Analysis of Isotopically-labeled Monogalactosyldiacylglycerol Molecular Species from [14C]Acetate-Labeled Tobacco Leaves.

Plant lipid metabolism is a dynamic network where synthesis of essential membrane lipids overlaps with synthesis of valuable storage lipids (e.g., vegetable oils). Monogalactosyldiacylglycerol (MGDG) is a key component of the chloroplast membrane system required for photosynthesis and is produced by multiple pathways within the lipid metabolic network. The bioengineering of plants to enhance oil production can alter lipid metabolism in unexpected ways which may not be apparent by static quantification of lipids, but changes to lipid metabolic flux can be traced with isotopic labeling commonly with [14C]acetate. Because lipid classes such as MGDG are composed of many different molecular species, full analysis of metabolically labeled lipids requires separation and quantification of the individually labeled molecular species which is traditionally performed by thin layer chromatography. Here we present a reverse phase HPLC method for the separation of MGDG molecular species from tobacco leaves in under 35 min. The quantification of each 14C-labeled molecular species was accomplished by an in-line flow radio detector. This method of analysis for [14C]Acetate labeled MGDG molecular species by radio-HPLC provides a rapid, high throughput, and reliable analytical approach to identify changes in MGDG metabolism due to bioengineering or other perturbations of metabolism.