Technical Advance: a novel technique for the sensitive quantification of acyl CoA esters from plant tissues.
We report a novel, highly sensitive and selective method for the extraction and quantification of acyl CoA esters from plant tissues. The method detects acyl CoA esters with acyl chain lengths from C4 to C20 down to concentrations as low as 6 fmol in extracts. Acyl CoA esters from standard solutions or plant extracts were derived to their fluorescent acyl etheno CoA esters in the presence of chloroacetaldehyde, separated by ion-paired reversed-phase high-performance liquid chromatography, and detected fluorometrically. This derivitization procedure circumvents the selectivity problems associated with previously published enzymatic methods, and methods that rely on acyl chain or thiol group modification for acyl CoA ester detection. The formation of acyl etheno CoA esters was verified by mass spectrometry, which was also used to identify unknown peaks from chromatograms of plant extracts. Using this method, we report the composition and concentration of the acyl CoA pool during lipid synthesis in maturing Brassica napus seeds and during storage lipid breakdown in 2-day-old Arabidopsis thaliana seedlings. The concentrations measured were in the 3--6 microM range for both tissue types. We also demonstrate the utility of acyl CoA profiling in a transgenic B. napus line that has high levels of lauric acid. To our knowledge, this is the first time that reliable estimates of acyl CoA ester concentrations have been made for higher plants, and the ability to profile these metabolites provides a valuable new tool for the investigation of gene function.
Larson
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TR
Centre for Novel Agricultural Products, Department of Biology, University of York, Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK. trl1@cnap.york.ac.uk
Graham
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IA
D000214:Acyl Coenzyme A / Q000032:analysis* / Q000302:isolation & purification; D017360:Arabidopsis / Q000737:chemistry; D001937:Brassica / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004952:Esters; D005230:Fatty Acids, Nonesterified / Q000032:analysis / Q000302:isolation & purification; D007202:Indicators and Reagents; D010944:Plants / Q000737:chemistry*; D012639:Seeds / Q000737:chemistry; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D000214:Acyl Coenzyme A; D004952:Esters; D005230:Fatty Acids, Nonesterified; D007202:Indicators and Reagents