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Rapid Kinetic Labeling of Arabidopsis Cell Suspension Cultures: Implications for Models of Lipid Export from Plastids

Tjellström, Henrik, Yang, Zhenle, Allen, Doug K., Ohlrogge, John B.
Plant physiology 2012 v.158 no.2 pp. 601
Arabidopsis thaliana, Pisum sativum, acetates, biosynthesis, cell suspension culture, chloroplasts, endoplasmic reticulum, fatty acids, fractionation, genes, glycerol, labeling, leaves, lipid composition, lysophosphatidylcholine, models, peas, plant tissues, protoplasts
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 [14C]acetate and [14C]glycerol. [14C]acetate was very rapidly incorporated into PC, preferentially at sn-2 and without an apparent precursor-product relationship to diacylglycerol (DAG). By contrast, [14C]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 [14C]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.