title: Rapid kinetic labeling of Arabidopsis cell suspension cultures: implications for models of lipid export from plastids.
abstract: Cell cultures allow rapid kinetic labeling experiments that can provide information on precursor-product relationships and intermediate pools. T-87 suspension cells are increasingly used in Arabidopsis (Arabidopsis thaliana) research, but there are no reports describing their lipid composition or biosynthesis. To facilitate application of T-87 cells for analysis of glycerolipid metabolism, including tests of gene functions, we determined composition and accumulation of lipids of light- and dark-grown cultures. Fatty acid synthesis in T-87 cells was 7- to 8-fold higher than in leaves. Similar to other plant tissues, phosphatidylcholine (PC) and phosphatidylethanolamine were major phospholipids, but galactolipid levels were 3- to 4-fold lower than Arabidopsis leaves. Triacylglycerol represented 10% of total acyl chains, a greater percentage than in most nonseed tissues. The initial steps in T-87 cell lipid assembly were evaluated by pulse labeling cultures with [(14)C]acetate and [(14)C]glycerol. [(14)C]acetate was very rapidly incorporated into PC, preferentially at sn-2 and without an apparent precursor-product relationship to diacylglycerol (DAG). By contrast, [(14)C]glycerol most rapidly labeled DAG. These results indicate that acyl editing of PC is the major pathway for initial incorporation of fatty acids into glycerolipids of cells derived from a 16:3 plant. A very short lag time (5.4 s) for [(14)C]acetate labeling of PC implied channeled incorporation of acyl chains without mixing with the bulk acyl-CoA pool. Subcellular fractionation of pea (Pisum sativum) leaf protoplasts indicated that 30% of lysophosphatidylcholine acyltransferase activity colocalized with chloroplasts. Together, these data support a model in which PC participates in trafficking of newly synthesized acyl chains from plastids to the endoplasmic reticulum.
authors: Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824, USA.
authors: Henrik
authors: H
authors: Tjellström
authors: Zhenle
authors: Z
authors: Yang
authors: Doug K
authors: DK
authors: Allen
authors: John B
authors: JB
authors: Ohlrogge
mesh_terms: D017360:Arabidopsis / Q000166:cytology* / Q000378:metabolism; D001692:Biological Transport; D002250:Carbon Radioisotopes; D018929:Cell Culture Techniques; D007700:Kinetics; D050356:Lipid Metabolism; D008954:Models, Biological; D018087:Plastids
chemical_list: D002250:Carbon Radioisotopes