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.

Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants.

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER-vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.

Arabidopsis thaliana GPAT8 and GPAT9 are localized to the ER and possess distinct ER retrieval signals: functional divergence of the dilysine ER retrieval motif in plant cells.

Glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the committed step in the production of glycerolipids, which are major components of cellular membranes, seed storage oils, and epicuticular wax coatings. While the biochemical activities of GPATs have been characterized in detail, the cellular features of these enzymes are only beginning to emerge. Here we characterized the phylogenetic relationships and cellular properties of two GPAT enzymes from the relatively large Arabidopsis thaliana GPAT family, including GPAT8, which is involved in cutin biosynthesis, and GPAT9, which is a new putative GPAT that has extensive homology with a GPAT from mammalian cells involved in storage oil formation and, thus, may have a similar role in plants. Immunofluorescence microscopy of transiently-expressed myc-epitope-tagged GPAT8 and GPAT9 revealed that both proteins were localized to the endoplasmic reticulum (ER), and differential permeabilization experiments indicated that their N- and C-termini were oriented towards the cytosol. However, these two proteins contained distinct types of ER retrieval signals, with GPAT8 possessing a divergent type of dilysine motif (-KK-COOH rather than the prototypic -KKXX-COOH or -KXKXX-COOH motif) and GPAT9 possessing a hydrophobic pentapeptide motif (-phi-X-X-K/R/D/E-phi-; where phi are large hydrophobic amino acid residues). Notably, the divergent dilysine motif in GPAT8 only functioned effectively when additional upstream residues were included to provide the proper protein context. Extensive mutational analyses of the divergent dilysine motif, based upon sequences present in the C-termini of other GPAT8s from various plant species, further expanded the functional definition of this molecular targeting signal, thereby providing insight to the targeting signals in other GPAT family members as well as other ER-resident membrane proteins within plant cells.

Engineering oilseeds for sustainable production of industrial and nutritional feedstocks: solving bottlenecks in fatty acid flux.

Oilseeds provide a unique platform for the production of high-value fatty acids that can replace non-sustainable petroleum and oceanic sources of specialty chemicals and aquaculture feed. However, recent efforts to engineer the seeds of crop and model plant species to produce new types of fatty acids, including hydroxy and conjugated fatty acids for industrial uses and long-chain omega-3 polyunsaturated fatty acids for farmed fish feed, have met with only modest success. The collective results from these studies point to metabolic 'bottlenecks' in the engineered plant seeds that substantially limit the efficient or selective flux of unusual fatty acids between different substrate pools and ultimately into storage triacylglycerol. Evidence is emerging that diacylglycerol acyltransferase 2, which catalyzes the final step in triacylglycerol assembly, is an important contributor to the synthesis of unusual fatty acid-containing oils, and is likely to be a key target for future oilseed metabolic engineering efforts.

Tung tree DGAT1 and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic reticulum.

Seeds of the tung tree (Vernicia fordii) produce large quantities of triacylglycerols (TAGs) containing approximately 80% eleostearic acid, an unusual conjugated fatty acid. We present a comparative analysis of the genetic, functional, and cellular properties of tung type 1 and type 2 diacylglycerol acyltransferases (DGAT1 and DGAT2), two unrelated enzymes that catalyze the committed step in TAG biosynthesis. We show that both enzymes are encoded by single genes and that DGAT1 is expressed at similar levels in various organs, whereas DGAT2 is strongly induced in developing seeds at the onset of oil biosynthesis. Expression of DGAT1 and DGAT2 in yeast produced different types and proportions of TAGs containing eleostearic acid, with DGAT2 possessing an enhanced propensity for the synthesis of trieleostearin, the main component of tung oil. Both DGAT1 and DGAT2 are located in distinct, dynamic regions of the endoplasmic reticulum (ER), and surprisingly, these regions do not overlap. Furthermore, although both DGAT1 and DGAT2 contain a similar C-terminal pentapeptide ER retrieval motif, this motif alone is not sufficient for their localization to specific regions of the ER. These data suggest that DGAT1 and DGAT2 have nonredundant functions in plants and that the production of storage oils, including those containing unusual fatty acids, occurs in distinct ER subdomains.