Tracking the metabolic pulse of plant lipid production with isotopic labeling and flux analyses: Past, present and future

Abstract

Metabolism is comprised of networks of chemical transformations, organized into integrated biochemical pathways that are the basis of cellular operation, and function to sustain life. Metabolism, and thus life, is not static. The rate of metabolites transitioning through biochemical pathways (i.e., flux) determines cellular phenotypes, and is constantly changing in response to genetic or environmental perturbations. Each change evokes a response in metabolic pathway flow, and the quantification of fluxes under varied conditions helps to elucidate major and minor routes, and regulatory aspects of metabolism. To measure fluxes requires experimental methods that assess the movements and transformations of metabolites without creating artifacts. Isotopic labeling fills this role and is a long-standing experimental approach to identify pathways and quantify their metabolic relevance in different tissues or under different conditions. The application of labeling techniques to plant science is however far from reaching it potential. In light of advances in genetics and molecular biology that provide a means to alter metabolism, and given recent improvements in instrumentation, computational tools and available isotopes, the use of isotopic labeling to probe metabolism is becoming more and more powerful. We review the principal analytical methods for isotopic labeling with a focus on seminal studies of pathways and fluxes in lipid metabolism and carbon partitioning through central metabolism. Central carbon metabolic steps are directly linked to lipid production by serving to generate the precursors for fatty acid biosynthesis and lipid assembly. Additionally some of the ideas for labeling techniques that may be most applicable for lipid metabolism in the future were originally developed to investigate other aspects of central metabolism. We conclude by describing recent advances that will play an important future role in quantifying flux and metabolic operation in plant tissues.

Transport and transcriptional regulation of oil production in plants

“…[22] PAs are essential for synthesis of various membrane lipids, which are crucial to the life of the cell, PA transport to various locations and their regulation are essential for plant growth and oil production. [5,20] Isotope labeling is a more powerful tool to trace the transport processes and identify the intermediates directly linked to lipid production by serving as precursors for fatty acid biosynthesis and lipid assembly. [5] The enzymes involved in degradation of various phospholipids, such as phospholipase A, D, and C (PLA, PLD, and PLC) have been extensively studied in plants.…”

“…[5,20] Isotope labeling is a more powerful tool to trace the transport processes and identify the intermediates directly linked to lipid production by serving as precursors for fatty acid biosynthesis and lipid assembly. [5] The enzymes involved in degradation of various phospholipids, such as phospholipase A, D, and C (PLA, PLD, and PLC) have been extensively studied in plants. For instance, research indicates that PLD-mediated phospholipid hydrolysis and PLD-generated PA plays a wide range of roles in lipid metabolism, such as TAG biosynthesis and acyl editing, plant response to abiotic and biotic stresses, and cellular dynamics such as vesicle trafficking and cytoskeleton dynamics.…”

“…[2] The importance of FAs and the major metabolic end products of TAGs and membrane glycerolipids in terms of life activity have led to increased demand for them in food, chemical, and medical industries. Though several reviews have discussed advances in the field previously, [3,4] significant progress has been made in our understanding of lipid biosynthesis, transport, [5,6] storage, [7] degradation, [8] and transcriptional regulation. [9] With the aim of updating new findings, particularly those in lipid transport and transcription regulation for metabolic engineering, this article reviews and discusses transport and transcriptional regulation mechanisms that may hold keys and have great potential to be used for improvement of plant oil production and composition.…”

A Novel Pathway for Triacylglycerol Biosynthesis Is Responsible for the Accumulation of Massive Quantities of Glycerolipids in the Surface Wax of Bayberry (Myrica pensylvanica) Fruit

“…Table 4). Notably, and unlike results from most oilseed labeling experiments (Bates et al, 2009;Allen et al, 2015), the initial kinetics of The rapid initial incorporation of [ 14 C]-acetate into the acyl chain of sn-2 MAG provided additional evidence that MAG is an early intermediate in Bayberry surface wax production. Because fatty acyl-CoA pools in plants are very small and would introduce a lag of less than 1 min (Larson and Graham, 2001;Tjellström et al, 2012), the labeling of [ 14 C]-MAG with no discernable lag is consistent with acylation of G3P by acyl-CoA.…”

“…[ 14 C]-glycerol is converted into G3P, the initial molecule that is acylated to produce glycerolipids, while [ 14 C]-acetate enters fatty acid synthesis and labels newly synthesized fatty acids (Slack et al, 1977). Both labels have been used extensively to study membrane and storage lipid synthesis in leaves and oilseeds and to infer metabolic precursor-product relationships, pool sizes, and fluxes (Harwood, 1988;Bates and Browse, 2012;Allen et al, 2015) but have not been used to identify pools of glycerolipid intermediates for cuticular lipid synthesis. Knobs isolated from freshly harvested fruits were incubated for up to 11 h with [ 14 C]-glycerol, and [ 14 C]-incorporation into lipids was analyzed by TLC at five time points.…”