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.

Genetic enhancement of oil content in potato tuber (Solanum tuberosum L.) through an integrated metabolic engineering strategy.

Potato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber-specific (patatin) and constitutive (CaMV-35S) promoters. This coordinated metabolic engineering approach resulted in over a 100-fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild-type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof-of-concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.

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.

Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves.

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co-expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications.

Kinetic study of the rearrangement of deuterium-labeled 4'-O-methylnorbelladine in Leucojum aestivum shoot cultures by mass spectrometry. Influence of precursor feeding on amaryllidaceae alkaloid accumulation.

Alkaloids from plants of the family Amaryllidaceae have important pharmacological properties and can be regarded as derivatives of the common precursor 4'-O-methylnorbelladine (6) via intramolecular oxidative phenol coupling. Their biosynthetic pathway, particularly in Leucojum aestivum, has not yet been totally elucidated. Therefore, shoot cultures of this plant were subcultured in medium containing the labeled precursor 4'-O-methyl-d(3)-norbelladine (3) at various concentrations (0.05, 0.10, and 0.20 g/L) and were incubated for various periods of time (15, 30, and 40 days). The aim of this work was to study the influence of this precursor on both labeled and native alkaloid accumulation. Biotransformation into galanthamine (1) and lycorine (2) in shoot cultures was demonstrated using HPLC coupled to mass spectrometry. A maximal amount of 0.16% of 1 referred to the dry weight was obtained at day 15 in shoots fed with 0.10 g/L of precursor. In addition, a 20.5% dry weight of 2 was reached after 40 days of feeding with 0.20 g/L of precursor.

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.

LCMS and GCMS for the screening of alkaloids in natural and in vitro extracts of Leucojum aestivum.

HPLC coupled to a mass spectrometer (MS) was used for the analysis of galanthamine and lycorine in natural extracts of Leucojum aestivum and in their in vitro cultures grown with a precursor (ACC), inhibitors (AgNO(3), STS), or an absorber (KMnO(4)) of ethylene. The maximum galanthamine (0.002%) and lycorine (0.02%) concentrations in tissue cultures were obtained in the presence of KMnO(4). GCMS was used to investigate underivatized alkaloid mixtures from L. aestivum. Seven alkaloids were identified in in vivo bulbs. KMnO(4) led to the highest diversity of alkaloids in tissue culture extracts.

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.

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.

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.

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.

Synergistic effect of WRI1 and DGAT1 coexpression on triacylglycerol biosynthesis in plants.

Metabolic engineering approaches to increase plant oil levels can generally be divided into categories which increase fatty acid biosynthesis ('Push'), are involved in TAG assembly ('Pull') or increase TAG storage/decrease breakdown ('Accumulation'). In this study, we describe the surprising synergy when Push (WRI1) and Pull (DGAT1) approaches are combined. Co-expression of these genes in the Nicotiana benthamiana transient leaf expression system resulted in TAG levels exceeding those expected from an additive effect and biochemical tracer studies confirmed increased flux of carbon through fatty acid and TAG synthesis pathways. Leaf fatty acid profile also synergistically shifts from polyunsaturated to monounsaturated fatty acids.

Recruiting a new substrate for triacylglycerol synthesis in plants: the monoacylglycerol acyltransferase pathway.

BACKGROUND: Monoacylglycerol acyltransferases (MGATs) are predominantly associated with lipid absorption and resynthesis in the animal intestine where they catalyse the first step in the monoacylglycerol (MAG) pathway by acylating MAG to form diacylglycerol (DAG). Typical plant triacylglycerol (TAG) biosynthesis routes such as the Kennedy pathway do not include an MGAT step. Rather, DAG and TAG are synthesised de novo from glycerol-3-phosphate (G-3-P) by a series of three subsequent acylation reactions although a complex interplay with membrane lipids exists. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that heterologous expression of a mouse MGAT acyltransferase in Nicotiana benthamiana significantly increases TAG accumulation in vegetative tissues despite the low levels of endogenous MAG substrate available. In addition, DAG produced by this acyltransferase can serve as a substrate for both native and coexpressed diacylglycerol acyltransferases (DGAT). Finally, we show that the Arabidopsis thaliana GPAT4 acyltransferase can produce MAG in Saccharomyces cerevisiae using oleoyl-CoA as the acyl-donor. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the concept of a new method of increasing oil content in vegetative tissues by using MAG as a substrate for TAG biosynthesis. Based on in vitro yeast assays and expression results in N. benthamiana, we propose that co-expression of a MAG synthesising enzyme such as A. thaliana GPAT4 and a MGAT or bifunctional M/DGAT can result in DAG and TAG synthesis from G-3-P via a route that is independent and complementary to the endogenous Kennedy pathway and other TAG synthesis routes.

New method for the study of Amaryllidaceae alkaloid biosynthesis using biotransformation of deuterium-labeled precursor in tissue cultures.

Biotransformation of deuterated-4'-O-methylnorbelladine into alkaloids galanthamine and lycorine in tissue cultures of Leucojum aestivum was demonstrated using HPLC coupled to mass spectrometry. GC-MS screening was also carried to investigate other native and deuterated alkaloids. A total of six labeled alkaloids were identified indicating that 4'-O-methyl-d(3)-norbelladine is incorporated into three different groups of Amaryllidaceae alkaloids that are biosynthesized by three modes of intramolecular oxidative phenol coupling.