Microsomal Phospholipid Molecular Species Alterations during Low Temperature Acclimation in Dunaliella

Abstract

(1 1, 14).In this communication, we describe the changes in microsomal membrane acyl chain composition of each major polar lipid of the green alga Dunaliella salina and demonstrate that the molecular species distribution within certain phospholipid classes is rapidly modified by low temperature stress. This analytical approach permits the recognition of structural and metabolic relationships not revealed by usual lipid analytical techniques. Similar analyses have also been carried out on chloroplast phospholipids and are presented in the accompanying paper.MATERIALS AND METHODS Culture Conditions. Axenic cultures of Dunaliella salina (UTEX 1644) were grown in synthetic medium under conditions previously described (7). Cultures were grown isothermally at 30°C or chilled slowly (over 2.5 h) to 12°C and maintained at 12°C for the designated time. Cells held at 12°C for longer than 100 h resumed logarithmic growth at low temperature and were considered as being 12°C-acclimated cells. It has been found that these cells have essentially the same lipid composition as cells grown at 12°C for much longer periods.Membrane Isolation. Cells were harvested in the middle to late logarithmic growth phase and fractionated as previously described (7) except that the cell suspension was allowed to equilibrate in the Parr bomb for 15 rather than 10 min. The 20,000g supernatant was centrifuged at 105,000g for 1 h to obtain the microsomal fraction.Lipid Analyses. Lipids were extracted using the procedure of Bligh and Dyer (2). Total phospholipids (including DGTH,3 a non-phosphorus-containing polar lipid [9]) were obtained by silicic acid column chromatography (9). The individual lipid classes (PC, PG, PE, and DGTH) were isolated by TLC on silica gel H using chloroform:acetic acid:methanol:water (70:25:5:2.2, v/v/v/v) as the solvent system. After flushing the plate with N2 for 2 to 5 min to remove the solvents, the lipid bands were detected by a brief exposure to iodine vapors. For molecular species and/or methyl ester analyses (8), the bands were immediately scraped off the plate, and the lipids were eluted from the silica gel using chloroform:methanol:water (3:5:1, v/v/v), then dried under N2. Alternatively, methyl esters were formed in the presence of silica gel by scraping the lipid bands directly into 3Abbreviations:

Effect of Osmotic Stress on Ion Transport Processes and Phospholipid Composition of Wheat (Triticum aestivum L.) Mitochondria

“…Often only lipid analyses of whole tissue or organs have been related to adaptation of a given plant species. A limited number of studies (5,6,9,17,20,30) have examined the relationship between lipid metabolism and stress tolerance based on a separate analysis of the organelle and an analysis of specific tissues. Accordingly, we analyzed the lipid composition of osmotic-stressed mitochondria of root and shoot tissue.…”

Diacylglycerol Metabolism in the Green Alga Dunaliella salina under Osmotic Stress

“…The lipids were resolved into FA, MAG, and unhydrolyzed DAG by TLC, and after visualizing the lipids with primulin the desired bands were extracted with chloroform/methanol (6:1, v/v). Following the addition of nonadecanoic acid internal standard, each sample was transmethylated and the FA methyl esters were analyzed by GC (15).…”

Structural analysis of phosphatidylcholines by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

“…PL molecular species are defined by the nature of the acyl (alkyl) residues attached to the C-1 (also called sn-1, using stereospecific numbering) and C-2 positions (sn-2) of the glycerol backbone. The molecular species are uniquely and differentially distributed among different tissues [1,2], and because of their importance in regulating development, function and adaptation [3][4][5], analysis of PL molecular species and their alterations is of considerable interest in many areas of biological research.Thin layer chromatography (TLC) [6 -8] and normal-phase high performance liquid chromatography (HPLC) [9,10] are well suited for the separation of PL classes, whereas the separation of molecular species within each class may be accomplished using gas chromatography (GC) [11,12] or reversed-phase HPLC [13][14][15] (see [5] for a review on lipid molecular species analysis). Conversion of PL species to GCsuitable derivatives is laborious and time consuming, and often results in analyte loss.…”

“…PL molecular species are defined by the nature of the acyl (alkyl) residues attached to the C-1 (also called sn-1, using stereospecific numbering) and C-2 positions (sn-2) of the glycerol backbone. The molecular species are uniquely and differentially distributed among different tissues [1,2], and because of their importance in regulating development, function and adaptation [3][4][5], analysis of PL molecular species and their alterations is of considerable interest in many areas of biological research.…”