Mouse Fat-Specific Protein 27 (FSP27) expressed in plant cells localizes to lipid droplets and promotes lipid droplet accumulation and fusion.

Fat-Specific Protein 27 (FSP27) belongs to a small group of vertebrate proteins containing a Cell-death Inducing DNA fragmentation factor-α-like Effector (CIDE)-C domain and is involved in lipid droplet (LD) accumulation and energy homeostasis. FSP27 is predominantly expressed in white and brown adipose tissues, as well as liver, and plays a key role in mediating LD-LD fusion. No orthologs have been identified in invertebrates or plants. In this study, we tested the function of mouse FSP27 in stably-transformed Arabidopsis thaliana leaves and seeds, as well as through transient expression in Nicotiana tabacum suspension-cultured cells and N. benthamiana leaves. Confocal microscopic analysis of plant cells revealed that, similar to ectopic expression in mammalian cells, FSP27 produced in plants 1) correctly localized to LDs, 2) accumulated at LD-LD contact sites, and 3) induced an increase in the number and size of LDs and also promoted LD clustering and fusion. Furthermore, FSP27 increased oil content in transgenic A. thaliana seeds. Given that plant oils have uses in human and animal nutrition as well as industrial uses such as biofuels and bioplastics, our results suggest that ectopic expression of FSP27 in plants represents a potential strategy for increasing oil content and energy density in bioenergy or oilseed crops.

CGI-58, a key regulator of lipid homeostasis and signaling in plants, also regulates polyamine metabolism.

Comparative Gene Identification-58 (CGI-58) is an α/ß hydrolase-type protein that regulates lipid homeostasis and signaling in eukaryotes by interacting with and stimulating the activity of several different types of proteins, including a lipase in mammalian cells and a peroxisomal ABC transporter (PXA1) in plant cells. Here we show that plant CGI-58 also interacts with spermidine synthase 1 (SPDS1), an enzyme that plays a central role in polyamine metabolism by converting putrescine into spermidine. Analysis of polyamine contents in Arabidopsis thaliana plants revealed that spermidine levels were significantly reduced, and putrescine increased, in both cgi-58 and cgi-58/pxa1 mutant plants, relative to pxa1 mutant or wild-type plants. Evaluation of polyamine-related gene expression levels, however, revealed similar increases in transcript abundance in all mutants, including cgi-58, pxa1, and cgi-58/pxa1, in comparison to wild type. Taken together, the data support a model whereby CGI-58 and PXA1 contribute to the regulation of polyamine metabolism at the transcriptional level, perhaps through a shared lipid-signaling pathway, and that CGI-58 also acts independently of PXA1 to increase spermidine content at a post-transcriptional level, possibly through protein-protein interaction with SPDS1.

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.

Lipid droplet-associated proteins (LDAPs) are involved in the compartmentalization of lipophilic compounds in plant cells.

While lipid droplets have traditionally been considered as inert sites for the storage of triacylglycerols and sterol esters, they are now recognized as dynamic and functionally diverse organelles involved in energy homeostasis, lipid signaling, and stress responses. Unlike most other organelles, lipid droplets are delineated by a half-unit membrane whose protein constituents are poorly understood, except in the specialized case of oleosins, which are associated with seed lipid droplets. Recently, we identified a new class of lipid-droplet associated proteins called LDAPs that localize specifically to the lipid droplet surface within plant cells and share extensive sequence similarity with the small rubber particle proteins (SRPPs) found in rubber-accumulating plants. Here, we provide additional evidence for a role of LDAPs in lipid accumulation in oil-rich fruit tissues, and further explore the functional relationships between LDAPs and SRPPs. In addition, we propose that the larger LDAP/SRPP protein family plays important roles in the compartmentalization of lipophilic compounds, including triacylglycerols and polyisoprenoids, into lipid droplets within plant cells. Potential roles in lipid droplet biogenesis and function of these proteins also are discussed.

Identification of a new class of lipid droplet-associated proteins in plants.

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 oleosin-mediated 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.

The α/ß hydrolase CGI-58 and peroxisomal transport protein PXA1 coregulate lipid homeostasis and signaling in Arabidopsis.

COMPARATIVE GENE IDENTIFICATION-58 (CGI-58) 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 ß-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.

Visualization of lipid droplet composition by direct organelle mass spectrometry.

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.

Disruption of the Arabidopsis CGI-58 homologue produces Chanarin-Dorfman-like lipid droplet accumulation in plants.

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 ß-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.