Plant Seed Oil Bodies Research Articles

Embryo-specific expression of soybean oleosin altered oil body morphogenesis and increased lipid content in transgenic rice seeds

Oleosin is the most abundant protein in the oil bodies of plant seeds, playing an important role in regulating oil body formation and lipid accumulation. To investigate whether lipid accumulation in transgenic rice seeds depends on the expression level of oleosin, we introduced two soybean oleosin genes encoding 24kDa proteins into rice under the control of an embryo-specific rice promoter REG-2. Overexpression of soybean oleosin in transgenic rice leads to an increase of seed lipid content up to 36.93 and 46.06% higher than that of the non-transgenic control, respectively, while the overall fatty acid profiles of triacylglycerols remained unchanged. The overexpression of soybean oleosin in transgenic rice seeds resulted in more numerous and smaller oil bodies compared with wild type, suggesting that an inverse relationship exists between oil body size and the total oleosin level. The increase in lipid content is accompanied by a reduction in the accumulation of total seed protein. Our results suggest that it is possible to increase rice seed oil content for food use and for use as a low-cost feedstock for biodiesel by overexpressing oleosin in rice seeds.

Caleosin-assembled oil bodies as a potential delivery nanocarrier

Encapsulation of hydrophobic agents with nanocarriers is challenging. Therefore, we have sought to use nanoscale artificial oil bodies (NOBs) as an alternative delivery carrier. To constitute NOBs, caleosin (Cal), a structural protein of plant seed oil bodies, was first fused with ZH2 (Cal-ZH2). ZH2 is a bivalent anti-HER2/neu affibody with a high affinity towards the HER2/neu receptor. After overproduction in Escherichia coli, insoluble Cal-ZH2 was isolated and used to assemble NOBs in one step. Consequently, resulting NOBs had a zeta potential around -49mV and ranged in size from 150 to 200nm. Upon loading with a hydrophobic fluorescence dye, NOBs were found to be selectively internalized into HER2/neu-positive tumor cells. Further analyses showed that more than 90% cells were invaded by dye-loaded NOBs and the cargo dye was released in cells with time. In addition, the in vitro assay revealed the release of the dye from NOBs in a slow and prolong manner. Overall, these results indicate the potential of Cal-based NOBs as a delivery vehicle.

New protein isoforms identified within Arabidopsis thaliana seed oil bodies combining chymotrypsin/trypsin digestion and peptide fragmentation analysis

Plant seed oil bodies, subcellular lipoprotein inclusions providing storage reserves, are composed of a neutral lipid core surrounded by a phospholipid monolayer with several integrated proteins that play a significant role in stabilization of the particles and probably also in lipid mobilization. Oil bodies' proteins are generally very hydrophobic, due to the long uncharged sequences anchoring them into the lipid core, which makes them extremely difficult to handle and to digest successfully. Although oil bodies have been intensively studied during last decades, not all their proteins have been identified yet. To overcome the problems connected with their identification, a method based on SDS-PAGE, in-gel digestion and LC-MS/MS analysis was used. Digestion was carried out with trypsin and chymotrypsin, single or in combination, which increased significantly the number of identified peptides, namely the hydrophobic ones. Thanks to this methodology it was possible to achieve an extensive coverage of proteins studied, to analyze their N-terminal modifications and moreover, to detect four new oil bodies' protein isoforms, which demonstrates the complexity of oil bodies' protein composition.

Selective internalization of self-assembled artificial oil bodies byHER2/neu-positive cells

A novel delivery carrier was developed using artificial oil bodies (AOBs). Plant seed oil bodies(OBs) consist of a triacylglycerol matrix surrounded by a monolayer of phospholipids embeddedwith the storage protein oleosin (Ole). Ole consists of a central hydrophobic domain withtwo amphiphatic arms that extrude from the surface of OBs. In this study, a bivalent anti-HER2/neu affibody domain (ZH2) was fused with Ole at the C terminus. After overproduction inEscherichia coli, the fusion protein (Ole-ZH2) was recovered to assemble AOBs. The size ofself-assembled AOBs was tailored by varying the oil/Ole-ZH2 ratio and pH toreach a nanoscale. Upon co-incubation with tumor cells, the nanoscale AOBsencapsulated with a hydrophobic fluorescence dye were selectively internalized byHER2/neu-overexpressing cells and displayed biocompatibility with the cells. In addition, theZH2-mediated endosomal entry of AOBs occurred in a time- and AOB dose-dependentmanner. The internalization efficiency was as high as 90%. The internalized AOBsdisintegrated at the non-permissive pH (e.g. in acidic endosomes) and the cargo dye wasreleased. Results of in vitro study revealed a sustained and prolonged release profile.Taken together, our findings indicate the potential of AOBs as a delivery carrier.

Gene family of oleosin isoforms and their structural stabilization in sesame seed oil bodies.

Oleosins are structural proteins sheltering the oil bodies of plant seeds. Two isoform classes termed H- and L-oleosin are present in diverse angiosperms. Two H-oleosins and one L-oleosin were identified in sesame oil bodies from the protein sequences deduced from their corresponding cDNA clones. Sequence analysis showed that the main difference between the H- and L-isoforms is an insertion of 18 residues in the C-terminal domain of H-oleosins. H-oleosin, presumably derived from L-oleosin, was duplicated independently in several species. All known oleosins can be classified as one of these two isoforms. Single copy or a low copy number was detected by Southern hybridization for each of the three oleosin genes in the sesame genome. Northern hybridization showed that the three oleosin genes were transcribed in maturing seeds where oil bodies are being assembled. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and sesame oleosin isoforms. The results indicated that reconstituted oil bodies could be stabilized by both isoforms, but L-oleosin gave slightly more structural stability than H-oleosin.

Cloning and secondary structure analysis of caleosin, a unique calcium-binding protein in oil bodies of plant seeds.

Plant seed oil bodies comprise a matrix of triacylglycerols surrounded by a monolayer of phospholipids embedded with abundant oleosins and some minor proteins. Three minor proteins, temporarily termed Sops 1-3, have been identified in sesame oil bodies. A cDNA sequence of Sop1 was obtained by PCR cloning using degenerate primers derived from two partial amino acid sequences, and subsequently confirmed via immunological recognition of its over-expressed protein in Escherichia coli. Alignment with four published homologous sequences suggests Sop1 as a putative calcium-binding protein. Immunological cross-recognition implies that this protein, tentatively named caleosin, exists in diverse seed oil bodies. Caleosin migrated faster in SDS-PAGE when incubated with Ca2+. A single copy of caleosin gene was found in sesame genome based on Southern hybridization. Northern hybridization revealed that both caleosin and oleosin genes were concurrently transcribed in maturing seeds where oil bodies are actively assembled. Hydropathy plot and secondary structure analysis suggest that caleosin comprises three structural domains, i.e., an N-terminal hydrophilic calcium-binding domain, a central hydrophobic anchoring domain, and a C-terminal hydrophilic phosphorylation domain. Compared with oleosin, a conserved proline knot-like motif is located in the central hydrophobic domain of caleosin and assumed to involve in protein assembly onto oil bodies.

Genomic cloning of 18 kDa oleosin and detection of triacylglycerols and oleosin isoforms in maturing rice and postgerminative seedlings.

Oleosins are hydrophobic proteins localized abundantly in the oil bodies of plant seeds. Two distinct oleosin isoforms of molecular masses 18 and 16 kDa are present in rice oil bodies. These isoforms were found in similar ratio in rice embryos and aleurone layers. To survey potential DNA sequences involved in the activation of oleosin genes, a genomic clone of rice 18 kDa oleosin was sequenced, and its 5'-flanking region was compared with that of the known rice 16 kDa oleosin gene. Corresponding mRNAs of the two rice oleosin isoforms appeared seven days after pollination and vanished in mature seeds. Triacylglycerols and oleosins were accumulated concomitantly in maturing rice reeds in accord with the active assembly of oil bodies, and partly mobilized in postgerminative seedlings. Approximately 60% of the stored triacylglycerols in rice were not utilized: while the majority of oil bodies in embryos were mobilized in five days after imbibition, those in aleurone layers remained intact in postgerminative seedlings.

Coexistence of both oleosin isoforms on the surface of seed oil bodies and their individual stabilization to the organelles.

The oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Two distinct oleosin isoforms with molecular masses of 18 and 16 kDa are present in rice oil bodies. Chicken antibodies raised against oleosin 18 kDa and rabbit antibodies raised against oleosin 16 kDa did not cross-recognize these two homologous isoforms. This peculiar non-cross recognition was used to locate the two oleosin isoforms on the surface of oil bodies via immunofluorescence detection using anti-chicken IgG conjugated with FITC (fluorescein isothiocyanate) and anti-rabbit IgG conjugated with Texas-Red. The results revealed that both oleosin isoforms resided on each oil body in vivo and in vitro. Artificial oil bodies were reconstituted via sonication using triacylglycerol, phospholipid, and oleosins. The results indicated that the two rice oleosin isoforms could stabilize artificial oil bodies individually whereas oleosin 16 kDa provided better stability to the organelles than oleosin 18 kDa.

Cloning, expression and isoform classification of a minor oleosin in sesame oil bodies.

The oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Two distinct oleosins are present in the oil bodies of diverse angiosperms, and classified as high and low Mr isoforms according to their relative molecular masses in each species. In sesame oil bodies, besides the two ubiquitous oleosin isoforms (17 and 15 kDa), an additional minor oleosin (15.5 kDa) was revealed on Tricine SDS-PAGE. A full-length cDNA fragment was cloned, sequenced and deduced to be a putative oleosin of 15,446 Da. The gene was constructed in a fusion or non-fusion vector and then over-expressed with different efficiency in Escherichia coli. All three oleosins purified from sesame oil bodies were subjected to immunoassaying using antibodies raised against the over-expressed oleosin. The results confirmed that this gene encodes the sesame 15.5 kDa oleosin. Sequence comparisons with other known oleosins revealed that sesame 15.5 kDa oleosin does not represent a new oleosin isoform class but may have been derived through gene duplication and truncation of sesame 17 kDa oleosin, and possesses the minimal structure of the high Mr oleosin isoform. A conserved amphipathic alpha-helix is predicted in sesame 15.5 kDa oleosin, which may imply a potential biological function associated with this isoform.

Oleosin of Plant Seed Oil Bodies Is Correctly Targeted to the Lipid Bodies in Transformed Yeast

Yeast (Saccharomyces cerevisiae) has been used extensively as a heterologous eukaryotic system to study the intracellular targeting of proteins to different organelles. The lipid bodies in yeast have not been previously subjected to such studies. These organelles are functionally equivalent to the subcellular storage oil bodies in plant seeds. A plant oil body has a matrix of oils (triacylglycerols) surrounded by a layer of phospholipids embedded with abundant structural proteins called oleosins. We tested whether plant oleosin could be correctly targeted to the lipid bodies in transformed yeast. The coding region of a maize (Zea mays L.) oleosin gene was incorporated into yeast high copy and low copy number plasmids in which its expression was under the control of GAL1 promoter. Yeast strains transformed with these plasmids produced oleosin when grown in a medium containing galactose but not glucose. The oleosin produced in yeast had a molecular mass slightly higher than that of the native protein in maize. Oleosin accumulated concomitantly with the storage lipids during growth of the transformed yeast, and it was not secreted. Subcellular fractionation of the cell extracts obtained by two different cell breakage procedures revealed that the oleosin was largely restricted to the lipid bodies. Oleosin apparently did not affect the lipid contents and composition of the transformed yeast lipid bodies but replaced some of the native proteins associated with the organelles. Immunocytochemistry of the transformed yeast cells showed that the oleosin was present mostly on the periphery of the lipid bodies. Oleosin isolated from maize or transformed yeast strain, alone or in the presence of phospholipids or SDS, did not bind to the yeast lipid bodies in vitro. We conclude that plant oleosin is correctly targeted to the lipid bodies in transformed yeast and that yeast may be used as a heterologous system to dissect the intracellular targeting signals in the oleosin.

Plant seed oil-bodies as carriers for foreign proteins.

Plant seeds frequently store oils (triglycerides) in discrete organelles called oil-bodies. These are normally surrounded by a phospholipid half-unit membrane equipped with specialized proteins called oleosins. Oleosins are highly lipophilic proteins, are expressed at high levels in many seeds and are specifically targeted to oil-bodies. We have investigated the potential of oleosins to act as carriers for recombinant proteins by the production of translational fusions between oleosins and genes encoding proteins foreign to plant cells. We have shown that a fusion comprising a complete oleosin coding domain and a beta-glucuronidase coding sequence may be expressed specifically in the seeds of the oilseed crop plant, Brassica napus, and its product is correctly targeted with approximately 80% of the activity partitioning with oil-bodies. Recombinant oil-bodies may be used to facilitate separation of a recombinant protein from other cellular proteins. Using this approach, the desired protein may be cleaved from the oil-bodies using an endoprotease and further purified. Alternatively, a fusion protein which is enzymatically active and resides on the oil-bodies may be used directly in heterogeneous catalysis. In this application, after a round of catalysis the oil-bodies may be recovered and re-used several times without loss of activity. Thus the oil-bodies act as an immobilization matrix. The fusion protein is stable in dry seeds for long periods and when extracted has a half-life of 3-4 weeks on oil-bodies. Finally, the production of these recombinant oil-bodies is extremely inexpensive, offering a novel route to the manufacture of recombinant proteins.

Structure of plant seed oil bodies

Plant seeds store triacylglycerols in intracellular oil bodies as food reserves for germination. Each spherical oil body has a diameter of about 0.5–2.0 μm, and consists of a triacylglycerol matrix surrounded by a layer of phospholipids embedded with abundant and unique proteins called oleosins. Oleosins have special secondary structures which interact with the surface phospholipid layer and the matrix triacylglycerols. They stabilize the oil bodies and possibly serve as recognition signals for lipase binding during seed germination.

Expression and characterization of the N-terminal domain of an oleosin protein from sunflower

Oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Although oleosins are amphipathic proteins, they are unlike bilayer membrane proteins since they are associated with a single lipid:water interface at the oil body surface. Oleosins are unusual proteins because they contain a 70-80-residue uninterrupted nonpolar domain, flanked by relative polar C- and N-terminal domains. In the present study, we report the expression of the N-terminal domain of the 18-kDa oleosin isoform from sunflower as a recombinant fusion protein in Escherichia coli and the determination of its secondary structure using CD and Fourier transform infrared spectroscopy either as a purified but partially denatured peptide or reconstituted into liposomes. The structure derived from physical studies was then compared and assigned with those predicted from analysis of the primary sequence of the N-terminal domain. Based on data derived from CD spectroscopy analysis of purified and partially renatured N-terminal polypeptide, it contains about 10% alpha-helical structure, 20-30% beta-strand structure, approximately 8% beta-turn structure, and 60% random coil structure. However, analysis of the polypeptide reconstituted into liposomes showed an increased content of alpha-helical structure to about 20% and an increased beta-strand structure content to about 30-40%. Data derived from Fourier transform infrared spectroscopy studies and compared with the data predicted from the primary sequence showed the peptide is well structured with some antiparallel beta-strand structure from residues 2-9, parallel beta-strand structure from residues 30-37 and/or 42-49, and alpha-helical structure from residues 10-23 and/or 43-49. There is potential amphipathic alpha-helix from residues 10-23. Based on these results, the following model for the secondary structure of the N-terminal domain of sunflower oleosin can be proposed. Residues 2-9 would produce amphipathic antiparallel beta-strand structure. Residues 10-23 would produce an amphipathic alpha-helical structure. Residues 30-37 and/or 42-49 would give parallel beta-strand structure, or residues 42-49 could form a nonpolar alpha-helical structure that would insert into the oil matrix.

Characterization of the charged components and their topology on the surface of plant seed oil bodies.

Oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins called oleosins. Oil bodies isolated from maize (Zea mays L.) in a medium of pH 7.2 maintained their entities but aggregated when the pH was lowered to 6.8 and 6.2. Aggregation did not lead to coalescence and was reversible with an elevation of the pH. Further decrease of the pH from 6.2 to 5.0 retarded the aggregation. Aggregation at pH 7.2 was induced with 2 mM CaCl2 or MgCl2 but not with NaCl. Aggregation at pH 6.8 was prevented by 10 microM sodium dodecyl sulfate but not with NaCl. We conclude that oil bodies have a negatively charged surface at pH 7.2 and an isoelectric point of about 6.0. This conclusion is supported by isoelectrofocusing results and by theoretical calculation of the positive charges in the oleosins and the negative charges in phosphatidylserine, phosphatidylinositol, and free fatty acids. Apparently, lowering of the pH from 7.2 to 6.2 protonates the histidine residues in the oleosins, and neutralizes the oil bodies. Further decrease of the pH to 5.0 likely protonates the free fatty acids and produces positively charged organelles. Similar charge properties were observed in the oil bodies isolated from rape, flax, and sesame seeds. An analysis of the oleosin secondary structures reveals an N-terminal amphipathic domain, a central hydrophobic anti-parallel beta-strand domain (not found in any other known protein), and a C-terminal amphipathic alpha-helical domain. In the two amphipathic domains, the positively charged residues are orientated toward the interior facing the negative charged lipids, whereas the negatively charged residues are exposed to the exterior. The negatively charged surface is a major factor in maintaining the oil bodies as stable individual entities.

Surface structure and properties of plant seed oil bodies.

Storage triacylglycerols (TAG) in plant seeds are present in small discrete intracellular organelles called oil bodies. An oil body has a matrix of TAG, which is surrounded by phospholipids (PL) and alkaline proteins, termed oleosins. Oil bodies isolated from mature maize (Zea mays) embryos maintained their discreteness, but coalesced after treatment with trypsin but not with phospholipase A2 or C. Phospholipase A2 or C exerted its activity on oil bodies only after the exposed portion of oleosins had been removed by trypsin. Attempts were made to reconstitute oil bodies from their constituents. TAG, either extracted from oil bodies or of a 1:2 molar mixture of triolein and trilinolein, in a dilute buffer were sonicated to produce droplets of sizes similar to those of oil bodies; these droplets were unstable and coalesced rapidly. Addition of oil body PL or dioleoyl phosphatidylcholine, with or without charged stearylamine/stearic acid, or oleosins, to the medium before sonication provided limited stabilization effects to the TAG droplets. High stability was achieved only when the TAG were sonicated with both oil body PL (or dioleoyl phosphatidylcholine) and oleosins of proportions similar to or higher than those in the native oil bodies. These stabilized droplets were similar to the isolated oil bodies in chemical properties, and can be considered as reconstituted oil bodies. Reconstituted oil bodies were also produced from TAG of a 1:2 molar mixture of triolein and trilinolein, dioleoyl phosphatidylcholine, and oleosins from rice (Oryza sativa), wheat (Triticum aestivum), rapeseed (Brassica napus), soybean (Glycine max), or jojoba (Simmondsia chinensis). It is concluded that both oleosins and PL are required to stabilize the oil bodies and that oleosins prevent oil bodies from coalescing by providing steric hindrance. A structural model of an oil body is presented. The current findings on seed oil bodies could be extended to the intracellular storage lipid particles present in diverse organisms.

Maize oleosin is correctly targeted to seed oil bodies in Brassica napus transformed with the maize oleosin gene.

Oleosins are small hydrophobic abundant proteins localized in the oil bodies of plant seeds. An oleosin gene from the monocotyledonous maize (Zea mays L.) was transferred into the dicotyledonous Brassica napus L. using Agrobacterium-mediated transformation. The maize oleosin gene was placed under the control of either its own promoter/terminator or the promoter/terminator of a Brassica seed storage protein (napin) gene. Southern blot analyses of individual transformed plants suggested that the oleosin gene from either construct was incorporated into the Brassica chromosomes without appreciable structural alterations. The amount of construct incorporated was from 1 to >10 copies per haploid genome, depending on the individual transformant. Maize oleosin mRNA and protein were detected only in the transformants containing the napin gene promoter/terminator constructs; these transformants were studied further. Northern blot analyses of RNA isolated from different tissues and seeds of different developmental stages indicated that the maize oleosin mRNA was present only in the maturing seed. Approximately 1% of the total protein in mature seed was represented by maize oleosin. Subcellular fractionation of the mature seed revealed that 90% or more of the maize oleosin, as well as the Brassica oleosin, was localized in the oil bodies. The results show that a monocotyledonous oleosin possesses sufficient targeting information for its proper intracellular transport in a dicotyledon and also suggest that the napin gene promoter/terminator of Brassica, or equivalent seed storage protein regulatory elements of other plant species, may be used to express genes for the genetic engineering of seed oils.