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.

Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosinmediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues.

CGI-58 is the defective gene in the human neutral lipid storage disease called Chanarin-Dorfman syndrome. This disorder causes intracellular lipid droplets to accumulate in nonadipose tissues, such as skin and blood cells. Here, disruption of the homologous CGI-58 gene in Arabidopsis thaliana resulted in the accumulation of neutral lipid droplets in mature leaves. Mass spectroscopy of isolated lipid droplets from cgi-58 loss-of-function mutants showed they contain triacylglycerols with common leaf-specific fatty acids. Leaves of mature cgi-58 plants exhibited a marked increase in absolute triacylglycerol levels, more than 10-fold higher than in wild-type plants. Lipid levels in the oil-storing seeds of cgi-58 loss-of-function plants were unchanged, and unlike mutations in β-oxidation, the cgi-58 seeds germinated and grew normally, requiring no rescue with sucrose. We conclude that the participation of CGI-58 in neutral lipid homeostasis of nonfat-storing tissues is similar, although not identical, between plant and animal species. This unique insight may have implications for designing a new generation of technologies that enhance the neutral lipid content and composition of crop plants.compartmentation | plant lipid metabolism P lants synthesize and store neutral lipids such as triacylglycerols (TAGs) primarily in cytosolic lipid droplets of maturing seeds (1, 2). In domesticated oilseeds, these stored TAGs represent a major source of calories for human and animal nutrition, an excellent feedstock for diesel fuels, and a reservoir for the deposition of industrially important fatty acids used as chemical feedstocks (3-6). Although not commonly appreciated, TAGs also are synthesized in nonseed tissues (7,8), but their abundance in these tissues is low, in part because of the metabolism of the cell and perhaps as a consequence of the continuous recycling of fatty acids for energy and membrane synthesis. Indeed, vegetative cells can incorporate radiolabeled precursors into TAG (7, 8), they express diacylglycerol acyltransferases [the only enzyme in the "Kennedy pathway" unique to TAG production (9)], and they can accumulate TAGs in β-oxidation mutants (2) and in some floral (10) and fruit (7) tissues. Thus, although plant vegetative cells appear to have the metabolic machinery to synthesize and accumulate neutral lipids, there are likely underlying regulatory mechanisms in place to minimize this process, none of which are understood.Chanarin-Dorfman syndrome is a neutral-lipid storage disease (11) caused by a defect in the protein CGI-58 (comparative gene identification-58, also called ABHD5 for α/β hydrolase-5). CGI-58 is a soluble enzyme that associates with cytosolic lipid droplets under certain metabolic conditions and appears to play a role in hydrolysis of stored lipids (11)(12)(13)(14). Several different mutations in this protein, including amino acid substitutions, premature stop codons, and defects in mRNA splicing, have been identified in various Chanarin-Dorfman patients, all of which result in a hyperac...

An expanding appreciation for the varied functions of neutral lipids in cellular organisms relies on a more detailed understanding of the mechanisms of lipid production and packaging into cytosolic lipid droplets (LDs). Conventional lipid profiling procedures involve the analysis of tissue extracts and consequently lack cellular or subcellular resolution. Here, we report an approach that combines the visualization of individual LDs, microphase extraction of lipid components from droplets, and the direct identification of lipid composition by nanospray mass spectrometry, even to the level of a single LD. The triacylglycerol (TAG) composition of LDs from several plant sources (mature cotton (Gossypium hirsutum) embryos, roots of cotton seedlings, and Arabidopsis thaliana seeds and leaves) were examined by direct organelle mass spectrometry and revealed the heterogeneity of LDs derived from different plant tissue sources. The analysis of individual LDs makes possible organellar resolution of molecular compositions and will facilitate new studies of LD biogenesis and functions, especially in combination with analysis of morphological and metabolic mutants. Furthermore, direct organelle mass spectrometry could be applied to the molecular analysis of other subcellular compartments and macromolecules. LDs4 are organelles that are specialized for the storage of neutral lipids and as such provide energy-rich reserves in all cellular organisms (1). Understanding LD ontogeny is of major importance to human physiology; on the one hand, seed oils, packaged in LDs, make up a growing proportion of daily caloric intake in most diets around the world, and on the other hand, the regulation of lipid storage and mobilization underlies significant human health issues: obesity, diabetes and cardiovascular disease.Although storage is considered the principal role of neutral lipids, LDs in nonfat storing tissues recently have become more appreciated for their dynamic nature and functional roles independent of storage. These roles include acyl reserves for phospholipid recycling (2), lipid signaling (3), membrane trafficking (2, 4), inflammation and cancer (5), and hostpathogen interactions (6, 7). These various functions attributed to LDs vary with cell type and likely are manifested by differences in droplet composition. The basic structural model of LDs in plant seeds provides a thermodynamically stable organization that is thought to be conserved throughout eukaryotes, although the nature of the lipids and proteins associated with droplets varies with cell/tissue type. The structure describes a neutral lipid core (triacylglycerols in plant seeds and/or steryl esters in other organisms or cell types) surrounded by a phospholipid monolayer with specific proteins associated with the LD surface (8). Although the endoplasmic reticulum is considered by most to be the major cellular location for LD biogenesis, droplets associate frequently with other subcellular compartments, presumably to carry out unique functions (9).The recent emphasis...

COMPARATIVE GENE ) is a key regulator of lipid metabolism and signaling in mammals, but its underlying mechanisms are unclear. Disruption of CGI-58 in either mammals or plants results in a significant increase in triacylglycerol (TAG), suggesting that CGI-58 activity is evolutionarily conserved. However, plants lack proteins that are important for CGI-58 activity in mammals. Here, we demonstrate that CGI-58 functions by interacting with the PEROXISOMAL ABC-TRANSPORTER1 (PXA1), a protein that transports a variety of substrates into peroxisomes for their subsequent metabolism by b-oxidation, including fatty acids and lipophilic hormone precursors of the jasmonate and auxin biosynthetic pathways. We also show that mutant cgi-58 plants display changes in jasmonate biosynthesis, auxin signaling, and lipid metabolism consistent with reduced PXA1 activity in planta and that, based on the double mutant cgi-58 pxa1, PXA1 is epistatic to CGI-58 in all of these processes. However, CGI-58 was not required for the PXA1-dependent breakdown of TAG in germinated seeds. Collectively, the results reveal that CGI-58 positively regulates many aspects of PXA1 activity in plants and that these two proteins function to coregulate lipid metabolism and signaling, particularly in nonseed vegetative tissues. Similarities and differences of CGI-58 activity in plants versus animals are discussed.