ABSTRACT
The effect of temperature and oxygen on the in vivo oleate desaturation and microsomal oleate desaturase (FAD2) activity was studied in peeled developing sunflower seeds. Using an oxygen concentration that was saturating for FAD2 enzyme, the amount of linoleic acid increased for all studied temperatures, being maximal at 20 degrees C. Under these conditions, FAD2 activity increased at the beginning of the incubation, remaining constant for the rest of the time, but reaching a lower level at 30 degrees C. Anoxia brought about a decrease in the FAD2 activity for all studied temperatures, becoming faster as the temperature increased. All these data suggest that temperature and oxygen control the level of FAD2 activity by separate mechanisms.
Subject(s)
Fatty Acid Desaturases/metabolism , Helianthus/physiology , Microsomes/enzymology , Seeds/enzymology , Aerobiosis , Kinetics , Oxidoreductases Acting on CH-CH Group Donors , Oxygen , TemperatureSubject(s)
Fatty Acids/chemistry , Helianthus/genetics , Mutation , Seeds/chemistry , Helianthus/metabolismABSTRACT
A procedure for the digestion of fresh tissue, transmethylation of lipids, and the extraction of fatty acid methyl esters (FAMES) in one step is described. Fresh tissues or isolated lipids are heated with a reagent containing methanol:heptane:benzene:2,2-dimethoxypropane:H2SO4 (37:36:20:5:2, by vol), methanol:heptane:toluene:2,2-dimethoxypropane:H2SO4 (39:34:20:5:2, by vol), or methanol:heptane:tetrahydrofuran:2,2-dimethoxypropane:H2SO4 (31:42:20:5:2; by vol). At 80 degrees C the simultaneous digestion and lipid transmethylation takes place in a single phase. After cooling at room temperature two phases are formed; the upper one contains the FAMES ready for GLC analysis. The procedure allows the determination of the fatty acid composition of lipids from tissues containing high proportions of triacylglycerols (oil seeds), water (leaves), or both (oil fruits). Due to the complete extraction of the lipids from the tissue, their content can be determined by adding an internal standard to the sample.
Subject(s)
Fatty Acids/isolation & purification , Lipids/isolation & purification , Plants/chemistry , Esters/isolation & purification , MethylationABSTRACT
The effect of temperature on oleate desaturation in developing sunflower (Helianthus annuus L.) seeds has been examined. When seeds from plants grown at low (20/10° C, day/night) temperature were transferred for 24 h to 10° C, an increase in the linoleate/oleate ratio in phosphatidylcholine and triacylglycerol was observed, but not when transfer was to 20 or 30° C. The same effect was observed in triacylglycerol, phosphatidylcholine and phosphatidylethanolamine in the newly synthesized lipids after in-vivo incubation with [1-(14)C]oleate at 10° C. The microsomal oleoyl phosphatidylcholine desaturase (ODS) activity of the seeds maintained at 10 C was also enhanced. The stimulation was observed after only 3 h in plants grown at high temperature (30/20° C). This effect was inhibited by cycloheximide, implying that the low-temperature stimulation of the ODS activity was caused by the synthesis of new enzyme. As a consequence, seeds from plants grown at low temperature had higher ODS activities and linoleate contents than those grown at high temperature. The microsomal ODS activity of seeds from plants grown at low temperature was dependent on incubation temperature and showed a maximum at 20° C. By contrast, this activity was almost temperature-insensitive in seeds from plants grown at high temperature. These results could explain how temperature regulates the fatty-acid composition in sunflower-seed lipids.
ABSTRACT
The kinetics of incorporation of [2-(14)C] acetate into lipids and acyl-CoAs in relation to added CoA and ATP by isolated spinach chloroplasts have been examined. The effect of the concentration of these cofactors on lipid and acyl-CoA synthesis was also studied. In the absence of cofactors, or when only one was present, the incorporation was very low and went mainly into lipids. When both cofactors were present a strong stimulation of both activities occurred. After 25 min, acyl-CoAs were more strongly labeled than lipids and both activities continued linearly for at least 60 min.
