Lipid Composition and Metabolism of Volvox carteri

1984

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The alterations in chloroplast phospholipid acyl chain composition and phospholipid molecular species composition of Dunalidla salina (UTEX 1644) it would seem that some membrane lipid structural parameter other than fatty acid composition must play a role in determining the key physical properties which control physiological activities.An alternative mechanism for bringing about increased membrane fluidity in response to low temperature stress has been detected in Tetrahymena (5,6). Detailed molecular species analyses using coupled GC-MS (6) suggested that rapid changes in the lipid physical properties were due to alterations in the phospholipid molecular species. Physical studies of pure phospholipids and simple phospholipid mixtures have demonstrated that the inter-and intramolecular exchange of acyl chains can markedly alter the physical properties of the lipids (4,15,16).In this communication, alterations in the acyl chain compositions of the individual lipid classes and the phospholipid molecular species compositions of chloroplast membranes during low temperature acclimation are described. Physical changes in the photosynthetic membrane associated with temperature acclimation are reflected in the threshold temperature of enhanced Chl fluorescence. Here, we present data which show that the extent and the time course of lipid changes in the chloroplast fraction differ from the microsomal patterns (see accompanying paper), and that certain molecular species changes reflect closely the changes in Chl fluorescence behavior. MATERIALS AND METHODSCulture Conditions. Cultures of Dunaliella salina (UTEX 1644) were grown as previously described (9). Cells were grown at 30°C or shifted to 12°C and maintained at that temperature for the designated time. Cells which had resumed logarithmic growth at low temperature (about 100 h following a temperature shift) were used as 12°C-acclimated cells.Membrane Isolation. The 2000g pellet obtained following cell disruption (9) was used as the starting membrane material. This pellet has already been shown to be highly enriched in chloroplasts with only minor cellular contamination (9).Lipid Analyses. Lipids were extracted using the procedure of Bligh and Dyer (3). The separation oflipids by silicic acid column chromatography ( 11) and TLC (accompanying paper) has been previously described. Details of the lipid analyses, including modifications of the procedures of Myher and Kuksis (12) for the analysis of molecular species, identification of molecular species, and determination ofacyl chain positional locations (20) are presented in the accompanying paper. Chl content and galactolipid ratios were determined as previously described (9). The technique for molecular analyses of MGDG3 and DGDG using HPLC has recently been presented (10).

Section: Methodsmentioning

confidence: 99%

Chloroplast Phospholipid Molecular Species Alterations during Low Temperature Acclimation in Dunaliella

Lynch¹,

1984

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“…The fatty acids 16:3 (isomer) and 16 (Table III), and these are distributed within the functionally different membrane systems in the usually observed proportions. Some distinctive constituents are present, the most prominent being DGTH, a lipid which has also been reported to occur in several other species of algae (7,10,19) and in a pathogenic fungus (29). The A more precise evaluation of the cell's response to low temperature can be gained by analyzing individual membrane types.…”

Section: Effects Ofmentioning

confidence: 99%

“…19). Employing the procedure described in "Material and Methods" using specific fatty acids as internal standards, the chloroplast DGTH content was estimated to be approximately 25% of the chloroplast PL content.…”

Section: Effects Ofmentioning

confidence: 99%

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Low Temperature-Induced Alterations in the Chloroplast and Microsomal Membranes of Dunaliella salina

Lynch

1982

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125

The metabolic regulation of membrane lipid composition has been examined using the cell wall-less, unicellular green alga Dunwaiela salina (UTEX 1644) as a model system. Low temperature stress was employed to initiate and study the We have undertaken a comprehensive study of membrane metabolism and physical properties in the eukaryotic plant cell, using the unicellular green alga Dunaliella salina as a model system. This alga has many unique properties which make it a useful experimental system. For example, it grows rapidly under axenic conditions to yield a population of very homogeneous cells. Being naturally wall-less, Dunaliella is easily disrupted, facilitating cell fractionation. The lipid composition of Dunaliella is typical of green algae and similar to that of higher plants. It is able to tolerate a wide range of temperatures and salinities (6), making it useful for investigating the role of the membrane in response to environmental stresses.A certain amount of information concerning the lipids of Dunaliella is already available (13). In this communication we report additional data of the type needed for a detailed analysis of intracellular membrane lipid dynamics. We have employed a shift in growth temperature as a means of altering membrane properties and lipid metabolism. Although a thorough understanding of the effects of low temperature on plant cells is lacking, it is clear that the cell membranes and their constituent lipids change in response to chilling (14). Studying the rate and the extent of lipid change in chloroplasts and microsomes during low temperature acclimation affords a sensitive tool for identifying the contributions of the two lipid synthesizing organelles to the plant cell's membrane economy.In recent years, much progress has been made in understanding the lipid metabolism, physical properties, and physiological role of biological membranes (25). Experimental evidence, derived mainly from microorganisms and higher animals, strongly suggests that the physiological functioning of a membrane is dependent upon its physical state (fluidity) and that the physical state is itself a function of the membrane lipid composition (24).Progress toward understanding the role of lipids in determinin'g properties of plant membranes has been more difficult for several reasons. The cellular heterogeneity of plant tissues and the pres-

“…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.…”

Section: Methodsmentioning

confidence: 99%

Microsomal Phospholipid Molecular Species Alterations during Low Temperature Acclimation in Dunaliella

Lynch¹,

1984

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(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: