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Title: Rapid kinetic labeling of Arabidopsis cell suspension cultures: Implications for models of lipid export from plastids

item TJELLSTROM, HENRIK - Michigan State University
item YANG, ZHENLE - Chinese Academy Of Sciences
item Allen, Douglas - Doug
item OHLROGGE, JOHN - Michigan State University

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/22/2011
Publication Date: 11/29/2011
Citation: Tjellstrom, H., Yang, Z., Allen, D.K., Ohlrogge, J.B. 2011. Rapid kinetic labeling of Arabidopsis cell suspension cultures: Implications for models of lipid export from plastids. Plant Physiology. 158:601-611.

Interpretive Summary: Lipids from plants represent an important renewable source of carbon for chemical feedstocks, biofuels and as consumable vegetable oils. Plant systems however are challenging to study with diverse cell types growing at different rates. Cell suspension cultures are much more uniform and they are commonly used as an important model for study of many aspects of cell biology, biochemistry and molecular biology of higher plants. These cells can therefore improve interpretation of biochemical and other studies. Additionally the suspension cells can be genetically altered easily, with straight-forward and less time-consuming generation and selection of large numbers of independent transgenic lines compared to whole-plant transformation. We characterized the utility of Arabidopsis T-87-cells as a model for lipid synthesis at the subcellular level. Lipid composition was analyzed and a set of experiments using radio-isotopes were conducted to investigate initial steps in storage oil biosynthesis. Specifically we fed developing cells 14C-acetate and 14C-glycerol. Glycerol and acetate can be easily taken up and incorporated by cells making oil and therefore provision of the 14C radioisotopes allowed us to observe oil biosynthetic metabolism. Our data indicates that phosphotidylcholine (PC), that is a primary component of membrane lipids, plays an important role in transferring fatty acids from one subcellular location to another in plant cells. In this case the acyl chains (i.e. fatty acids) are made in the plastid and transported outside of this organelle so that they can be used for oil production. Direct in vivo evidence for this transport has previously been lacking. These findings are important because they improve our understanding of lipid biosynthesis and will aid metabolic engineering attempts at increased oil production. In addition, development and characterization of the cell system will speed advancements in biotechnology by allowing more rapid testing of genes that regulate lipid biosynthesis.

Technical Abstract: T-87 suspension cell cultures are increasingly used in Arabidopsis research, but there are no reports describing their lipid composition or biosynthesis. To evaluate if T-87 cell cultures as a model system for analysis of lipid metabolism, including tests of gene candidate functions, we have determined lipid composition in light and dark grown cultures. The net rate of fatty acid synthesis in T-87 cells was 18 nmol carbon per hour per mg fresh weight, approximately 7-8 fold higher than measured rates in leaves. Similar to other plant tissues, phosphatidylcholine (PC) and phosphatidylethanolamine were the major phospholipids but compared to Arabidopsis leaves galactolipid levels were 3-4 fold lower. Unlike most plant tissues, triacylglycerol represented approximately 10% of total acyl chains. Rapid pulse labeling of T-87 cultures with [14C]-acetate and [14C]-glycerol allowed evaluation of initial steps in lipid assembly. [14C]-acetate was rapidly incorporated into PC, preferentially at sn-2, and without any apparent precursor-product relationship to diacylglycerol (DAG). In contrast, [14C]-glycerol predominantly labeled DAG. Together these data implicate ‘acyl editing’ of PC as a major pathway for initial incorporation of fatty acids into glycerolipids and extend previous observations by Bates et al 2007; 2009 on acyl editing in 18:3 plants to a 16:3 plant. We also considered if initial incorporation of acyl chains into PC might be associated with chloroplast envelopes. Subcellular fractionation indicated that 30% of pea leaf lysophosphatidylcholine acyltransferase activity co-localized with chloroplasts. These data are consistent with a model in which chloroplast PC may participate in trafficking of newly synthesized acyl chains from plastids to the ER.