane Fraction of Dunaliella salina Grown in Media of Varying Salinity1 Thomas C. Peeler, Martha B. Stephenson, Kregg J. Einspahr, and Guy A. Thompson, Jr.* Department of Botany, University of Texas, Austin, Texas 78713 high salinity. It is generally accepted that the high NaCl concentrations in which Dunaliella lives would disrupt membrane processes in the majority of organisms by inhibiting enzyme activities and disordering membrane structure. Cytoplasmic enzymes isolated from Dunaliella cells are inhibited by high concentrations of NaCl (7), indicating that the plasma membrane of Dunaliella must not only remain functional under the high external concentrations of NaCl, but also maintain a permeability barrier against the high NaCl concentration outside of the cell. Indeed, internal Na+ concentrations of less than 100 mm have been reported in Dunaliella cells grown in 0.5 to 4 M NaCl (16). The plasma membrane must also enable the cell to sense changes in the surrounding osmotic conditions so that it can adjust its internal glycerol concentration to achieve osmotic equilibrium. We have adapted an aqueous two-phase system (19) that allows us to purify D. salina plasma membrane more quickly and completely than was possible with earlier methods (28). Utilizing this method, we obtained an enriched plasma membrane fraction from D. salina grown in our standard growth medium containing 1.7 M NaCl, and from cells grown in media containing one-half the amount of NaCl (0.85 M) and twice the amount of NaCl (3.4 M). The enriched plasma membrane fractions were then characterized as to lipid composition. Several significant differences were observed which may be responsible for the tolerance of D. salina for a wide range of osmotic conditions. ABSTRACT We have developed a rapid procedure for isolating a fraction enriched in plasma membrane from DunalielIa salUna using an aqueous two-phase system (dextran/polyethylene glycol, 6.7%/ 6.7%). An enriched plasma membrane fraction, free of chloroplast and mitochondrial contamination, could be obtained in 2.5 hours. Plasma membrane proteins, which accounted for approximately 1% of the total membrane protein, contained a number of unique proteins compared with the other cell fractions, as shown by gel electrophoresis. The lipids of the plasma membrane fraction from 1.7 molar NaCI-grown cells were extracted and characterized. Phosphafidylethanolamine and phosphatidylcholine were the two most prevalent phospholipids, at 20.6% and 6.0% of the total lipid, respectively. In addition, inositol phospholipids were a significant component of the D. salina plasma membrane fraction. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5bisphosphate accounted for 5.2% and 1.5% of the plasma membrane phospholipid, respectively. Diacylglyceryltrimethylhomoserine accounted for 7.9% of the plasma membrane total lipid. Free sterols were the major component of the plasma membrane fraction, at 55% of the total lipid, and consisted of ergosterol and 7-dehydroporiferasterol. Sterol peroxides were not present in the plasma membrane fraction. The lipid composition of enriched plasma membrane fractions from cells grown at 0.85 molar NaCI and 3.4 molar NaCI were compared with those grown at 1.7 molar NaCI. The concentration of diacylglyceryltrimethylhomoserine and the degree of plasma membrane fatty acid saturation increased in 3.4 molar plasma membranes. The relative concentration of sterols in the plasma membrane fraction was similar in all three NaCI concentrations tested. MATERIALS AND METHODS Cell Culture Axenic cultures of Dunaliella salina (UTEX 1644) were grown in synthetic medium under conditions previously described (22). The cultures were maintained at 30C in continuous light (100 gmol m-2s-'). Cell population density was measured using a Coulter Counter model ZB. Dunaliella salina is a single-celled alga that is extraordinarily tolerant to salt stress. It can withstand a wide variety of NaCl concentrations (0.86 to 4.3 M) (1 1) and is one of the few organisms that can survive in extreme environments such as the Great Salt Lake. D. salina can also survive rapid fluctations in external NaCl concentrations. The cell osmoregulates by rapidly increasing or decreasing the internal concentration of glycerol to osmotically balance the external concentration of NaCl (4, 7). The plasma membrane of Dunaliella must therefore have particular characteristics which allow the cell to tolerate such Plasma Membrane Purification Cells were harvested and fractionated as previously described (22, 23), except that the cell disruption buffer (400 mM mannitol, 2 mm EDTA, 1 mM MgCl2, 100 mm NaPO4 [pH 8.0] contained phosphate instead of Tris-HCl. After disruption in the Parr bomb, the suspension of broken cells was centrifuged at 2639g for 3.5 min to pellet chloroplasts and cellular debris. The supernatant over the chloroplast pellet 'This study was supported in part by grants from the Robert A. Welch Foundation (F-350), the National Science Foundation (DMB8506750), and the Texas Advanced Technology Research Program. 970 Downloaded from on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1989 American Society of Plant Biologists. All rights reserved. PLASMA MEMBRANE COMPOSITION AND NaCI ACCLIMATION was then added to a dextran-PEG aqueous two-phase system to partition the plasma membrane away from the remainder of the organelles. The final concentrations of each of the components of the two-phase system were; 6.7% Dextran T 500 (w/w), 6.7% PEG 3350 (w/w), 178 mM mannitol, 0.89 mM EDTA, 0.44 mm MgCl2, 44.6 mM NaPO4 (pH 8.0). In addition, approximately 30 mM NaCl was added so that the two-phase system partitioned properly. The NaCl that remained with the pelleted cells (from the growth medium) was also necessary for plasma membrane purification. Membranes from cells washed in NaCl-free media did not partition properly. In a typical experiment, 1.5 x 109 cells were disrupted in 40 mL of phosphate disruption buffer, and the subsequent supernatant was divided between two separate tubes of the dextran-PEG system. Each 17.5 mL aliquot of the supernatant was added to 21.7 mL of the dextran-PEG system. The complete two-phase system was mixed by shaking and centrifuged at 660g for 10 min in a Sorvall HB-4 swinging-bucket rotor. The upper phase (PEG-rich) was removed and centrifuged at 150,000g for 1 h in a Beckman SW-41 swinging bucket rotor. The resulting pellet was enriched in plasma membrane. The remaining lower phase (dextran-rich) was diluted with buffer and also centrifuged at 1 50,000g for 1 h. This lower phase pellet was enriched in cellular membranes other than the plasma membrane and chloroplasts. All steps of this isolation procedure were carried out at 4°C. Standard Assays for Fraction Characterization Proteins were measured using the bicinchoninic acid protein assay (Pierce) (29). Lipid phosphorus was quantified using the method of Bartlett (3). Chl was measured according to Arnon (2). Nucleic acid was precipitated with 2.5 vol ethanol, and the concentration determined at A260 nm (24). ATPase activity was measured at 37°C by the method of Sandstrom et al. (27). The sensitivity of the ATPase activity to vanadate was assayed by adding 0.5 mM Na3VO4 to inhibit the activity. Cyt c oxidase and antimycin A-insensitive NAD(P)H Cyt c reductase were assayed according to Hodges and Leonard (14). Antimycin A (1.0 nmol) was added to the reaction mixture for measuring antimycin A-insensitive NAD(P)H Cyt c reductase. The values reported are from a single representative experiment. Samples utilized for gel electrophoresis were boiled in SDS sample buffer containing 50 mM Tris (pH 6.8), 1% SDS, and 2% f3-mercaptoethanol, and electrophoresed on a discontinous Tris-glycine-buffered polyacrylamide gel system using a 17.5% resolving gel and a 3% stacker (18). Lipid Extraction and Characterization Lipids were extracted from cell fractions using the procedure of Bligh and Dyer (5). Plasma membrane lipids were separated by two-dimensional TLC on precoated silica gel plates (Kieselgel 60 F254, Merck), using acetone/ benzene/methanol/H20 (91:30:20:8, v/v/v/v) for the first dimension, and chloroform/acetone/methanol/acetic acid/ H20 (50:20:10:10:5, v/v/v/v/v) for the second dimension. Following development, lipid spots were located under ultraviolet light after spraying with primulin. Individual lipids were 971 identified by their TLC mobility, by specific spray reagents, and by comparison with authentic standards. After the addition of a known amount of heptadecanoic acid as an internal standard, lipid spots were scraped from the TLC plate. Fatty acid methyl esters were made from the lipids by adding 14% BF3 in methanol (Sigma) and heating for 10 min at 100CC in a sealed ampule. The fatty acid methyl esters were extracted in hexane and quantified by GC on a Varian model 3700 gas chromatograph using the procedures of Lynch and Thompson (23). Concentrations of the individual phospholipids were calculated by comparison with the internal standard. Polar lipid analyses were performed 5 times for 1.7 M plasma membranes, and 2 times each for 0.85 and 3.4 M plasma membranes. The values reported are from a single representative experiment. Sterol Characterization Lipids were extracted following the procedure of Bligh and Dyer (5). Whole cell and chloroplast extracts were concentrated and separated by TLC using Silica Gel H in a solvent system consisting of petroleum ether/diethyl ether/acetic acid (70:30:1, v/v/v). After drying the plate under N2, the free sterol-containing areas were immediately scraped into conical centrifuge tubes, and sterols were eluted two times with 5 mL of chloroform/methanol (2:1, v/v). The supernatants were combined and concentrated to dryness under N2. To remove any traces of nonsterol lipids, the residue was saponified by adding 5 mL of 1 N KOH in 90% methanol, 10% H20 and incubating the mixture under N2 and in darkness for 3 h at 25°C. After adding water, the nonsaponifiable products were extracted with hexane, and the washed hexane extracts were concentrated to dryness and redissolved in chloroform/methanol 6:1 (v/v) for storage in darkness under N2 at -20°C. For use as a standard, ergosterol peroxide was prepared from ergosterol following the procedure of Gunatilaka et al. (12). The peroxide was purified by TLC, using Silica Gel H in a solvent system consisting of benzene/diethyl ether/ ethanol/acetic acid (50:40:2:0.2, v/v/v/v). Elution from silica gel and preparation for HPLC analysis was performed in the same manner as described for the sterols. Individual sterols and sterol peroxides were resolved by HPLC, using a Waters model 510 dual piston pump equipped with a Waters model U6K universal injector and a C18, 25 cm x 4.6 mm, 5 gm Rainin Microsorb ODS reverse phase column. The mobile phase consisted of methanol/H20 (98:2, v/v). Samples were prepared for analysis following the procedure of Bulder et al. (8). After adding an appropriate amount of internal standard (fucosterol), samples were injected in 50 gL of isopropanol. The identity of individual sterols was based upon comparison with authentic standards using GC, HPLC, and GC-MS. Because the amounts of lipid obtained from the aqueous two-phase separations were very small, these were injected directly onto the HPLC column without TLC purification or saponification. Sterols separated by HPLC were quantified with a Tracor Instruments 945 Flame Ionization LC Detector. Sterol fractions were analyzed three times for each of the plasma membrane and whole cell samples. The values reported are from a single representative experiment. Downloaded from on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1989 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 89,1989 PEELER ET AL. 972 Acidic Extraction The acidic lipid extraction procedure used to quantify PIP2 and PIP2 is described in Einspahr et al. (10). RESULTS Purification of Plasma Membrane A standard cell fractionation procedure was used to break D. salina cells and pellet the chloroplasts and other cellular debris from the remainder of the cell membranes. The membranes in the supernatant over the chloroplast pellet were partitioned directly, without first pelleting them, by a dextranPEG two-phase system. After testing a number of different concentrations, 6.7% dextran and 6.7% PEG were found to partition mainly plasma membrane into the upper phase of the two-phase system, as judged by the criteria described below. The concentrations ofphosphate buffer and NaCl were also of critical importance to the success of the plasma membrane enrichment. Phosphate ions partition preferentially into the lower (dextran) phase, causing the lower phase to become negatively charged, and the upper phase to be positively charged (19). This difference in charge is believed to be responsible for the selective partitioning of plasma membrane into the upper phase. Other ions, such as Nae and Cl-, also influence the charge differential between the two phases. Following partitioning of membranes between the two phases, the membranes of each phase were collected by centrifugation at higher speeds. The optimized procedure for obtaining an enriched plasma membrane fraction from D. salina took approximately 2.5 h. Three membrane pellets were subsequently used for analysis: the initial chloroplast pellet, the upper phase pellet (plasma membrane), and the lower phase pellet (mitochondria, endoplasmic reticulum, and other remaining membranes). Assay of Purity of the Isolated Plasma Membrane The isolation procedure and characterization of the plasma membrane fraction was first performed on cells grown in standard growth medium containing 1.7 M NaCl. Analysis of the three major fractions obtained in this isolation procedure (Table I) showed that the plasma membrane fraction accounted for approximately 1% of the total membrane protein in the sample, was free of detectable chlorophyll, and had a relatively low nucleic acid/protein ratio compared with the other fractions. Enzyme activities characteristic for specific organelles and membranes were measured in each of the three fractions (Table II). The plasma membrane fraction was enriched in an ATPase activity that was stimulated by K+ and inhibited by vanadate. This type of ATPase activity is considered a specific marker for plasma membrane in higher plants (14). Thus the 2 Abbreviations: PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol; PI, phosphatidylinositol; DGTS, diacylglyceryltrimethylhomoserine; SL, sulfoquinovosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol. Table 1. Characterization of the Three Membrane Fractions Isolated from D. salina Membrane protein (,gg/ 1 06 cells) Percent of total membrane protein Lipid P (1gmol)/protein (mg) Chl (Ag)/protein (mg) Nucleic acid (mg)/protein (mg) aND = not detected. Chloroplast Plasma Membrane Lower phase Membranes 0.4 3.5 28.2 1.2 10.9 87.8 Membranes 0.28 0.15 0.07 ND" 0.07 0.03 0.31 0.86 0.15 Table II. Marker Enzyme Activities in the Three Membrane Fractions Isolated from D. salina Plasma Lower phase Chloroplast Membrane Membranes Membranes Arnol/mg protein/min ATPase Basal +K+ K+ + vanadate Cyt c oxidase NAD(P)H Cyt c reductase a ND = not detected. 0.61 0.97 0.28 NDa 0.039 0.29 0.38 0.32 0.14 0.017 0.1 0.11 0.1 0.08 ND plasma membrane of D. salina was selectively partitioned to the upper phase during the two-phase isolation procedure. Cyt c oxidase activity was absent in the plasma membrane fraction, demonstrating that mitochondrial contamination was insignificant. NAD(P)H-Cyt c reductase activity has been considered characteristic of ER in many plant species (15). This enzyme activity was present in the plasma membrane fraction, indicating that there may have been some ER contamination of the plasma membrane preparation. However, the majority of the activity remained in the supernatants of both the upper and lower phases, suggesting that this activity may be soluble and not be specific to ER in D. salina. In addition, two recent reviews (20, 25) have noted that NAD(P)H-reductases are often associated with higher plant plasma membranes purified by aqueous two-phase systems, and are not reliable markers for ER. Proteins of the three membrane fractions were resolved by gel electrophoresis. Characteristic protein patterns are apparent in the Coomassie blue-stained gels (Fig. 1). The proteins present in the plasma membrane fraction indicate a number of bands unique to that fraction, as well as some bands that are common to other fractions. The identity of specific proteins was not determined. Lipid Characterization of the Plasma Membrane Fractions An overall comparison of the amounts of polar lipids and fatty acids present in the plasma membrane of cells grown in Downloaded from on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1989 American Society of Plant Biologists. All rights reserved. PLASMA MEMBRANE COMPOSITION AND NaCI ACCLIMATION ab .88 .i. .iii _1 1 1 46 - .I .p~ 14 A.W Figure 1. SDS-PAGE of the proteins from the three fractions isolated from D. salina. Each sample lane contains approximately 100 'gg of protein. Lane a, protein standards (kilodaltons); b, plasma membrane; c, lower phase membranes; d, chloroplast membranes. 0.85, 1.7, and 3.4 M NaCl is shown in Table III. The plasma membrane polar lipids from Bligh and Dyer extractions were quantified by scraping individual lipid classes from two-dimensional TLC plates and analyzing fatty acid methyl esters following the addition of an internal standard. PE was the phospholipid present in the highest amounts at each NaCl concentration, followed by PC, PG, and PI. DGTS and SL were the only polar lipids in the plasma membrane fraction that did not contain phosphorus. DGTS was present as a major component of the plasma membrane at each NaCl concentration, and was more concentrated in cells grown in 3.4 M NaCl than in 0.85 M or our standard 1.7 M medium. The concentration of SL was relatively low at all NaCl concentrations. The fatty acid compositions of the polar lipids extracted by the Bligh and Dyer procedure are also shown in Table III. There was a large amount of 16:0 present in all of the polar lipids of the plasma membrane. PE contained a large percentage of 14:2, accounting for all that was present in the plasma membrane. PG was responsible for all of the 18:2 (A6,9) found in the plasma membrane fraction. At 3.4 M NaCl, there was a decrease in 18:3 and an increase in 18:2 (A9,12) (DGTS) and 18:1 (PC), compared with plasma membranes from cells grown at the lower NaCl concentrations. Phospholipids were also quantified by extracting lipids from 973 membrane fractions by an acidic extraction procedure which maximizes the recovery of charged inositol phospholipids. In these experiments 1.7 M cells were labeled with 32PO4 for 48 h in order to achieve isotopic equilibrium. PIP and PIP2 were present in the plasma membrane fraction in significant quantities, accounting for 5.2% and 1.5%, respectively, ofthe total phospholipid, which amounted to a 6-fold (PIP) and a 7-fold (PIP2) enrichment of the polyphosphoinositides over the amounts found in whole cell lipids (10). In addition, a slightly higher percentage of PI was recovered by this method than by the Bligh and Dyer procedure. The relative amounts of PE, PC, and PG were similar in the two extraction procedures. Analysis of the neutral lipid components of plasma membrane fractions purified from 0.85, 1.7, and 3.4 M cells revealed sterol/phospholipid molar ratios of approximately 1.7. The principal sterols, 7-dehydroporiferasterol and ergosterol (Table IV), were enriched in the plasma membrane fractions compared to whole cell values (Table V). The relative amount of individual sterols in the plasma membrane fraction changed only slightly over the range of NaCl conditions, in contrast to whole cell sterols, where the proportions of individual sterols varied with changes in NaCl growth conditions. The mol% of 7-dehydroporiferasterol increased from 25% of the total in 0.85 M cells to 45% of the total in 3.4 M cells. The percentage of whole cell sterol peroxides changed in an opposite fashion, decreasing from 44% of the total in 0.85 M to 17% of the total in 3.4 M cells. Ergosterol peroxide was identified by comparison to a known standard. We believe that a second peak, with a retention time slightly longer than that of ergosterol peroxide, was 7-dehydroporiferasterol peroxide, although no standard could be obtained for comparison. Unlike previous findings (28), we did not find sterol peroxides to be present in significant amounts in plasma membrane preparations. In contrast, sterol peroxides accounted for 30 mol% of the sterols plus sterol peroxides extracted from whole 1.7 M cells. A preferential enrichment of sterol peroxide in the lower phase of the two-phase system was apparent following TLC oflipids extracted from the upper phase (plasma membrane-enriched) and the lower phase (remaining nonchloroplast membranes). DISCUSSION Purification and General Characterization of the Plasma Membrane The aqueous two-phase system for purifying plasma membranes described here has several advantages over previous methods for purifying plasma membrane. It is relatively fast, requiring 2.5 h for enrichment of D. salina plasma membranes, as compared to approximately 6 h for isolation by a previous method not using a dextran-PEG system (28). We were able to reduce the time required for purification by not pelleting the non-chloroplast membrane fraction before applying it to the two-phase system. Although this increased the volume of the two-phase system necessary to partition the plasma membranes from the other microsomal components, it did not affect the quality of the purification. The two-phase method that we used partitioned plasma membrane into the upper (PEG) phase, as measured by Downloaded from on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1989 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 89,1989 PEELER ET AL. 974 Table Ill. Polar Lipid and Fatty Acid Composition of D. salina Plasma Membranes Grown at 3.4, 1.7, and 0.85 M NaCI Percent of L N 14-2 14-1* Polar Lipid NaCI Concn total 16-0 18:1 59.9 64.1 65.3 32.9 32.0 5.8 6.4 8.8 12.6 4.0 39.3 52.9 50.1 52.8 33.0 38.2 47.8 45.9 48.6 53.4 70.8 61.4 64.0 5.3 7.9 7.2 9.5 42.6 34.6 47.1 6.5 3.4 3.8 11.3 12.4 16.7 18:1 18:2 All A9 18:2 A9,12 18:3 A6,9 18:3 A9,12,15 A6,9,12 8.1 5.6 2.2 2.3 3.7 34.8 36.8 1.0 0.7 1.1 10.8 17.2 0.4 0.8 0.8 4.4 4.1 33.4 14.5 3.4 wt % mol % M 27.9 32.3 3.2 3.4 24.0 42.4 2.1 1.7 41.2 3.7 16.4 0.85 3.4 12.6 PC 17.8 1.7 0.85 12.6 13.4 PG 3.4 1.7 10.7 0.85 17.0 Pi 3.4 6.2 1.7 6.3 0.85 4.9 3.4 26.8 DGTS 1.7 16.0 0.85 19.4 3.4 SL 6.9 1.7 7.1 4.9 0.85 a A blank space indicates the fatty acid was not detected. PE 39.2 42.7 37.6 20.1 20.6 4.3 6.6 4.4 2.1 29.1 21.9 11.3 15.3 2.8 8.7 20.2 9.2 10.8 11.6 16.1 8.7 15.4 7.7 6.5 5.2 Table IV. Sterol Composition of Plasma Membranes Isolated from D. salina Grown at 0.85, 1.7, and 3.4 M NaCI 1.7 M 3.4 M Sterol 0.85 M 7-Dehydroporiferasterol Ergosterol moP/O Sterol (m/ol)/ MOO/O 1.07 0.58 65 35 1.08 0.62 64 36 P (p.MOl) 1.05 0.70 mo/O 3.4 M Sterol (mo/)/ Table V. Sterol Composition of D. salina Whole Cells 0.85 M Sterol P (4smol) 1.7 M Sterol (m/)ol)/ mo/0 P (AMI)P (uml) Sterol (Pmo)/ 7-Dehydroporiferasterol Ergosterol f3-Sitosterol Peroxides (total) Unknown Sterol (Aroly Sterol (m/ol)/ P (pfMOl) 0.08 0.05 0.04 0.14 0.01 25 16 12 44 3 several different assays. The enrichment of a K+-stimulated, vanadate-inhibited ATPase in the plasma membrane fraction demonstrates that the plasma membrane partitioned preferentially into the upper phase. The complete absence of Chl in the plasma membrane fraction indicates that no chloroplast contamination was present. Mitochondrial contamination was ruled out be the absence of Cyt c oxidase activity in the plasma membrane pellet. Endoplasmic reticulum may have been present, as reflected by the presence of NAD(P)H Cyt c reductase activity in the plasma membrane fraction. However, the unusual distribution of the total NAD(P)H Cyt c reductase activity in the plasma membrane fraction. However, the unusual distribution of the total NAD(P)H Cyt c reductase activity, that is, with the majority of the activity in the soluble 0.07 0.05 0.03 0.07 0.01 P 30 22 13 30 4 (pumol) 0.15 0.07 0.04 0.06 0.02 moP 60 40 mO/ ml 45 20 12 17 6 fractions, suggests that this activity may not be a specific marker for the presence of ER in this species. In fact, NAD(P)H Cyt c reductase has been reported to be associated with the plasma membrane (20, 25). Some nucleic acids were present in the plasma membrane fraction, which could have resulted from ribosomes on contaminating RER. However, the amount of nucleic acid was small compared to the total present in all fractions, suggesting that any contamination by RER was minor. Phospholipids accounted for a 34.6 mol% of the total plasma membrane fraction lipids in D. salina grown in 1.7 M NaCl. PE was the most abundant phospholipid, followed by PC and PG. The fact that PC is not the prevalent phospholipid here, as it is in the plasma membrane of a number of other Downloaded from on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1989 American Society of Plant Biologists. All rights reserved. PLASMA MEMBRANE COMPOSITION AND NaCI ACCLIMATION species (21, 26), may be related to the presence of the unusual lipid DGTS in large amounts. DGTS, although not a phospholipid, is, like PC, a zwitterionic compound and therefore may fulfill certain roles ordinarily played by PC. The inositol phospholipids (PI, PIP, and PIP2) were present, with PIP and PIP2 being highly enriched in the plasma membrane, as they are in the plasma membrane of several animal cell types (1), and in fusogenic carrot cells (30). Inositol phospholipids are involved in signal transduction in a number of cell types, and levels of inositol phospholipids respond dramatically to osmotic shock in D. salina (9, 10). SL was also a component of the plasma membrane fraction. However, its presence in the plasma membrane in even small amounts is unusual, because it is normally associated with the chloroplast. Its presence in the plasma membrane fraction is not likely to be simply the result of chloroplast contamination, however, since the other characteristic chloroplast galactolipids MGDG and DGDG were not detectable in the plasma membrane fraction. This implies that SL is a genuine component of the plasma membrane. With the exception of PC and DGTS, which were quite similar in their fatty acid compositions, the major plasma membrane fraction polar lipids each had a characteristic fatty acid pattern. The fatty acid patterns for each lipid class in the plasma membrane strongly resembled the distributions reported for D. salina microsomes, although the relative proportions of the lipid classes in the two membrane types differed (22, 23). The plasma membrane fraction SL fatty acid pattern resembled that reported for chloroplast SL (22). Sterols were present in the plasma membrane fraction (55% of the total lipid) entirely in the form of free sterols. The composition of the plasma membrane sterols was quite different from that found in higher plants, in agreement with previous characterizations of D. salina sterols (28). Higher plant plasma membranes have also been found to contain modified sterols, such as acylated steryl glucosides and steryl glucosides (21, 26), but these lipids appeared to be completely absent from even heavily loaded TLC plates of D. salina lipids. Likewise, there was no evidence for the presence of cerebrosides in the D. salina plasma membrane fraction. An earlier paper by Sheffer et al. (28) reported the lipid composition of a D. salina plasma membrane fraction purified by a different method. They ruptured D. salina cells under hypoosmotic conditions, and used a sucrose density gradient to separate plasma membranes from other organelles and membranes. There are several differences between their results and those reported here. Sheffer et al. found more PC than PE in their plasma membrane fraction (which was the reverse of our results), and they found DGTS to be the most prevalent polar lipid, accounting for 23.5% of the total lipid (compared to 7.9% in our plasma membrane fraction). They also found a small amount of MGDG and DGDG (6.0% of the total lipid), which was not present in our plasma membrane fraction. These galactolipids may indicate that there was some chloroplast contamination in their plasma membrane preparation. The most striking difference between the two plasma membrane preparations concerns the presence of sterol peroxides. Sheffer et al. found that sterol peroxides were 21.8% of the total plasma membrane lipid, while free sterols 975 were only 4.6% of the lipid present. In our membrane fraction free sterols accounted for 55% of the total, and sterol peroxides were not present. Perhaps the more rapid isolation procedure that we employed reduced the percentage of plasma membrane sterols that were oxidized to sterol peroxides. Sterols such as ergosterol and 7-dehydroporiferasterol, which contain A5,7 unsaturation, are very prone to peroxidation, as confirmed by our observation that authentic ergosterol was rapidly converted to ergosterol peroxide by refluxing in ethanol in the presence of light and a trace of eosin ( 12). A recent review of Dunaliella has called into question some of the species designations used in various laboratories (1 1). The possibility exists that even though we both have reported on the plasma membrane composition of D. salina, we may indeed be using two different species of Dunaliella. Perhaps this could explain the differences between our results and those reported by Sheffer et al. Plasma Membrane Lipid Changes in Response to Varying Salinity Polar lipid changes in plasma membranes from cells grown in various NaCl concentrations were relatively minor. The most notable change was the 10% increase in the concentration of DGTS in 3.4 M plasma membranes compared to 1.7 M cells, with a concomitant loss of PE in the 3.4 M plasma membranes. The capacity to regulate DGTS concentration in the plasma membrane may play a role in the ability of D. salina to grow in a variety of salinities. The fatty acid compositions of the polar lipids also showed small but significant changes in response to NaCl concentration. There was a reduction in 18:3 in both PC and DGTS in plasma membranes from cells grown at 3.4 M NaCl, with an increase in 18:2 in DGTS and 18:1 in PC. Similar changes have been seen in higher plant species in response to high concentrations of NaCl ( 13). Increases in the degree of saturation of membrane-associated fatty acids have been hypothesized to make the membrane less permeable to NaCl (17). Although the sterol composition of whole cells changed with increasing NaCl concentration, the sterol composition of the plasma membrane fraction changed only slightly. The plasma membrane fraction contained a high proportion of sterol (55% of the total lipid), which may be responsible for some of the unusual permeability characteristics of D. salina plasma membranes. Membrane permeability is affected by the proportion of sterol in the lipid bilayer (6). In conclusion, the lipid composition of D. salina plasma membranes remained relatively constant in cells grown at varying NaCl concentrations. There were several significant differences, however, including an increase in the amount of DGTS at 3.4 M NaCl, and a decrease in the degree of fatty acid unsaturation in prevalent phospholipids at 3.4 M NaCl. The relative stability of the plasma membrane composition under a wide range of osmoticums may enable D. salina to adapt so successfully to sudden changes in the external NaCl concentration, because major changes in lipid composition are not necessary to maintain plasma membrane integrity after osmotic shock. Downloaded from on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1989 American Society of Plant Biologists. All rights reserved. 976 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. PEELER ET AL. 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