Sesamum indicum Oleosin L improves oil packaging in Nicotiana benthamiana leaves.

Plant oil production has been increasing continuously in the past decade. There has been significant investment in the production of high biomass plants with elevated oil content. We recently showed that the expression of Arabidopsis thaliana WRI1 and DGAT1 genes increase oil content by up to 15% in leaf dry weight tissue. However, triacylglycerols in leaf tissue are subject to degradation during senescence. In order to better package the oil, we expressed a series of lipid droplet proteins isolated from bacterial and plant sources in Nicotiana benthamiana leaf tissue. We observed further increases in leaf oil content of up to 2.3-fold when we co-expressed Sesamum indicum Oleosin L with AtWRI1 and AtDGAT1. Biochemical assays and lipid droplet visualization with confocal microscopy confirmed the increase in oil content and revealed a significant change in the size and abundance of lipid droplets.

Development of a Brassica napus (Canola) Crop Containing Fish Oil-Like Levels of DHA in the Seed Oil.

Plant seeds have long been promoted as a production platform for novel fatty acids such as the ω3 long-chain (≥ C20) polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) commonly found in fish oil. In this article we describe the creation of a canola (Brassica napus) variety producing fish oil-like levels of DHA in the seed. This was achieved by the introduction of a microalgal/yeast transgenic pathway of seven consecutive enzymatic steps which converted the native substrate oleic acid to α-linolenic acid and, subsequently, to EPA, docosapentaenoic acid (DPA) and DHA. This paper describes construct design and evaluation, plant transformation, event selection, field testing in a wide range of environments, and oil profile stability of the transgenic seed. The stable, high-performing event NS-B50027-4 produced fish oil-like levels of DHA (9-11%) in open field trials of T3 to T7 generation plants in several locations in Australia and Canada. This study also describes the highest seed DHA levels reported thus far and is one of the first examples of a deregulated genetically modified crop with clear health benefits to the consumer.

Consensus Mutagenesis and Ancestral Reconstruction Provide Insight into the Substrate Specificity and Evolution of the Front-End Δ6-Desaturase Family.

Marine algae are a major source of ω-3 long-chain polyunsaturated fatty acids (ω3-LCPUFAs), which are conditionally essential nutrients in humans and a target for industrial production. The biosynthesis of these molecules in marine algae requires the desaturation of fatty acids by Δ6-desaturases, and enzymes from different species display a range of specificities toward ω3- and ω6-LCPUFA precursors. In the absence of a molecular structure, the structural basis for the variable substrate specificity of Δ6-desaturases is poorly understood. Here we have conducted a consensus mutagenesis and ancestral protein reconstruction-based analysis of the Δ6-desaturase family, focusing on the ω3-specific Δ6-desaturase from Micromonas pusilla (MpΔ6des) and the bispecific (ω3/ω6) Δ6-desaturase from Ostreococcus tauri (OtΔ6des). Our characterization of consensus amino acid substitutions in MpΔ6des revealed that residues in diverse regions of the protein, such as the N-terminal cytochrome b5 domain, can make important contributions to determining substrate specificity. Ancestral protein reconstruction also suggests that some extant Δ6-desaturases, such as OtΔ6des, could have adapted to different environmental conditions by losing specificity for ω3-LCPUFAs. This data set provides a map of regions within Δ6-desaturases that contribute to substrate specificity and could facilitate future attempts to engineer these proteins for use in biotechnology.

Proteomics reveals the in vitro protein digestibility of seven transmembrane enzymes from the docosahexaenoic acid biosynthesis pathway.

The measurement of protein digestibility is one of the key steps in determining the safety of a genetically modified crop that has been traditionally accomplished using antibodies. Membrane proteins are often extremely difficult to express with replicated authentic tertiary structure in heterologous systems. As a result raising antibodies for use in safety assessment may not be feasible. In this study, LC-MS based proteomics was used to characterise seven transmembrane enzymes from the docosahexaenoic acid biosynthetic pathway that had been introduced into canola. The application of a two-stage digestion strategy involving simulated gastric fluid followed by trypsin enabled the measurement of protein digestibility in vitro. Tryptic peptide markers that spanned the length of each desaturase protein were monitored and showed that these proteins were readily degraded (>95% within 5 min) and highlighted regions of the elongase enzymes that showed limited resistance to simulated gastric digestion. Traditional gel-based and Western blotting analysis of ω3-desaturase and Δ6-elongase revealed rapid hydrolysis of the intact proteins within seconds and no fragments (>3 kDa) remained after 60 min, complementing the novel approach described herein. The LC-MS approach was sensitive, selective and did not require the use of purified proteins.

Quantitation of seven transmembrane proteins from the DHA biosynthesis pathway in genetically engineered canola by targeted mass spectrometry.

Examining tissue-specific expression and the measurement of protein abundance are important steps when assessing the performance of genetically engineered crops. Liquid chromatography-mass spectrometry offers many advantages over traditional methods for protein quantitation, especially when dealing with transmembrane proteins that are often difficult to express or generate antibodies against. In this study, discovery proteomics was used to detect the seven transgenic membrane-bound enzymes from the docosahexaenoic acid (DHA) biosynthetic pathway that had been engineered into canola. Subsequently, a targeted LC-MS/MS method for absolute quantitation was developed and applied to the simultaneous measurement of the seven DHA biosynthetic pathway enzymes in genetically modified canola grown across three sites. The results of this study demonstrated that the enzymatic proteins that drive the production of DHA using seed-specific promoters were detected only in mature and developing seed of DHA canola. None of the DHA biosynthesis pathway proteins were detected in wild-type canola planted in the same site or in the non-seed tissues of the transgenic canola, irrespective of the sampling time or the tissues tested. This study describes a streamlined approach to simultaneously measure multiple membrane-bound proteins in planta.

Metabolic engineering for enhanced oil in biomass.

The world is hungry for energy. Plant oils in the form of triacylglycerol (TAG) are one of the most reduced storage forms of carbon found in nature and hence represent an excellent source of energy. The myriad of applications for plant oils range across foods, feeds, biofuels, and chemical feedstocks as a unique substitute for petroleum derivatives. Traditionally, plant oils are sourced either from oilseeds or tissues surrounding the seed (mesocarp). Most vegetative tissues, such as leaves and stems, however, accumulate relatively low levels of TAG. Since non-seed tissues constitute the majority of the plant biomass, metabolic engineering to improve their low-intrinsic TAG-biosynthetic capacity has recently attracted significant attention as a novel, sustainable and potentially high-yielding oil production platform. While initial attempts predominantly targeted single genes, recent combinatorial metabolic engineering strategies have focused on the simultaneous optimization of oil synthesis, packaging and degradation pathways (i.e., 'push, pull, package and protect'). This holistic approach has resulted in dramatic, seed-like TAG levels in vegetative tissues. With the first proof of concept hurdle addressed, new challenges and opportunities emerge, including engineering fatty acid profile, translation into agronomic crops, extraction, and downstream processing to deliver accessible and sustainable bioenergy.

Identification of Genes Involved in Lipid Biosynthesis through de novo Transcriptome Assembly from Cocos nucifera Developing Endosperm.

Cocos nucifera (coconut), a member of the Arecaceae family, is an economically important woody palm that is widely grown in tropical and subtropical regions. The coconut palm is well known for its ability to accumulate large amounts of oil, approximately 63% of the seed weight. Coconut oil varies significantly from other vegetable oils as it contains a high proportion of medium-chain fatty acids (MCFA; 85%). The unique composition of coconut oil raises interest in understanding how the coconut palm produces oil of a high saturated MCFA content, and if such an oil profile could be replicated via biotechnology interventions. Although some gene discovery work has been performed there is still a significant gap in the knowledge associated with coconut's oil production pathways. In this study, a de novo transcriptome was assembled for developing coconut endosperm to identify genes involved in the synthesis of lipids, particularly triacylglycerol. Of particular interest were thioesterases, acyltransferases and oleosins because of their involvement in the processes of releasing fatty acids for assembly, esterification of fatty acids into glycerolipids and protecting oils from degradation, respectively. It is hypothesized that some of these genes may exhibit a strong substrate preference for MCFA and hence may assist the future development of vegetable oils with an enriched MCFA composition. In this study, we identified and confirmed functionality of five candidate genes from the gene families of interest. This study will benefit future work in areas of increasing vegetable oil production and the tailoring of oil fatty acid compositions.

Up-regulation of lipid biosynthesis increases the oil content in leaves of Sorghum bicolor.

Synthesis and accumulation of the storage lipid triacylglycerol in vegetative plant tissues has emerged as a promising strategy to meet the world's future need for vegetable oil. Sorghum (Sorghum bicolor) is a particularly attractive target crop given its high biomass, drought resistance and C4 photosynthesis. While oilseed-like triacylglycerol levels have been engineered in the C3 model plant tobacco, progress in C4 monocot crops has been lagging behind. In this study, we report the accumulation of triacylglycerol in sorghum leaf tissues to levels between 3 and 8.4% on a dry weight basis depending on leaf and plant developmental stage. This was achieved by the combined overexpression of genes encoding the Zea mays WRI1 transcription factor, Umbelopsis ramanniana UrDGAT2a acyltransferase and Sesamum indicum Oleosin-L oil body protein. Increased oil content was visible as lipid droplets, primarily in the leaf mesophyll cells. A comparison between a constitutive and mesophyll-specific promoter driving WRI1 expression revealed distinct changes in the overall leaf lipidome as well as transitory starch and soluble sugar levels. Metabolome profiling uncovered changes in the abundance of various amino acids and dicarboxylic acids. The results presented here are a first step forward towards the development of sorghum as a dedicated biomass oil crop and provide a basis for further combinatorial metabolic engineering.

Increased DHA Production in Seed Oil Using a Selective Lysophosphatidic Acid Acyltransferase.

Metabolic engineering of the omega-3 (ω3) long chain polyunsaturated fatty acid biosynthesis pathway has generated fish oil-like levels of pharmaceutically and nutritionally important docosahexaenoic acid (DHA) in plant seeds. However, the majority of DHA has been accumulated at the sn-1 and sn-3 positions of triacylglycerol (TAG) in these engineered seeds, leaving only a minor amount (∼10%) at sn-2 position and indicating a strong discrimination (or, a very poor specificity) for DHA by seed lysophosphatidic acid acyltransferases (LPAATs), which mediate the acylation of sn-2 position of glycerol backbone. In order to increase the level of DHA at sn-2 position of TAG and to increase overall DHA level in seeds, we attempted to discover DHA-preferring LPAATs. Several LPAATs for acylation of the sn-2 position of the TAG glycerol backbone were investigated for substrate preference for DHA. In transiently expressing these LPAATs in Nicotiana benthamiana, a Mortierella alpina LPAAT had the highest substrate specificity for accumulating DHA onto oleoyl-lysophosphatidic acid (oleoyl-LPA), while the plant LPAATs tested showed lower preference for DHA. In a competition assay with a pool of four ω3 acyl-Coenzyme A (CoA) substrates involved in the DHA biosynthesis pathway, LPAATs from both M. alpina and Emiliania huxleyi showed a high preference for DHA-CoA acylation onto oleoyl-LPA. When docosahexaenoyl-LPA was used as the acyl receiver, M. alpina LPAAT also showed a high preference for DHA-CoA. Stable overexpression of M. alpina LPAAT in an Arabidopsis line that expressed the DHA biosynthesis pathway significantly increased both the total DHA levels and the distribution of DHA onto the sn-2 position of seed TAG. LC-MS analysis of the seed TAG species also confirmed that overexpression of M. alpina LPAAT increased di-DHA and tri-DHA TAGs, suggesting that the M. alpina LPAAT could enrich DHA at the TAG sn-2 position, leading to a metabolic engineering of oil seed for channeling DHA into the sn-2 position of TAG and to a higher DHA level.

Comparative Lipidomics and Proteomics of Lipid Droplets in the Mesocarp and Seed Tissues of Chinese Tallow (Triadica sebifera).

Lipid droplets (LDs) are composed of a monolayer of phospholipids (PLs), surrounding a core of non-polar lipids that consist mostly of triacylglycerols (TAGs) and to a lesser extent diacylglycerols. In this study, lipidome analysis illustrated striking differences in non-polar lipids and PL species between LDs derived from Triadica sebifera seed kernels and mesocarp. In mesocarp LDs, the most abundant species of TAG contained one C18:1 and two C16:0 and fatty acids, while TAGs containing three C18 fatty acids with higher level of unsaturation were dominant in the seed kernel LDs. This reflects the distinct differences in fatty acid composition of mesocarp (palmitate-rich) and seed-derived oil (α-linoleneate-rich) in T. sebifera. Major PLs in seed LDs were found to be rich in polyunsaturated fatty acids, in contrast to those with relatively shorter carbon chain and lower level of unsaturation in mesocarp LDs. The LD proteome analysis in T. sebifera identified 207 proteins from mesocarp, and 54 proteins from seed kernel, which belong to various functional classes including lipid metabolism, transcription and translation, trafficking and transport, cytoskeleton, chaperones, and signal transduction. Oleosin and lipid droplets associated proteins (LDAP) were found to be the predominant proteins associated with LDs in seed and mesocarp tissues, respectively. We also show that LDs appear to be in close proximity to a number of organelles including the endoplasmic reticulum, mitochondria, peroxisomes, and Golgi apparatus. This comparative study between seed and mesocarp LDs may shed some light on the structure of plant LDs and improve our understanding of their functionality and cellular metabolic networks in oleaginous plant tissues.

A reconfigured Kennedy pathway which promotes efficient accumulation of medium-chain fatty acids in leaf oils.

Medium-chain fatty acids (MCFA, C6-14 fatty acids) are an ideal feedstock for biodiesel and broader oleochemicals. In recent decades, several studies have used transgenic engineering to produce MCFA in seeds oils, although these modifications result in unbalance membrane lipid profiles that impair oil yields and agronomic performance. Given the ability to engineer nonseed organs to produce oils, we have previously demonstrated that MCFA profiles can be produced in leaves, but this also results in unbalanced membrane lipid profiles and undesirable chlorosis and cell death. Here we demonstrate that the introduction of a diacylglycerol acyltransferase from oil palm, EgDGAT1, was necessary to channel nascent MCFA directly into leaf oils and therefore bypassing MCFA residing in membrane lipids. This pathway resulted in increased flux towards MCFA rich leaf oils, reduced MCFA in leaf membrane lipids and, crucially, the alleviation of chlorosis. Deep sequencing of African oil palm (Elaeis guineensis) and coconut palm (Cocos nucifera) generated candidate genes of interest, which were then tested for their ability to improve oil accumulation. Thioesterases were explored for the production of lauric acid (C12:0) and myristic (C14:0). The thioesterases from Umbellularia californica and Cinnamomum camphora produced a total of 52% C12:0 and 40% C14:0, respectively, in transient leaf assays. This study demonstrated that the introduction of a complete acyl-CoA-dependent pathway for the synthesis of MFCA-rich oils avoided disturbing membrane homoeostasis and cell death phenotypes. This study outlines a transgenic strategy for the engineering of biomass crops with high levels of MCFA rich leaf oils.

Step changes in leaf oil accumulation via iterative metabolic engineering.

Synthesis and accumulation of plant oils in the entire vegetative biomass offers the potential to deliver yields surpassing those of oilseed crops. However, current levels still fall well short of those typically found in oilseeds. Here we show how transcriptome and biochemical analyses pointed to a futile cycle in a previously established Nicotiana tabacum line, accumulating up to 15% (dry weight) of the storage lipid triacylglycerol in leaf tissue. To overcome this metabolic bottleneck, we either silenced the SDP1 lipase or overexpressed the Arabidopsis thaliana LEC2 transcription factor in this transgenic background. Both strategies independently resulted in the accumulation of 30-33% triacylglycerol in leaf tissues. Our results demonstrate that the combined optimization of de novo fatty acid biosynthesis, storage lipid assembly and lipid turnover in leaf tissue results in a major overhaul of the plant central carbon allocation and lipid metabolism. The resulting further step changes in oil accumulation in the entire plant biomass offers the possibility of delivering yields that outperform current oilseed crops.

Thioesterase overexpression in Nicotiana benthamiana leaf increases the fatty acid flux into triacylgycerol.

Increasing the oil content of leafy biomass is emerging as a sustainable source of vegetable oil to meet global demand. Transient gene expression in leaf provides a reproducible platform to study the effect of transgenes on lipid biosynthesis. We first generated a transgenic Nicotiana benthamiana line containing high levels of triacylglycerol in the leaf tissue (31.4% by dry weight) by stably expressing WRI1, DGAT1 and OLEOSIN. We then used this line as a platform to test the effect of three Arabidopsis thaliana thioesterases (FATA1, FATA2 and FATB). Further increases in leaf oil content were observed with biochemical and lipid assays revealing an increase in the export of fatty acids from the chloroplast and a modification in the oil profile.

Reduced Triacylglycerol Mobilization during Seed Germination and Early Seedling Growth in Arabidopsis Containing Nutritionally Important Polyunsaturated Fatty Acids.

There are now several examples of plant species engineered to synthesize and accumulate nutritionally important polyunsaturated fatty acids in their seed triacylglycerols (TAG). The utilization of TAG in germinating seeds of such transgenic plants was unknown. In this study, we examined the TAG utilization efficiency during seed germination in transgenic Arabidopsis seeds containing several examples of these fatty acids. Seed TAG species with native fatty acids had higher utilization rate than the TAG species containing transgenically produced polyunsaturated fatty acids. Conversely, quantification of the fatty acid components remaining in the total TAG after early stages of seed germination revealed that the undigested TAGs tended to contain elevated levels of the engineered polyunsaturated fatty acids (PUFA). LC-MS analysis further revealed asymmetrical mobilization rates for the individual TAG species. TAGs which contained multiple PUFA fatty acids were mobilized slower than the species containing single PUFA. The mobilized engineered fatty acids were used in de novo membrane lipid synthesis during seedling development.

Deep Sequencing of the Fruit Transcriptome and Lipid Accumulation in a Non-Seed Tissue of Chinese Tallow, a Potential Biofuel Crop.

Chinese tallow (Triadica sebifera) is a valuable oilseed-producing tree that can grow in a variety of conditions without competing for food production, and is a promising biofuel feedstock candidate. The fruits are unique in that they contain both saturated and unsaturated fat present in the tallow and seed layer, respectively. The tallow layer is poorly studied and is considered only as an external fatty deposition secreted from the seed. In this study we show that tallow is in fact a non-seed cellular tissue capable of triglyceride synthesis. Knowledge of lipid synthesis and storage mechanisms in tissues other than seed is limited but essential to generate oil-rich biomass crops. Here, we describe the annotated transcriptome assembly generated from the fruit coat, tallow and seed tissues of Chinese tallow. The final assembly was functionally annotated, allowing for the identification of candidate genes and reconstruction of lipid pathways. A tallow tissue-specific paralog for the transcription factor gene WRINKLED1 (WRI1) and lipid droplet-associated protein genes, distinct from those expressed in seed tissue, were found to be active in tallow, underpinning the mode of oil synthesis and packaging in this tissue. Our data have established an excellent knowledge base that can provide genetic and biochemical insights for engineering non-seed tissues to accumulate large amounts of oil. In addition to the large data set of annotated transcripts, the study also provides gene-based simple sequence repeat and single nucleotide polymorphism markers.

Metabolic engineering of medium-chain fatty acid biosynthesis in Nicotiana benthamiana plant leaf lipids.

Various research groups are investigating the production of oil in non-seed biomass such as leaves. Recently, high levels of oil accumulation have been achieved in plant biomass using a combination of biotechnological approaches which also resulted in significant changes to the fatty acid composition of the leaf oil. In this study, we were interested to determine whether medium-chain fatty acids (MCFA) could be accumulated in leaf oil. MCFA are an ideal feedstock for biodiesel and a range of oleochemical products including lubricants, coatings, and detergents. In this study, we explore the synthesis, accumulation, and glycerolipid head-group distribution of MCFA in leaves of Nicotiana benthamiana after transient transgenic expression of C12:0-, C14:0-, and C16:0-ACP thioesterase genes. We demonstrate that the production of these MCFA in leaf is increased by the co-expression of the WRINKLED1 (WRI1) transcription factor, with the lysophosphatidic acid acyltransferase (LPAAT) from Cocos nucifera being required for the assembly of tri-MCFA TAG species. We also demonstrate that the newly-produced MCFA are incorporated into the triacylglycerol of leaves in which WRI1 + diacylglycerol acyltransferase1 (DGAT1) genes are co-expressed for increased oil accumulation.

Expression of Mouse MGAT in Arabidopsis Results in Increased Lipid Accumulation in Seeds.

Worldwide demand for vegetable oil is projected to double within the next 30 years due to increasing food, fuel, and industrial requirements. There is therefore great interest in metabolic engineering strategies that boost oil accumulation in plant tissues, however, efforts to date have only achieved levels of storage lipid accumulation in plant tissues far below the benchmark to meet demand. Monoacylglycerol acyltransferase (MGAT) is predominantly associated with lipid absorption and resynthesis in the animal intestine where it catalyzes monoacylglycerol (MAG) to form diacylglycerol (DAG), and then triacylglycerol (TAG). In contrast plant lipid biosynthesis routes do not include MGAT. Rather, DAG and TAG are either synthesized from glycerol-3-phosphate by a series of three subsequent acylation reactions, or originated from phospholipids via an acyl editing pathway. Mouse MGATs 1 and 2 have been shown to increase oil content transiently in Nicotiana benthamiana leaf tissue by 2.6 fold. Here we explore the feasibility of this approach to increase TAG in Arabidopsis thaliana seed. The stable MGAT2 expression resulted in a significant increase in seed oil content by 1.32 fold. We also report evidence of the MGAT2 activity based on in vitro assays. Up to 3.9 fold increase of radiolabeled DAG were produced in seed lysate which suggest that the transgenic MGAT activity can result in DAG re-synthesis by salvaging the MAG product of lipid breakdown. The expression of MGAT2 therefore creates an independent and complementary TAG biosynthesis route to the endogenous Kennedy pathway and other glycerolipid synthesis routes.

Lipidomic analysis of Arabidopsis seed genetically engineered to contain DHA.

Metabolic engineering of omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) in oilseeds has been one of the key targets in recent years. By expressing a transgenic pathway for enhancing the synthesis of the ω3 LC-PUFA docosahexaenoic acid (DHA) from endogenous α-linolenic acid (ALA), we obtained the production of fish oil-like proportions of DHA in Arabidopsis seed oil. Liquid chromatography-mass spectrometry (LC-MS) was used to characterize the triacylglycerol (TAG), diacylglycerol (DAG) and phospholipid (PL) lipid classes in the transgenic and wild type Arabidopsis seeds at both developing and mature stages. The analysis identified the appearance of several abundant DHA-containing phosphatidylcholine (PC), DAG and TAG molecular species in mature seeds. The relative abundances of PL, DAG, and TAG species showed a preferred combination of LC-PUFA with ALA in the transgenic seeds, where LC-PUFA were esterified in positions usually occupied by 20:1ω9. Trace amounts of di-DHA PC and tri-DHA TAG were identified and confirmed by high resolution MS/MS. Studying the lipidome in transgenic seeds provided insights into where DHA accumulated and combined with other fatty acids of neutral and phospholipids from the developing and mature seeds.

Transcriptional and biochemical responses of monoacylglycerol acyltransferase-mediated oil synthesis and associated senescence-like responses in Nicotiana benthamiana.

Triacylglycerol (TAG) accumulates in plant seeds as a major renewable source of carbon for food, fuel and industrial feedstock. Approaches to enhance TAG content by altering lipid pathways and genes in vegetative parts have gained significant attention for biofuel and other applications. However, consequences of these modifications are not always studied in detail. In an attempt to increase TAG levels in leaves we previously demonstrated that a novel substrate, monoacylglycerol (MAG), can be used for the biosynthesis of diacylglycerol (DAG) and TAG. Transient expression of the Mus musculus monoacylglycerol acyltransferases MGAT1 and 2 in the model plant Nicotiana benthamiana increased TAG levels at 5 days post-infiltration (dpi). Here we show that increased TAG and DAG levels can be achieved as early as 2 dpi. In addition, the MGAT1 infiltrated areas showed senescence-like symptoms from 3 dpi onwards. To unravel underlying molecular mechanisms, Illumina deep sequencing was carried out (a) for de-novo assembling and annotation of N. benthamiana leaf transcripts and (b) to characterize MGAT1-responsive transcriptome. We found that MGAT1-responsive genes are involved in several processes including TAG biosynthesis, photosynthesis, cell-wall, cutin, suberin, wax and mucilage biosynthesis, lipid and hormone metabolism. Comparative analysis with transcript profiles from other senescence studies identified characteristic gene expression changes involved in senescence induction. We confirmed that increased TAG and observed senescence-symptoms are due to the MAG depletion caused by MGAT1 activity and suggest a mechanism for MGAT1 induced TAG increase and senescence-like symptoms. The data generated will serve as a valuable resource for oil and senescence related studies and for future N. benthamiana transcriptome studies.