Identification of triacylglycerol remodeling mechanism to synthesize unusual fatty acid containing oils.

Typical plant membranes and storage lipids are comprised of five common fatty acids yet over 450 unusual fatty acids accumulate in seed oils of various plant species. Plant oils are important human and animal nutrients, while some unusual fatty acids such as hydroxylated fatty acids (HFA) are used in the chemical industry (lubricants, paints, polymers, cosmetics, etc.). Most unusual fatty acids are extracted from non-agronomic crops leading to high production costs. Attempts to engineer HFA into crops are unsuccessful due to bottlenecks in the overlapping pathways of oil and membrane lipid synthesis where HFA are not compatible. Physaria fendleri naturally overcomes these bottlenecks through a triacylglycerol (TAG) remodeling mechanism where HFA are incorporated into TAG after initial synthesis. TAG remodeling involves a unique TAG lipase and two diacylglycerol acyltransferases (DGAT) that are selective for different stereochemical and acyl-containing species of diacylglycerol within a synthesis, partial degradation, and resynthesis cycle. The TAG lipase interacts with DGAT1, localizes to the endoplasmic reticulum (with the DGATs) and to puncta around the lipid droplet, likely forming a TAG remodeling metabolon near the lipid droplet-ER junction. Each characterized DGAT and TAG lipase can increase HFA accumulation in engineered seed oils.

Triacylglycerol stability limits futile cycles and inhibition of carbon capture in oil-accumulating leaves.

Engineering plant vegetative tissue to accumulate triacylglycerols (TAG, e.g., oil) can increase the amount of oil harvested per acre to levels that exceed current oilseed crops. Engineered tobacco (Nicotiana tabacum) lines that accumulate 15% to 30% oil of leaf dry weight resulted in starkly different metabolic phenotypes. In-depth analysis of the leaf lipid accumulation and 14CO2 tracking describe metabolic adaptations to the leaf oil engineering. An oil-for-membrane lipid tradeoff in the 15% oil line (referred to as HO) was surprisingly not further exacerbated when lipid production was enhanced to 30% (LEC2 line). The HO line exhibited a futile cycle that limited TAG yield through exchange with starch, altered carbon flux into various metabolite pools and end products, and suggested interference of the glyoxylate cycle with photorespiration that limited CO2 assimilation by 50%. In contrast, inclusion of the LEAFY COTYLEDON 2 (LEC2) transcription factor in tobacco improved TAG stability, alleviated the TAG-to-starch futile cycle, and recovered CO2 assimilation and plant growth comparable to wild type but with much higher lipid levels in leaves. Thus, the unstable production of storage reserves and futile cycling limit vegetative oil engineering approaches. The capacity to overcome futile cycles and maintain enhanced stable TAG levels in LEC2 demonstrated the importance of considering unanticipated metabolic adaptations while engineering vegetative oil crops.

Comparison of TLC, HPLC, and direct-infusion ESI-MS methods for the identification and quantification of diacylglycerol molecular species.

Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent cellular signaling molecules. The different DAG molecular species that accumulate over development or in different tissues reflect the changing aspects of cellular lipid metabolism. Consequently, an accurate determination of DAG molecular species in biological samples is essential to understand various metabolic processes and their diagnostic relevance. However, quantification of DAG molecular species in various biological samples represents a challenging task because of their low abundance, hydrophobicity, and instability. This chapter describes the most common chromatographic (TLC and HPLC) and mass spectrometry (MS) methods used to analyze DAG molecular species. In addition, we directly compared the three methods using DAG obtained by phospholipase C hydrolysis of phosphatidylcholine purified from a Nicotiana benthamiana leaf extract. We conclude that each method identified similar major molecular species, however, the exact levels of those varied mainly due to sensitivity of the technique, differences in sample preparation, and processing. This chapter provides three different methods to analyze DAG molecular species, and the discussion of the benefits and challenges of each technique will aid in choosing the right method for your analysis.

Assessing the biotechnological potential of cotton type-1 and type-2 diacylglycerol acyltransferases in transgenic systems.

The chemical and physical properties of vegetable oils are largely dictated by the ratios of 4-6 common fatty acids contained within each oil. However, examples of plant species that accumulate from trace amounts to >90% of certain unusual fatty acids in seed triacylglycerols have been reported. Many of the general enzymatic reactions that drive both common and unusual fatty acid biosynthesis and accumulation in stored lipids are known, but which isozymes have evolved to specifically fill this role and how they coordinate in vivo is still poorly understood. Cotton (Gossypium sp.) is the very rare example of a commodity oilseed that produces biologically relevant amounts of unusual fatty acids in its seeds and other organs. In this case, unusual cyclopropyl fatty acids (named after the cyclopropane and cyclopropene moieties within the fatty acids) are found in membrane and storage glycerolipids (e.g. seed oils). Such fatty acids are useful in the synthesis of lubricants, coatings, and other types of valuable industrial feedstocks. To characterize the role of cotton acyltransferases in cyclopropyl fatty acid accumulation for bioengineering applications, we cloned and characterized type-1 and type-2 diacylglycerol acyltransferases from cotton and compared their biochemical properties to that of litchi (Litchi chinensis), another cyclopropyl fatty acid-producing plant. The results presented from transgenic microbes and plants indicate both cotton DGAT1 and DGAT2 isozymes efficiently utilize cyclopropyl fatty acid-containing substrates, which helps to alleviate biosynthetic bottlenecks and enhances total cyclopropyl fatty acid accumulation in the seed oil.

Brassica napus Plants Gain Improved Salt-Stress Tolerance and Increased Storage Oil Biosynthesis by Interfering with CRL3BPM Activities.

Generating new strategies to improve plant performance and yield in crop plants becomes increasingly relevant with ongoing and predicted global climate changes. E3 ligases that function as key regulators within the ubiquitin proteasome pathway often are involved in abiotic stress responses, development, and metabolism in plants. The aim of this research was to transiently downregulate an E3 ligase that uses BTB/POZ-MATH proteins as substrate adaptors in a tissue-specific manner. Interfering with the E3 ligase at the seedling stage and in developing seeds results in increased salt-stress tolerance and elevated fatty acid levels, respectively. This novel approach can help to improve specific traits in crop plants to maintain sustainable agriculture.

Suppression of Physaria fendleri SDP1 Increased Seed Oil and Hydroxy Fatty Acid Content While Maintaining Oil Biosynthesis Through Triacylglycerol Remodeling.

Physaria fendleri is a burgeoning oilseed crop that accumulates the hydroxy fatty acid (HFA), lesquerolic acid, and can be a non-toxic alternative crop to castor for production of industrially valuable HFA. Recently, P. fendleri was proposed to utilize a unique seed oil biosynthetic pathway coined "triacylglycerol (TAG) remodeling" that utilizes a TAG lipase to remove common fatty acids from TAG allowing the subsequent incorporation of HFA after initial TAG synthesis, yet the lipase involved is unknown. SUGAR DEPENDENT 1 (SDP1) has been characterized as the dominant TAG lipase involved in TAG turnover during oilseed maturation and germination. Here, we characterized the role of a putative PfeSDP1 in both TAG turnover and TAG remodeling. In vitro assays confirmed that PfeSDP1 is a TAG lipase and demonstrated a preference for HFA-containing TAG species. Seed-specific RNAi knockdown of PfeSDP1 resulted in a 12%-16% increase in seed weight and 14%-19% increase in total seed oil content with no major effect on seedling establishment. The increase in total oil content was primarily due to ~4.7% to ~14.8% increase in TAG molecular species containing two HFA (2HFA-TAG), and when combined with a smaller decrease in 1HFA-TAG content the proportion of total HFA in seed lipids increased 4%-6%. The results are consistent with PfeSDP1 involved in TAG turnover but not TAG remodeling to produce 2HFA-TAG. Interestingly, the concomitant reduction of 1HFA-TAG in PfeSDP1 knockdown lines suggests PfeSDP1 may have a role in reverse TAG remodeling during seed maturation that produces 1HFA-TAG from 2HFA-TAG. Overall, our results provide a novel strategy to enhance the total amount of industrially valuable lesquerolic acid in P. fendleri seeds.

Metabolic flux analysis of the non-transitory starch tradeoff for lipid production in mature tobacco leaves.

The metabolic plasticity of tobacco leaves has been demonstrated via the generation of transgenic plants that can accumulate over 30% dry weight as triacylglycerols. In investigating the changes in carbon partitioning in these high lipid-producing (HLP) leaves, foliar lipids accumulated stepwise over development. Interestingly, non-transient starch was observed to accumulate with plant age in WT but not HLP leaves, with a drop in foliar starch concurrent with an increase in lipid content. The metabolic carbon tradeoff between starch and lipid was studied using 13CO2-labeling experiments and isotopically nonstationary metabolic flux analysis, not previously applied to the mature leaves of a crop. Fatty acid synthesis was investigated through assessment of acyl-acyl carrier proteins using a recently derived quantification method that was extended to accommodate isotopic labeling. Analysis of labeling patterns and flux modeling indicated the continued production of unlabeled starch, sucrose cycling, and a significant contribution of NADP-malic enzyme to plastidic pyruvate production for the production of lipids in HLP leaves, with the latter verified by enzyme activity assays. The results suggest an inherent capacity for a developmentally regulated carbon sink in tobacco leaves and may in part explain the uniquely successful leaf lipid engineering efforts in this crop.

Triacylglycerol remodeling in Physaria fendleri indicates oil accumulation is dynamic and not a metabolic endpoint.

Oilseed plants accumulate triacylglycerol (TAG) up to 80% of seed weight with the TAG fatty acid composition determining its nutritional value or use in the biofuel or chemical industries. Two major pathways for production of diacylglycerol (DAG), the immediate precursor to TAG, have been identified in plants: de novo DAG synthesis and conversion of the membrane lipid phosphatidylcholine (PC) to DAG, with each pathway producing distinct TAG compositions. However, neither pathway fits with previous biochemical and transcriptomic results from developing Physaria fendleri seeds for accumulation of TAG containing >60% lesquerolic acid (an unusual 20 carbon hydroxylated fatty acid), which accumulates at only the sn-1 and sn-3 positions of TAG. Isotopic tracing of developing P. fendleri seed lipid metabolism identified that PC-derived DAG is utilized to initially produce TAG with only one lesquerolic acid. Subsequently a nonhydroxylated fatty acid is removed from TAG (transiently reproducing DAG) and a second lesquerolic acid is incorporated. Thus, a dynamic TAG remodeling process involving anabolic and catabolic reactions controls the final TAG fatty acid composition. Reinterpretation of P. fendleri transcriptomic data identified potential genes involved in TAG remodeling that could provide a new approach for oilseed engineering by altering oil fatty acid composition after initial TAG synthesis; and the comparison of current results to that of related Brassicaceae species in the literature suggests the possibility of TAG remodeling involved in incorporation of very long-chain fatty acids into the TAG sn-1 position in various plants.

14C-Tracing of Lipid Metabolism.

Lipids are produced through a dynamic metabolic network involving branch points, cycles, reversible reactions, parallel reactions in different subcellular compartments, and distinct pools of the same lipid class involved in different parts of the network. For example, diacylglycerol (DAG) is a biosynthetic and catabolic intermediate of many different lipid classes. Triacylglycerol can be synthesized from DAG assembled de novo, or from DAG produced by catabolism of membrane lipids, most commonly phosphatidylcholine. Quantification of lipids provides a snapshot of the lipid abundance at the time they were extracted from the given tissue. However, quantification alone does not provide information on the path of carbon flux through the metabolic network to synthesize each lipid. Understanding lipid metabolic flux requires tracing lipid metabolism with isotopically labeled substrates over time in living tissue. [14C]acetate and [14C]glycerol are commonly utilized substrates to measure the flux of nascent fatty acids and glycerol backbones through the lipid metabolic network in vivo. When combined with mutant or transgenic plants, tracing of lipid metabolism can provide information on the molecular control of lipid metabolic flux. This chapter provides a method for tracing in vivo lipid metabolism in developing Arabidopsis thaliana seeds, including analysis of 14C labeled lipid classes and fatty acid regiochemistry through both thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) approaches.

Combinatorial reprogramming of lipid metabolism in plants: a way towards mass-production of bio-fortified arbuscular mycorrhizal fungi inoculants.

Arbuscular mycorrhizal fungi (AMF) are among the most ancient, widespread and functionally important symbioses on Earth that help feed the world. Yet, mass-production of clean (i.e. in vitro produced), safe and robust inoculum at affordable costs remains a critical challenge. Very recently, Luginbuehl et al. (2017) found that plants supply lipids to the symbiotic partner, thus 'providing the AMF with a robust source of carbon for their metabolic needs'. Hence, engineering plants for enhanced delivery of lipids to AMF could represent an innovative avenue to produce a novel generation of high-quality and cost-effective bio-fortified AMF inoculants for application in agro-ecosystems.

Oil-Producing Metabolons Containing DGAT1 Use Separate Substrate Pools from those Containing DGAT2 or PDAT.

Seed triacylglycerol (TAG) biosynthesis involves a metabolic network containing multiple different diacylglycerol (DAG) and acyl donor substrate pools. This network of pathways overlaps with those for essential membrane lipid synthesis and utilizes multiple different classes of TAG biosynthetic enzymes. Acyl flux through this network ultimately dictates the final oil fatty acid composition. Most strategies to alter seed oil composition involve the overexpression of lipid biosynthetic enzymes, but how these enzymes are assembled into metabolons and which substrate pools are used by each is still not well understood. To understand the roles of different classes of TAG biosynthetic acyltransferases in seed oil biosynthesis, we utilized the Arabidopsis (Arabidopsis thaliana) diacylglycerol acyltransferase mutant dgat1-1 (in which phosphatidylcholine:diacylglycerol acyltransferase (AtPDAT1) is the major TAG biosynthetic enzyme), and enhanced TAG biosynthesis by expression of Arabidopsis acyltransferases AtDGAT1 and AtDGAT2, as well as the DGAT2 enzymes from soybean (Glycine max), and castor (Ricinus communis), followed by isotopic tracing of glycerol flux through the lipid metabolic network in developing seeds. The results indicate each acyltransferase has a unique effect on seed oil composition. AtDGAT1 produces TAG from a rapidly produced phosphatidylcholine-derived DAG pool. However, AtPDAT1 and plant DGAT2 enzymes utilize a different and larger bulk phosphatidylcholine-derived DAG pool that is more slowly turned over for TAG biosynthesis. Based on metabolic fluxes and protein:protein interactions, our model of TAG synthesis suggests that substrate channeling to select enzymes and spatial separation of different acyltransferases into separate metabolons affect efficient TAG production and oil fatty acid composition.

The BADC and BCCP subunits of chloroplast acetyl-CoA carboxylase sense the pH changes of the light-dark cycle.

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a "fly-casting mechanism." We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, Arabidopsis badc1 badc3 mutant lines grown in a light-dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity.

Normal phase HPLC method for combined separation of both polar and neutral lipid classes with application to lipid metabolic flux.

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 14C based metabolic tracing of lipid metabolism in leaves and seeds.

Analysis of Isotopically-labeled Monogalactosyldiacylglycerol Molecular Species from [14C]Acetate-Labeled Tobacco Leaves.

Plant lipid metabolism is a dynamic network where synthesis of essential membrane lipids overlaps with synthesis of valuable storage lipids (e.g., vegetable oils). Monogalactosyldiacylglycerol (MGDG) is a key component of the chloroplast membrane system required for photosynthesis and is produced by multiple pathways within the lipid metabolic network. The bioengineering of plants to enhance oil production can alter lipid metabolism in unexpected ways which may not be apparent by static quantification of lipids, but changes to lipid metabolic flux can be traced with isotopic labeling commonly with [14C]acetate. Because lipid classes such as MGDG are composed of many different molecular species, full analysis of metabolically labeled lipids requires separation and quantification of the individually labeled molecular species which is traditionally performed by thin layer chromatography. Here we present a reverse phase HPLC method for the separation of MGDG molecular species from tobacco leaves in under 35 min. The quantification of each 14C-labeled molecular species was accomplished by an in-line flow radio detector. This method of analysis for [14C]Acetate labeled MGDG molecular species by radio-HPLC provides a rapid, high throughput, and reliable analytical approach to identify changes in MGDG metabolism due to bioengineering or other perturbations of metabolism.

Reorganization of Acyl Flux through the Lipid Metabolic Network in Oil-Accumulating Tobacco Leaves.

The triacylglycerols (TAGs; i.e. oils) that accumulate in plants represent the most energy-dense form of biological carbon storage, and are used for food, fuels, and chemicals. The increasing human population and decreasing amount of arable land have amplified the need to produce plant oil more efficiently. Engineering plants to accumulate oils in vegetative tissues is a novel strategy, because most plants only accumulate large amounts of lipids in the seeds. Recently, tobacco (Nicotiana tabacum) leaves were engineered to accumulate oil at 15% of dry weight due to a push (increased fatty acid synthesis)-and-pull (increased final step of TAG biosynthesis) engineering strategy. However, to accumulate both TAG and essential membrane lipids, fatty acid flux through nonengineered reactions of the endogenous metabolic network must also adapt, which is not evident from total oil analysis. To increase our understanding of endogenous leaf lipid metabolism and its ability to adapt to metabolic engineering, we utilized a series of in vitro and in vivo experiments to characterize the path of acyl flux in wild-type and transgenic oil-accumulating tobacco leaves. Acyl flux around the phosphatidylcholine acyl editing cycle was the largest acyl flux reaction in wild-type and engineered tobacco leaves. In oil-accumulating leaves, acyl flux into the eukaryotic pathway of glycerolipid assembly was enhanced at the expense of the prokaryotic pathway. However, a direct Kennedy pathway of TAG biosynthesis was not detected, as acyl flux through phosphatidylcholine preceded the incorporation into TAG. These results provide insight into the plasticity and control of acyl lipid metabolism in leaves.

Metabolically Distinct Pools of Phosphatidylcholine Are Involved in Trafficking of Fatty Acids out of and into the Chloroplast for Membrane Production.

The eukaryotic pathway of galactolipid synthesis involves fatty acid synthesis in the chloroplast, followed by assembly of phosphatidylcholine (PC) in the endoplasmic reticulum (ER), and then turnover of PC to provide a substrate for chloroplast galactolipid synthesis. However, the mechanisms and classes of lipids transported between the chloroplast and the ER are unclear. PC, PC-derived diacylglycerol, phosphatidic acid, and lyso-phosphatidylcholine (LPC) have all been implicated in ER-to-chloroplast lipid transfer. LPC transport requires lysophosphatidylcholine acyltransferase (LPCAT) activity at the chloroplast to form PC before conversion to galactolipids. However, LPCAT has also been implicated in the opposite chloroplast-to-ER trafficking of newly synthesized fatty acids through PC acyl editing. To understand the role of LPC and LPCAT in acyl trafficking we produced and analyzed the Arabidopsis (Arabidopsis thaliana) act1 lpcat1 lpcat2 triple mutant. LPCAT1 and LPCAT2 encode the major lysophospholipid acyltransferase activity of the chloroplast, and it is predominantly for incorporation of nascent fatty acids exported form the chloroplast into PC by acyl editing. In vivo acyl flux analysis revealed eukaryotic galactolipid synthesis is not impaired in act1 lpcat1 lpcat2 and uses a PC pool distinct from that of PC acyl editing. We present a model for the eukaryotic pathway with metabolically distinct pools of PC, suggesting an underlying spatial organization of PC metabolism as part of the ER-chloroplast metabolic interactions.

Quantitative Proteomic Analysis of Low Linolenic Acid Transgenic Soybean Reveals Perturbations of Fatty Acid Metabolic Pathways.

To understand the effect of fatty acid desaturase gene (GmFAD3) silencing on perturbation of fatty acid (FA) metabolic pathways, the changes are compared in protein profiling in control and low linolenic acid transgenic soybeans using tandem mass tag based mass spectrometry. Protein profiling of the transgenic line unveiled changes in several key enzymes of FA metabolism. This includes enzymes of lower abundance; fabH, fabF, and thioestrase associated with FA initiation, elongation, and desaturation processes and LOX1_5, ACOX, ACAA1, MFP2 associated with ß-oxidation of α-linolenic acids pathways. In addition, the GmFAD3 silencing results in a significant reduction in one of the major allergens, Gly m 4 (C6T3L5). These results are important for exploring how plants adjust in their biological processes when certain changes are induced in the genetic makeup. A complete understanding of these processes will aid researchers to alter genes for developing value-added soybeans.

Specialized lysophosphatidic acid acyltransferases contribute to unusual fatty acid accumulation in exotic Euphorbiaceae seed oils.

MAIN CONCLUSION: In vivo and in vitro analyses of Euphorbiaceae species' triacylglycerol assembly enzymes substrate selectivity are consistent with the co-evolution of seed-specific unusual fatty acid production and suggest that many of these genes will be useful for biotechnological production of designer oils. Many exotic Euphorbiaceae species, including tung tree (Vernicia fordii), castor bean (Ricinus communis), Bernardia pulchella, and Euphorbia lagascae, accumulate unusual fatty acids in their seed oils, many of which have valuable properties for the chemical industry. However, various adverse plant characteristics including low seed yields, production of toxic compounds, limited growth range, and poor resistance to abiotic stresses have limited full agronomic exploitation of these plants. Biotechnological production of these unusual fatty acids (UFA) in high yielding non-food oil crops would provide new robust sources for these valuable bio-chemicals. Previous research has shown that expression of the primary UFA biosynthetic gene alone is not enough for high-level accumulation in transgenic seed oils; other genes must be included to drive selective UFA incorporation into oils. Here, we use a series of in planta molecular genetic studies and in vitro biochemical measurements to demonstrate that lysophosphatidic acid acyltransferases from two Euphorbiaceae species have high selectivity for incorporation of their respective unusual fatty acids into the phosphatidic acid intermediate of oil biosynthesis. These results are consistent with the hypothesis that unusual fatty acid accumulation arose in part via co-evolution of multiple oil biosynthesis and assembly enzymes that cooperate to enhance selective fatty acid incorporation into seed oils over that of the common fatty acids found in membrane lipids.