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Uncovering the Molecular Mechanisms behind Triacylglycerol Biosynthesis in Flaxseed (Linum usitatissimum L.) Open Access


Other title
alpha-linolenic acid
evolutionary analysis
polyunsaturated fatty acid
enzyme coupling
Type of item
Degree grantor
University of Alberta
Author or creator
Pan, Xue
Supervisor and department
Weselake, Randall J (AFNS)
Examining committee member and department
Ohlrogge, John B (Department of Plant Biology, Michigan State University)
Ozga, Jocelyn (AFNS)
Strelkov, Stephen (AFNS)
Gaenzle, Michael (AFNS)
Department of Agricultural, Food, and Nutritional Science
Plant Science
Date accepted
Graduation date
Doctor of Philosophy
Degree level
Flax (Linum usitatissimum L.) seed oil contains approximately 45%-65% of α-linolenic acid (ALA) and 15-29 % of linoleic acid (LA), and is one of the richest natural sources of these polyunsaturated fatty acids (PUFAs). Due to its high amounts of PUFAs, especially ALA, flax oil has a number of applications in the manufacture of food, feed and industrial products. In developing oilseeds, PUFAs are mainly synthesized on phosphatidylcholine (PC). To accumulate large amounts of PUFAs, flax may contain efficient mechanisms for transferring PUFAs from PC into triacylglycerols (TAGs), which are the main components of seed oils. Understanding these molecular mechanisms has the potential to develop new strategies to improve the quality and increase the value of flax seed oil. The present doctoral thesis includes four related studies, which aimed to use a multi-disciplinary approach, including biochemistry, molecular biology and bioinformatics, to investigate the TAG biosynthetic machinery in flax. The first study isolated and functionally characterized enzymes involved in the final step of TAG synthesis from flax. Two copies of ACYL COA:DIACYLGLYCEROL ACYLTRANSFERASE 1(DGAT1), three copies of DGAT2, two copies of DEFECTIVE IN CUTICULAR RIDGE (DCR), and six copies of PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) genes have been identified in the flax genome. Expression analyses along with functional characterization of candidate genes in yeast and Arabidopsis heterologous expression systems revealed that flax contains unique forms of PDATs that can efficiently channel ALA into TAG. Intrigued by the finding that multiple PDAT paralogs from flax are differentially expressed and encode enzymes with different TAG-forming capacities, the second study was designed to investigate the evolutionary relationship of the PDAT gene family across green plants. The results showed that the PDAT gene family is widely present in plant species and can be divided into seven major clades. The study further revealed that there is a eudicot-wide PDAT gene expansion. The ancient gene duplication and divergent selective pressures may have led to the diversification of PDAT paralogs in the core eudicots. The third study investigated another mechanism for PUFA enrichment in TAG, which involves the biochemical coupling of the reverse reaction of acyl-CoA:lysophosphatidylcholine acyltransferase to the forward reaction of DGAT1. Data obtained from in vivo and in vitro experiments support that PUFAs can be transferred from PC into the acyl-CoA pool by the reverse reaction of LPCAT, and then be utilized by the DGAT1-catalyzed reaction for TAG synthesis. A higher amount of PUFA-containing TAGs was produced by the DGAT1 reaction in the presence than in the absence of LPCAT. The final study was designed to provide direct evidence showing the role of PDAT and DGAT1 in seed oil and ALA accumulation in native flax plants via an RNA interference approach. T1 transgenic seeds were obtained. More analyses need to be carried out. Overall, the present doctoral studies revealed important mechanisms for PUFA-rich TAG production in flax and discovered an interesting evolutionary pattern of the plant PDAT gene family. The knowledge obtained from these studies provides important insights into the seed oil biosynthesis in flax and will benefit future initiatives aimed at producing TAGs with increased PUFA content in plants.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Pan X, Siloto RMP, Wickramarathna AD, Mietkiewska E, Weselake RJ (2013) Identification of a Pair of Phospholipid:Diacylglycerol Acyltransferases from Developing Flax (Linum usitatissimum L.) Seed Catalyzing the Selective Production of Trilinolenin. J Biol Chem 288: 24173–24188Pan X, Peng FY, Weselake RJ (2015) Genome-Wide Analysis of PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) Genes in Plants Reveals the Eudicot-Wide PDAT Gene Expansion and Altered Selective Pressures Acting on the Core Eudicot PDAT Paralogs. Plant Physiol 167: 887-904.Pan X, Chen G, Kazachkov M, Greer MS, Zou J, Weselake RJ (2015) In vivo and In vitro Evidence for Biochemical Coupling of Reactions Catalyzed by Lysophosphatidylcholine Acyltransferase and Diacylglycerol Acyltransferase. J Biol Chem [Epub ahead of print]

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