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

Author:
Tjellström, Henrik, Yang, Zhenle, Allen, Doug K., Ohlrogge, John B.
Source:
Plant physiology 2012 v.158 no.2 pp. 601
ISSN:
0032-0889
Subject:
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
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 [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.
Agid:
55193
Handle:
10113/55193