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Three normal phase HPLC methods were produced to separate lipid classes on a PVA-Sil stationary phase including: 9 polar lipids (method 1); 13 combined polar and neutral lipids (method 2); and a combined method that further separates the neutral lipids into 2-4 subclasses based on the presence of fatty acids containing a polar functional group (e.g. hydroxyl) for a total of 20 lipid classes and subclasses separated in a single run (method 3). Polar lipids separated include: the phosphoglycerolipids PG, PE, PI, PS, PC and LPC; the galactoglycerolipids MGDG and DGDG; and a sulfoglycerolipid SQDG. Neutral lipids include TAG, DAG, and MAG classes and sub-classes containing 0-3, 0-2, and 0-1 hydroxy fatty acids, respectively. The hexane/isopropanol/methanol/aqueous system separates polar lipids without the use of chloroform such that it is suitable for radioactivity analysis by in-line flow scintillation counting. Each method was optimized using the natural lipid standards comprised of diverse molecular species that were detected by ELSD. All molecular species of each lipid class eluted together as single peak detected by ELSD. The methods were demonstrated to be suitable for resolving lipid extracts from animal, microbial, and plant sources as well as application to 14 C based metabolic tracing of lipid metabolism in leaves and seeds., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In this paper, we report a method for the separation of hydroxy fatty acid and non-hydroxy fatty acid containing neutral lipid classes via normal phase HPLC with UV detection on a PVA-Sil column. The hexane/isopropanol/methanol/water based method separates all the neutral lipids in 21 min, and subsequently flushes through the polar lipids by 27 min such that prefractionation of neutral and polar lipids are not required, and the column is re-equilibrated for the next run in 15 min, for a total run time of 45 min per sample. The separation was demonstrated at both 1.0 mL/min and 1.5 mL/min for added applicability for fraction collection or inline analysis. Separation of various hydroxy fatty acid containing lipids was demonstrated from three different plant species Ricinus communis, Physaria fendleri, and engineered Arabidopsis thaliana. Additionally, we have combined this method with an in-line liquid scintillation counter for the separation and quantification of 14 C labeled lipids obtained from in vivo metabolic flux experiments conducted in the developing seeds of Arabidopsis thaliana., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Plant oil biosynthesis involves a complex metabolic network with multiple subcellular compartments, parallel pathways, cycles, and pathways that have a dual function to produce essential membrane lipids and triacylglycerol. Modern molecular biology techniques provide tools to alter plant oil compositions through bioengineering, however with few exceptions the final composition of triacylglycerol cannot be predicted. One reason for limited success in oilseed bioengineering is the inadequate understanding of how to control the flux of fatty acids through various fatty acid modification, and triacylglycerol assembly pathways of the lipid metabolic network. This review focuses on the mechanisms of acyl flux through the lipid metabolic network, and highlights where uncertainty resides in our understanding of seed oil biosynthesis. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Degradation of unusual fatty acids through β-oxidation within transgenic plants has long been hypothesized as a major factor limiting the production of industrially useful unusual fatty acids in seed oils. Arabidopsis seeds expressing the castor fatty acid hydroxylase accumulate hydroxylated fatty acids up to 17% of total fatty acids in seed triacylglycerols; however, total seed oil is also reduced up to 50%. Investigations into the cause of the reduced oil phenotype through in vivo [(14)C]acetate and [(3)H]2O metabolic labeling of developing seeds surprisingly revealed that the rate of de novo fatty acid synthesis within the transgenic seeds was approximately half that of control seeds. RNAseq analysis indicated no changes in expression of fatty acid synthesis genes in hydroxylase-expressing plants. However, differential [(14)C]acetate and [(14)C]malonate metabolic labeling of hydroxylase-expressing seeds indicated the in vivo acetyl-CoA carboxylase activity was reduced to approximately half that of control seeds. Therefore, the reduction of oil content in the transgenic seeds is consistent with reduced de novo fatty acid synthesis in the plastid rather than fatty acid degradation. Intriguingly, the coexpression of triacylglycerol synthesis isozymes from castor along with the fatty acid hydroxylase alleviated the reduced acetyl-CoA carboxylase activity, restored the rate of fatty acid synthesis, and the accumulation of seed oil was substantially recovered. Together these results suggest a previously unidentified mechanism that detects inefficient utilization of unusual fatty acids within the endoplasmic reticulum and activates an endogenous pathway for posttranslational reduction of fatty acid synthesis within the plastid.