ISRN BotanyVolume 2012 (2012), Article ID 173954, 16 pagesdoi:10.5402/2012/173954

Review Article

Integral Proteins in Plant Oil Bodies

Jason T. C. Tzen

Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan

Received 11 September 2012; Accepted 3 October 2012

Academic Editors: G. T. Maatooq and Y. Yamauchi

Copyright © 2012 Jason T. C. Tzen. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Hydrophobic storage neutral lipids are stably preserved in specialized organelles termed oil bodies in the aqueous cytosolic compartment of plant cells via encapsulation with surfactant molecules including phospholipids and integral proteins. To date, three classes of integral proteins, termed oleosin, caleosin, and steroleosin, have been identified in oil bodies of angiosperm seeds. Proposed structures, targeting traffic routes, and biological functions of these three integral oil-body proteins were summarized and discussed. In the viewpoint of evolution, isoforms of oleosin and caleosin are found in oil bodies of pollens as well as those of more primitive species; moreover, caleosin- and steroleosin-like proteins are also present in other subcellular locations besides oil bodies. Technically, artificial oil bodies of structural stability similar to native ones were successfully constituted and seemed to serve as a useful tool for both basic research studies and biotechnological applications.

1. Introduction

The stored energy in plant tissues is occasionally preserved in the form of proteins, yet much more commonly in the form of carbohydrates or lipids. Plant cells deposit storage resources of carbohydrates, proteins, and neutral lipids in subcellular particles termed starch granules, protein bodies, and oil bodies, respectively. In contrast with the active studies of protein bodies and starch granules [1–6], research progress on oil bodies is relatively late and slow presumably due to less research input and inevitable technical problems caused by the hydrophobic features of these lipid-storage organelles.

Oil bodies are intracellular organelles for storing neutral lipids, mainly triacylglycerols and sterol esters, and they are also referred to as lipid bodies, lipid droplets, oil globules, oleosomes, and spherosomes. These organelles have been found across a wide range of plant cells, from microalgae to the most complex angiosperms; among them, oil bodies obtained from seed cells have been studied most intensively [7]. According to the cumulative research outcome in the past three decades, it is generally assumed that an oil body is composed of a neutral lipid matrix surrounded by a monolayer of phospholipids embedded with some unique integral proteins [8–10]. This paper focused on the integral proteins of oil bodies in terms of their proposed structures, organelle targeting, biological functions, homologous isoforms, and utilization of artificial oil bodies.

2. Identification of Integral Proteins in Oil Bodies of Angiosperm Seeds

Constituents of oil bodies in angiosperm species, particularly those in oily seeds, have been continually investigated in the past three decades [11–13]. Research approaches by using tools of molecular biology and protein chemistry were relatively active in this research area in the past two decades.

2.1. Structural Components of Seed Oil Bodies

Vegetable cooking oils commonly extracted from various oily seeds are triacylglycerol molecules that tend to segregate from aqueous solution and form a transparent layer on the top. These hydrophobic triacylglycerol molecules are originally assembled in specialized organelles termed oil bodies, and these lipid storage organelles are stably packed in aqueous environments, that is, the cytosolic compartment of seed cells, with sizes mostly ranging from 0.5 to 2 m (Figure 1(a)) [14, 15]. Intact oil bodies isolated from oily seeds, such as sesame, form a milky layer on top of the solution after centrifugation (Figure 1(b)), and they look drastically different from the transparent vegetable cooking oils that are generally extracted from seed oil bodies under relatively stringent conditions (high temperature or organic solvent). The isolated oil bodies remained maintaining their structural integrity and stability when they were suspended in an aqueous solution as observed under a light microscope (Figure 1(c)). Evidently, oil bodies are remarkably stable both in vivo and in vitro as compressed oil bodies in cells of a mature seed or in the milky layer during isolation never coalesce or aggregate. The remarkable stability of oil bodies in aqueous environments implies that surfactant molecules are present on the surface of oil bodies [16–19]. Chemical analyses suggested that phospholipids and proteins might be minor constituents (one to a few percent by weight) of oil bodies and served as surfactants to encapsulate abundant hydrophobic neutral lipids into many relatively small hydrophilic particles.

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Figure 1: Visualizations of oil bodies. (a) Electron microscopy of a seed cell of mature sesame (adopted and modified from Figure 5 of Peng and Tzen, [14]). The most abundant gray spherical particles are oil bodies. (b) A photo of isolated sesame oil bodies. Purified oil bodies floated on the top and formed a milky layer after centrifugation. (c) Light microscopy of isolated sesame oil bodies. Isolated oil bodies were kept in 0.1 M sodium phosphate buffer, pH 7.5 for 30 min before taking the photo.

2.2. Identification of a Major Integral Protein in Seed Oil Bodies

According to the chemical detection of phospholipids in oil bodies as well as the observation of one single boundary line on the surface of oil bodies under an electron microscope, it is generally accepted that the matrix neutral lipids of seed oil bodies are encapsulated by a monolayer of phospholipids [20–24]. In contrast, the presence of unique integral proteins, rather than nonspecifically associated contaminants of purification, in seed oil bodies was not confirmed until the striking discovery of oleosin, a major surfactant protein in seed oil bodies of maize (Zea mays L.) [25]. The amino acid sequences of two maize oleosins deduced from their cDNA clones show a conservative central hydrophobic domain of approximately 70 residues that is the longest hydrophobic segment found in natural proteins so far, and apparently responsible for the anchorage of the proteins on the surface of oil bodies [26]. Oleosin was named in 1990 taking its meaning of an oil (oleo-) protein (-sin). Right after the kick-off studies on maize oleosins, homologous oleosin isoforms were subsequently identified in oil bodies of rapeseed (Brassica napus), soybean (Glycine max L.), carrot (Daucus carota), sunflower (Helianthus annuus), Arabidopsis thaliana, and cotton (Gossypium hirsutum) with their corresponding cDNA fragments cloned [27–34]. It seems that oleosin isoforms are universally present in oil bodies of angiosperm seeds including both monocotyledonous and dicotyledonous species [35–39]. Furthermore, oleosin isoforms found as the major proteins (approximately 80–90%) in seed oil bodies were demonstrated to shield the whole surface of the organelles in the company of phospholipids [40]. The structural integrity and stability of seed oil bodies were assumed to be provided by abundant oleosins via two factors, steric hindrance and electronegative repulsion [41].

2.3. Identification of Two Minor Integral Proteins in Seed Oil Bodies

After the identification of the major protein, oleosin in seed oil bodies, whether minor integral proteins could also be found in these organelles became the next challenge. The approach to this challenge was severely impeded since many non-specifically associated proteins were contaminated in the preparation of oil bodies. The impediment was overcome by the development of a purification protocol that removed almost all the contaminated proteins on the surface of isolated oil bodies by washing harshly with detergent, high salt, chaotropic agent, and hexane [42]. As exemplified by seed oil bodies of sesame (Sesamum indicum L.), besides abundant oleosin isoforms, three minor protein bands of relatively high molecular masses were found after the harsh washing [43]. These three minor proteins were later confirmed as two classes of integral oil-body proteins named caleosin and steroleosin [44, 45]. Caleosin was named in 1999 taking its meaning of a calcium-binding (cal-) oil protein (-leosin) [46], and steroleosin was named in 2002 as a sterol-regulatory (sterol-) oil protein (-leosin) [47]. Taken together, a structural model of seed oil body was depicted in Figure 2.

Figure 2: A structural model of an oil body with three types of integral oil-body proteins. An oil body is proposed to contain a triacylglycerol (TAG) matrix surrounded by a monolayer of phospholipids (PL) embedded with three classes of proteins, oleosin, caleosin, and steroleosin.

2.4. Identification of Other Potential Oil-Body Proteins

Searching for more oil-body proteins has been approached by subproteomic analysis under the assistance of liquid chromatography electrospray ionization tandem mass spectrometry in the past few years [48–54]. In addition to the three known integral oil-body proteins, oleosin, caleosin, and steroleosin, several proteins were detected as potential oil-body proteins in seed oil bodies of maize and rapeseed. However, none of these newly identified proteins have been confirmed as integral oil-body proteins or peripheral proteins associated with some surface components of oil bodies for particular physiological functions. It remains to be clarified if proteins other than oleosin, caleosin, and steroleosin are embedded or peripherally associated on the surface of seed oil bodies.

3. Proposed Structures of Integral Oil-Body Proteins

Due to the insolubility of oleosin, caleosin, and steroleosin possibly caused by their hydrophobic oil-body anchoring domains, no three-dimensional structures derived from X-ray or NMR are available at the present time. Proposed structures of these three oil-body proteins are predicted based on their sequence analyses and spectrometric determination.

3.1. Proposed Oleosin Structure

An oleosin molecule is proposed to comprise three structural domains: an N-terminal amphipathic domain, a central hydrophobic oil-body anchoring domain, and a C-terminal amphipathic α-helical domain (Figure 3(a)) [55]. The N-terminus of oleosin is blocked with acetylation after the removal of the first methionine, a cotranslational modification presumably related to the enhancement of protein structural stability to fulfill the long-term storage of oil bodies within seed cells [56]. Both N- and C-terminal domains are not conserved among oleosins of diverse species, and even their lengths are quite variable in different oleosin isoforms. It is generally agreed that these two domains are putatively resided on the surface of oil bodies and stabilize the organelles via steric hindrance and electronegative repulsion [57–59]. In contrast, the central anchoring domain of oleosins is highly conserved among diverse species, particularly in a relatively hydrophilic proline knot motif at the middle of the sequence [60–63]. To date, controversial secondary structures of this domain have been proposed or determined in the past two decades, yet the contents of -helical and -stranded structures in these proposed models are extremely different [41, 64–72]. The controversy among these proposed structures does not seem to be receded unless a convincing three-dimensional structure of oleosin or at least its central hydrophobic domain is resolved.

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Figure 3: Secondary structure organizations of integral oil-body proteins on the surface of an oil body. Proposed secondary structures of oleosin (a), caleosin (b), and steroleosin (c) are adopted and modified from Figure 5 of Tzen et al., [55], Figure 7 of Chen et al., [46], and Figure 6 of Lin et al., [47], respectively.

3.2. Proposed Caleosin Structure

A caleosin molecule is also proposed to comprise three structural domains: an N-terminal hydrophilic calcium-binding domain, a central hydrophobic oil-body anchoring domain, and a C-terminal hydrophilic phosphorylation domain (Figure 3(b)) [46]. The N-terminus of caleosin is also blocked with acetylation after the removal of the first methionine, presumably modified by the same co-translational mechanism found in oleosin [56]. The N-terminal hydrophilic domain consists of an EF hand calcium-binding motif of 28 residues including an invariable glycine residue as a structural turning point and five conserved oxygen-containing residues as calcium-binding ligands [23, 46]. Owing to the presence of this calcium-binding domain, calcium ion affected electrophoretic mobility of native and recombinant caleosin on SDS-PAGE [46, 73, 74]. The calcium-binding capacity of caleosin is in agreement with the observation of calcium staining on the surface of oil bodies in electron microscopy prior to the discovery of caleosin in these lipid storage organelles [75]. The central hydrophobic domain of caleosin is relatively short in comparison with that of oleosin, and comprises an amphipathic -helix and an anchoring region. The amphipathic -helix is assumed to be located in the interface between hydrophobic and hydrophilic environments while the anchoring region is predicted to comprise a pair of anti-parallel β-strands connected with a proline knot motif. The similarity of proline knot motifs of oleosin and caleosin seems to imply a significant role associated with this unique motif, such as protein folding, intermolecular assembly, or specific targeting to oil bodies [76–78]. The C-terminal hydrophilic domain of caleosin contains several potential phosphorylation sites. The native caleosin in seed oil bodies of Arabidopsis thaliana, but not bacterially expressed caleosin, has been found partially phosphrylated [79]. An invariable cysteine residue is present near the C-terminus of caleosin and unlikely involved in any interdisulfide linkage with another caleosin molecule or other proteins on the surface of oil bodies.

3.3. Proposed Steroleosin Structure

A steroleosin molecule is proposed to comprise a relatively small N-terminal oil-body anchoring domain and a relatively large soluble sterol-binding dehydrogenase domain (Figure 3(c)) [47]. Free N-terminus occurs in steroleosin with the translation-initiating methionine as the first residue, in contrast with the acetylation-blocked N-termini of oleosin and caleosin [56]. So far, less research investigation has been executed on the structure of steroleosin in comparison with that of oleosin or caleosin. The N-terminal anchoring segment comprises two amphipathic α-helices (12 residues in each helix) connected by a hydrophobic sequence of 14 residues bordered by 1-2 proline residues at each end. The relatively hydrophilic proline residues located in both ends of the 14-residue hydrophobic sequence are proposed to aggregate in the hydrophobic surroundings and form a unique structure, termed proline knob motif, for the integrity and stability of steroleosin anchorage on the surface of oil bodies. The soluble sterol-binding dehydrogenase domain contains an NADPH-binding subdomain, an active site region, and a sterol-binding subdomain. Three-dimensional structure of the dehydrogenase domain has been simulated by homology modeling [47]. The modeling structure reveals that the NADPH-binding region, active site, and sterol-binding region are located in the C-terminal ends of a parallel β-sheet and that the NADPH-binding region is expectably located in the crevice region, termed topological switch point, as observed in all similar α/β structures [80].

4. Possible Biological Functions of Integral Oil-Body Proteins

Except for the structural role, it is reasonable that oil-body proteins may exert some biological functions related to the synthesis or degradation of oil bodies, for example, signaling for the formation, assembly, fusion, or mobilization of these lipid storage organelles. Indeed, some physiological functions have been proposed for oil-body proteins in the past decade [81]. Further verification of these proposed physiological functions is anticipated in the come-up research progress.

4.1. Proposed Functions of Oleosin

Oleosin has been proven to play a key role in the stability of seed oil bodies via electronegative repulsion and steric hindrance [41]. This structural role prevents coalescence of oil bodies during seed desiccation and maintains them as discrete and relatively small organelles. It is demonstrated that the structural role of oleosin also protects Arabidopsis thaliana seeds against freeze/thaw-induced damage of their cells in vivo [82]. The contents of oleosins are found to determine sizes of seed oil bodies; presumably the ratio of oleosin over triacylglycerol is inversely proportional to the sizes of oil bodies [83–88]. The stability of isolated oil bodies could be substantially enhanced after their surface proteins were cross-linked by linker molecules, such as glutaraldehyde or genipin [87]. Being structural proteins, oleosin isoforms as well as caleosin ones are partially degraded by a special thiol-protease, thioredoxin h, after germination, and this specific degradation of oil-body structural proteins is proposed to be associated with mobilization of oil bodies in seedlings [89–92].

Furthermore, oleosin is suggested to be a bifunctional enzyme that has both monoacylglycerol acyltransferase and phospholipase activities during seed germination [93]. The regulation of these distinct dual activities seems to be controlled by the phosphorylation of oleosin presumably by a serine/threonine/tyrosine protein kinase, and the oleosin phosphorylation is also found to be activated by phosphatidylcholine and diacylglycerol, but inhibited by lysophosphatidylcholine, oleic acid, and calcium ion [94]. It will be interesting to see if these two enzymatic activities are universally detectable in oleosin isoforms of diverse species since both N- and C-terminal domains are not conserved among these oleosin isoforms. Meanwhile, most oleosins are relative small proteins of 15–20 kDa, particularly their N- and C-terminal domains are quite tiny (3 to 5 kDa for each domain; Figure 3(a)). In terms of structure-function relationship, it is a challenging task to reveal how the small structural domain(s) of oleosins construct the three dimensional active sites for the two observed enzymatic activities.

4.2. Proposed Functions of Caleosin

Having a structural organization and oil-body anchorage similar to oleosin, caleosin has been demonstrated to stabilize seed oil bodies as efficiently as oleosin [76]. The structural role of caleosin is clearly verified by the observation of stable cycad (Cycas revoluta) seed oil bodies that are mainly sheltered by caleosin without the presence of any oleosin isoform [95]. The investigation also invalidates a prevalent concept in this research area for two decades, declaring that oleosin is an essential constituent and can be regarded as a marker protein of plant oil bodies.

Caleosin comprises a calcium-binding motif and several potential phosphorylation sites, that is, well-known candidates involved in signal transduction, and thus may possess biological function(s) in addition to its structural role for the stability of oil bodies. According to the characterization of two independent insertion mutants lacking caleosin, it was proposed that caleosin might play a role in the degradation of storage lipids in oil bodies by inducing the interaction of oil bodies with vacuoles during germination [96]. Putative interaction between oil bodies and vacuoles was also observed in pollen cells after germination under electron microscopy; and the pollen oil bodies were presumably surrounded by tubular membrane structures and encapsulated in the vacuoles after germination [97–99]. The detailed molecular interaction between caleosin on the surface of oil bodies and its specific partner protein on the membrane of vacuoles remains to be studied.

Caleosin isoforms or caleosin-like proteins are not only localized in oil bodies but also found as membrane-bound proteins in other subcellular fractions, such as microsomal membrane; moreover, they were demonstrated to possess different biological functions, such as peroxygenase activity in biotic and abiotic stress responses in their phosphorylated forms [100–102]. Site-directed mutagenesis studies revealed that the peroxygenase catalytic activity of caleosin, an original heme-oxygenase, was dependent on two highly conserved histidines [100]. It was proposed that caleosin-like proteins might be involved in the plant-pathogen recognition, symptom development, and the basal tolerance to biotic and abiotic stresses through the salicylic acid signaling pathway [102]. In Arabidopsis, a stress-responsive caleosin-like protein, AtCLO4, was demonstrated to act as a negative regulator of ABA responses [103], whereas another caleosin-like protein, RD20, was involved in ABA-mediated inhibition of germination but did not response to biotic or abiotic stresses [104]. Recently, a wheat caleosin-like protein was proposed to play a role in the -triggered feedback regulation of both the canonical Gα subunit of the heterotrimeric G protein complex and phosphoinositide-specific phospholipase C [105], and a noncanonical caleosin from Arabidopsis was found to epoxidize unsaturated fatty acids efficiently with complete stereoselectivity [106]. Taken together, the recent research progress on the identification of caleosin functions is encouraging. However, it also raises a puzzle how the highly conserved caleosin isoforms execute several diverse functions that may require a well-structured active site for an enzymatic reaction and a well-featured binding surface for a specific protein-protein interaction.

4.3. Proposed Functions of Steroleosin

Steroleosin possesses a sterol-regulatory dehydrogenase domain that belongs to a superfamily of presignal proteins involved in signal transduction via activation of its partner receptor after binding to a regulatory sterol [45]. Besides dehydrogenase activity, no other biological functions have been experimentally proven for steroleosin so far [44, 47]. As caleosin and steroleosin are minor integral proteins of comparable contents in oil bodies of sesame seeds, it is speculated that caleosin may be regulated by a pre-signal partner, such as a sterol-activated steroleosin, to serve as a receptor or signaling molecule on the surface of oil bodies. Meanwhile, two steroleosin isoforms having distinct sterol-binding sites are found in oil bodies of angiosperm seeds, and may be involved in the activation of sterol signal transduction that regulates specialized biological functions related to the mobilization of oil bodies during seed germination.

5. Targeting of Integral Oil-Body Proteins

Targeting of oil-body proteins, particularly oleosin, has been extensively investigated via in vivo systems using transgenic techniques and in vitro systems using microsomal membranes for integration of translated proteins [107–111]. The signal segment for specific targeting of oleosin to oil bodies is apparently located within the protein itself since recombinant oleosins are able to target correctly oil bodies in different transgenic plants and yeasts [112–114]. The central hydrophobic domain, particularly the conservative proline knot motif, of oleosin or caleosin has been demonstrated to play an essential role for the protein targeting oil bodies [60–63, 78]. According to the studies with an in vitro system using microsomal membranes as targets for integration of translated proteins, it has been suggested that oleosin might target endoplasmic reticulum (ER) under the assistance of the signal recognition particle (SRP) prior to transportation to maturing oil bodies [115–119]. However, neither oleosin nor caleosin possesses a cleavable or noncleavable N-terminal signal sequence required for the SRP-dependent pathway of targeting to the ER [120, 121]. Thus, it is proposed that oleosin contains several segments that are capable of interacting with SRP to direct the protein to the ER membrane [122].

An in vitro system was established to evaluate the targeting traffic routes of oleosin, caleosin, and steroleosin by constituting artificial oil emulsions (generated by sonication of triacylglycerol and phospholipid in a buffer solution) to mimic maturing oil bodies for integration of translated oil-body proteins [9, 78]. The results suggest that steroleosin and caleosin/oleosin may be assembled to maturing oil bodies through different locations of ER membrane, that is, caleosin/oleosin directly target maturing oil bodies where ER membranes are enlarged with deposited triacylglycerol molecules, whereas steroleosin is recognized by SRP and guided to integrate into the phospholipid bilayer of ER membrane prior to its lateral migration to maturing oil bodies (Figure 4). The distinct targeting traffic routes between steroleosin and caleosin/oleosin are in agreement with the following two observations. Firstly, steroleosin and caleosin/oleosin target and anchor to oil bodies via different structural organizations. Steroleosin possesses a non-cleavable N-terminal signal sequence putatively responsible for ER targeting via SRP dependent pathway, and its anchoring to oil bodies lies mainly in the N-terminal hydrophobic domain; caleosin and oleosin, lacking an N-terminal signal sequence, target/anchor to oil bodies via their central hydrophobic domains. Secondly, steroleosin possesses a free methionine at its N-terminus while caleosin and oleosin are N-terminally blocked by acetylation after the removal of the first methionine residue [56]. Presumably, the N-terminus of steroleosin is protected by SRP complex and/or embedded in ER membrane during its synthesis and targeting while the N-termini of caleosin and oleosin are freely exposed to cytosol during their synthesis and targeting to maturing oil bodies via central hydrophobic domains.

Figure 4: Targeting model of oleosin, caleosin, and steroleosin to a maturing oil body. Oleosin and caleosin are proposed to target directly a maturing oil body. In contrast, steroleosin is assumed to be integrated into the phospholipid bilayer of endoplasmic reticulum (ER) membrane prior to its lateral migration to a maturing oil body (adopted and modified from Figure 7 of Chen and Tzen, [78]).

Negatively charged phospholipids (phosphatidylserine and phosphatidylinositol) are present in a consistent amount (30–40%) in the phospholipids of oil bodies from diverse seeds [15]. According to an in vitro targeting study, inclusion of negatively charged phospholipids in artificial oil emulsions substantially enhanced the targeting efficiency of oleosin and caleosin to these emulsions [78]. It is assumed that negatively charged phospholipids in the surface area of oil bodies are involved in targeting or assembling of oil-body proteins to these organelles. Definitely, it is an important task to figure out the specific targeting interaction between the unique segments of oil-body proteins and the negatively charged phospholipids of maturing oil bodies.

6. Isoforms of Integral Oil-Body Proteins in Evolution

6.1. Oleosin Isoforms

Oleosin is an alkaline protein unique to oil bodies and has been found exclusively in plant species. Two distinct classes, H- and L- (high and low molecular weight) oleosins, are present in seed oil bodies of diverse angiosperms, and one or more isoforms may occur in each oleosin class of the same species [123]. It has been shown that H- and L-oleosins coexist on the surface of each oil body in seed cells of sesame [124]. The main difference between these two oleosin isoforms is an insertion of 18 residues in the C-terminal domain of H-oleosin, accounting for a 2 kDa difference in mass between the two classes found in many species [125–127]. The physiological significance of the presence of these two oleosin isoforms in oil bodies of angiosperm seeds remains to be elucidated. L-oleosin but not H-oleosin is found in megagametophytes of two gymnosperm species, pine (Pinus koraiensis) and ginkgo (Ginkgo biloba); it may imply that L-oleosin is a more primitive isoform class, with H-oleosin derived from L-oleosin before the divergence of monocot and dicot species during evolution [128]. Moreover, cDNA fragments encoding putative oleosin isoforms were found in pollen of rapeseed [129, 130]. Recently, stable oil bodies were successfully isolated from lily (Lilium longiflorum Thunb.) pollen, and a unique P-oleosin was found as the major integral oil-body protein [97]. Three oleosin genes, representative of early trends in evolution, were found in the model moss, Physcomitrella with a complex pattern of expression based on gene splicing [131]. Moreover, oleosin-like proteins, forming a distinct class termed oleopollenin, were found in tapetum and external surfaces of pollen grains [132–134].

6.2. Caleosin Isoforms

In angiosperm species, sequence alignment shows that caleosins in monocot seed oil bodies seem to possess an additional N-terminal appendix of approximately 40–70 residues, and thus are larger than those in dicotyledonous seed oil bodies [135–137]. Recently, a distinct P-caleosin isoform was also identified in pollen oil bodies of lily and olive (Olea europaea L.) [97, 98, 138]. Caleosin is also found in more primitive species, such as cycad and microalgae, and thus is assumed to be an oil-body protein more primitive than oleosin in evolution [95, 139]. Phylogenetic tree analysis supports that microalgal caleosin is the most primitive caleosin found in oil bodies to date [8, 23, 139]. The additional N-terminal appendix found in monocot caleosins is not present in pollen or cycad caleosin. Therefore, the additional N-terminal appendix found in monocot caleosins seems to be resulted from an insertion mutation in monocot seed caleosin in evolution. Of course, this hypothetic evolutionary event should be verified by further molecular evidence. Since caleosin is more primitive than oleosin, it is reasonable to speculate that caleosin is an essential integral protein of plant oil bodies [76]. However, this speculation has been invalidated as stable oil bodies located in rice aleurone layer are composed of H- and L-oleosin but not caleosin [135]. Interestingly, those rice oil bodies lacking caleosin are not mobilized after germination [39]. It remains to be studied whether caleosin is indispensable for the mobilization of oil bodies. In contrast with oleosin isoforms that are unique to oil bodies, caleosin isoforms or caleosin-like proteins are possibly present in other cellular locations, for example, ER membrane [140]. Moreover, the same caleosin isoform is possibly present in both seed oil bodies and membrane-bound fractions of other tissues [99].

6.3. Steroleosin Isoforms

Limited research progress has been advanced in the identification of steroleosin isoforms so far. Similar to caleosin, steroleosin isoforms or steroleosin-like proteins are possibly present not only in oil bodies but also in other subcellular locations [44]. Homologous proteins of steroleosin are presumably present in all kinds of living organisms including bacteria and humans [47]. Most steroleosin-like proteins are lack of the N-terminal hydrophobic anchoring domain, and they all possess the highly conservative NADPH-binding subdomain and active site, but diverse sterol-binding subdomains. Diverse sterol-binding subdomains are also found in the two steroleosin isoforms located in sesame oil bodies, implying that different sterols may regulate these two steroleosin isoforms to conduct distinct biological functions related to the formation or degradation of seed oil bodies [9].

7. Artificial Oil Bodies in Basic Research Studies

Artificial oil bodies of similar sizes (0.5–2 μm) and structural stability have been successfully reconstituted with triacylglycerols, phospholipids, and integral oil-body proteins under the same proportions as they are found in native oil bodies [125]. The sizes of artificial oil bodies could be controlled by changing the ratio of triacylglycerol over oil-body protein, whereas both thermostability and structural stability of artificial oil bodies decreased as their size increased, and vice versa [87]. For encapsulation of artificial oil bodies, recombinant oleosins expressed in Escherichia coli were found comparable to native oleosins isolated from seed oil bodies [141, 142]. It has been demonstrated that artificial oil bodies could be stabilized by oleosin or caleosin, but not steroleosin, and the average sizes (50–200 nm) of artificial oil bodies constituted with caleosin were 10-times smaller than those (0.5–2 m) constituted with oleosin (Figure 5) [143].

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Figure 5: Constitution of artificial oil bodies. A cartoon diagram showed the preparation of artificial oil bodies by sonication (a). Artificial oil bodies (AOB) were generated with triacylglycerol (TAG) and phospholipid (PL) in the presence of oil-body protein, oleosin (b), or caleosin (c). Photos of (b) and (c) are adopted from Figure 3 of Chen et al., [143].

Since stable artificial oil bodies could be simply generated with triacylglycerol, phospholipid, and oil-body protein, but not any two of them, it is apparent that these three constituents are essential components for the construction of oil bodies [41]. Artificial oil bodies could be stabilized by the combination of sesame oleosin isoforms or any oleosin isoform alone, that is, H1-oleosin, H2-oleosin, or L-oleosin; however, a slightly better structural stability was observed in artificial oil bodies constituted with L-oleosin than those constituted with either of the two H-oleosin isoforms [125]. Similar results were observed for the artificial oil bodies constituted with L-oleosin or H-oleosin extracted from oil bodies of rice seeds [124]. Obviously, L-oleosin is a better oil-body structural protein than H-oleosin. The relative small artificial oil bodies constituted with caleosin possessed a better thermostability (up to 70°C) than native oil bodies or artificial oil bodies stabilized with oleosin (lower than 50°C) [143]. The observation was in accordance with a later investigation on the relatively high thermostability (up to 70°C) of small cycad oil bodies that were mainly sheltered by a unique caleosin [95]. Evidently, caleosin is a better oil-body structural protein than oleosin in terms of thermal tolerance.

Artificial oil bodies constituted with truncated oleosin and caleosin have been utilized to evaluate the segments responsible for the structural stability of oil bodies. Artificial oil bodies constituted with truncated oleosins of the central hydrophobic domain longer than 36 residues were as stable as native sesame oil bodies, and those constituted with truncated oleosins lacking more than half of the original central hydrophobic domain inclined to coalesce upon collision or aggregation [60]. Both structural stability and thermostability of artificial oil bodies were slightly or severely reduced when the amphiphatic α-helix (15 residues) or proline-knot subdomain (21 residues) of recombinant caleosin was truncated, and thus the whole central hydrophobic domain of 36 () residues is crucial for the stability of oil bodies [77]. Taken together, the minimal length of hydrophobic domain to serve as an oil-body anchoring segment is approximately 36 residues (mainly the proline-knot regions shown in the secondary structures of oleosin and caleosin in Figure 3).

8. Artificial Oil Bodies in Biotechnological Applications

Several biotechnological applications have been developed by using the unique characteristics of oil bodies, such as new ingredients for flavoring or emulsifying agents, affinity matrices for enzyme fixation/purification, and expression/purification systems for producing recombinant proteins via transgenic plants [144–154]. Many applications related to the utilization of oil bodies have been patented [155–163]. These applications of seed oil bodies can be authentically applied to the utilization of artificial oil bodies. Moreover, some novel usages of artificial particles have also been developed in the past decade.

8.1. Protein Expression/Purification System

A bacterial expression/purification system to produce recombinant proteins was developed by using artificial oil bodies [141, 164–169]. In this system, a target protein was first overexpressed as an insoluble oleosin-fused polypeptide, collected from the pellet of cell lysate simply by centrifugation, assembled into artificial oil bodies, separated from oleosin, and then harvested by concentrating the ultimate supernatant. This technique offers a powerful and competitive option to replace the conventional affinity chromatography used for protein purification. However, the requirement of using a relatively expensive endopeptidase, for example, factor Xa, for specific release of the target protein from the recombinant oleosin-fused polypeptide raises the processing cost substantially, and thus severely restricts its potential applications. To cost down this process, an improved system was developed by replacing the specific proteolytic cleavage sequence between oleosin and the target protein with an intein (an inducible self-splicing polypeptide) linker. In this revised system, the target protein was released from artificial oil bodies via self-splicing of the intein linker, induced by temperature alteration or dithiothreitol supplement, without using the expensive endopeptidase.

8.2. Matrix for Enzyme Immobilization

A new technique of enzyme fixation was designed to achieve, in one step, protein refolding and immobilization by linking a target enzyme, for example, D-hydantoinase, to oleosin on the surface of artificial oil bodies [170, 171]. The immobilized enzyme remained stable for at least 15 days when stored at 4°C, and its conversion yield exceeded 80% after 7 cycles of repeated use. Apparently, the simple and effective system by fixing target enzymes on the surface of artificial oil bodies is practical and useful for the routine operation of industrial enzymatic reactions.

8.3. Formula for Encapsulation of Bacteria

Numerous healthy and nutritional benefits have been ascribed to probiotics, such as lactic acid bacteria. Since probiotics may not survive in sufficient number to retain their functionality in human gastrointestinal tract, many approaches have been explored to increase their viability when used as food supplements. A technique was developed to protect lactic acid bacteria against simulated gastrointestinal conditions by encapsulation of bacterial cells within artificial oil bodies [172]. Compared with nonencapsulated cells, the entrapped bacteria demonstrated a significant increase (approximately 10,000 times) in survival rate in the presence of simulated high acid gastric or bile salt conditions. It is recommended that artificial oil bodies may represent a suitable formula of biocapsule to encapsulate bacteria for commercial utilization in dairy products.

8.4. Carrier for Drug Delivery

Relatively small artificial oil bodies stabilized with caleosin have been used to develop an oral delivery system for hydrophobic drugs, for example, cyclosporine A, a drug commonly utilized as a clinical immunosuppressant to prevent transplant rejection and to treat several autoimmune diseases [173]. Cyclosporine A efficiently encapsulated in artificial oil bodies stabilized with caleosin could be stably stored for weeks at 4°C. An oral delivery formulation with cyclosporine A in artificial oil bodies was demonstrated to exhibit satisfactory bioavailability in an animal test [173]. This drug delivery system or its improved formula may also be used as an adequate carrier for many other hydrophobic drugs, such as antitumor drugs [174–177].

8.5. Antibody Generation System

Recently, a system of generating antibodies against small molecules (haptens) was established under the assistance of artificial oil bodies [178]. To develop this system, a series of recombinant caleosins were engineered with more Lys residues to link and render small molecules on the surface of artificial oil bodies for antibody production. In this design, covalently conjugated haptens were anticipated to cover the whole surface of artificial oil bodies constituted with hapten-charged caleosins. The results indicate that engineered Lys-rich caleosins are suitable carrier proteins for the production of monospecific antibodies against small molecules, such as drug, herbal compounds, pesticides, herbicides, antibiotics, and hormones.

9. Perspective

In the past three decades, continual research advancement has confirmed the presence of three classes of integral proteins, oleosin caleosin, and steroleosin on the surface of oil bodies. A lot of potential oil-body proteins have been recently screened by the subproteomic approaches under the assistance of mass spectrometry; though a few of them seem to be contaminants apparently, some candidate proteins are waiting for further verification to see if they are real integral oil-body proteins or peripheral proteins associated with some surface components of oil bodies for particular physiological functions. Controversial structures have been proposed for oleosin, and the controversy cannot be receded until a convincing three-dimensional structure of oleosin is determined. Several physiological functions other than structural role have been actively demonstrated or proposed for oleosin and caleosin in the past decade. Taken together, it seems unlikely that these two relatively small proteins are capable of executing several diverse biological functions jointly, and thus some of the proposed functions may not be correct and should be ruled out in the follow-up researches. Oleosin- and caleosin-stabilized artificial oil bodies have been successfully constituted and used to develop various systems for biotechnological applications. Further investigation and technical improvement will create novel artificial oil bodies as versatile vehicles to fulfill many other requirements for specialized applications.

Acknowledgments

The work was supported by Grants from the National Science Council, Taiwan, ROC (NSC 100-2313-B-005-012-MY3 and NSC 100-2313-B-005-015-MY3 to JTC Tzen). The author cordially expresses appreciation to all the colleagues, particularly graduate students, contributing to this research area synergistically in the past two decades.

References

V. Ibl and E. Stoger, “The formation, function and fate of protein storage compartments in seeds,” Protoplasma, vol. 249, no. 2, pp. 379–392, 2012.

M. R. Tandang-Silvas, E. M. Tecson-Mendoza, B. Mikami, S. Utsumi, and N. Maruyama, “Molecular design of seed storage proteins for enhanced food physicochemical properties,” Annual Review of Food Science and Technology, vol. 2, pp. 59–73, 2011.

T. Kawakatsu and F. Takaiwa, “Cereal seed storage protein synthesis: fundamental processes for recombinant protein production in cereal grains,” Plant Biotechnology Journal, vol. 8, no. 9, pp. 939–953, 2010.

S. Pérez and E. Bertoft, “The molecular structures of starch components and their contribution to the architecture of starch granules: a comprehensive review,” Starch/Staerke, vol. 62, no. 8, pp. 389–420, 2010.

S. G. Ball and M. K. Morell, “From bacterial glycogen to starch: understanding the biogenesis of the plant starch granule,” Annual Review of Plant Biology, vol. 54, pp. 207–233, 2003.

A. M. Smith, “The biosynthesis of starch granules,” Biomacromolecules, vol. 2, no. 2, pp. 335–341, 2001.

K. D. Chapman, J. M. Dyer, and R. T. Mullen, “Biogenesis and functions of lipid droplets in plants: Thematic Review Series: Lipid Droplet Synthesis and Metabolism: from Yeast to Man,” Journal of Lipid Research, vol. 53, no. 2, pp. 215–226, 2012.

D. J. Murphy, “The dynamic roles of intracellular lipid droplets: from archaea to mammals,” Protoplasma, vol. 249, no. 3, pp. 541–585, 2011.

J. T. C. Tzen, “Seed oil bodies of sesame and their surface proteins, oleosin, caleosin, and steroleosin,” in Sesame, the Genus Sesamum, D. Bedigian, Ed., vol. 48, chapter 10, pp. 187–200, CRC Press, London, UK, 1st edition, 2011.

A. H. C. Huang, “Oleosins and oil bodies in seeds and other organs,” Plant Physiology, vol. 110, no. 4, pp. 1055–1061, 1996.

D. L. Brasaemle and N. E. Wolins, “Packaging of fat: an evolving model of lipid droplet assembly and expansion,” Journal of Biological Chemistry, vol. 287, no. 4, pp. 2273–2279, 2012.

D. Zweytick, K. Athenstaedt, and G. Daum, “Intracellular lipid particles of eukaryotic cells,” Biochimica et Biophysica Acta, vol. 1469, no. 2, pp. 101–120, 2000.

J. A. Napier, A. K. Stobart, and P. R. Shewry, “The structure and biogenesis of plant oil bodies: the role of the ER membrane and the oleosin class of proteins,” Plant Molecular Biology, vol. 31, no. 5, pp. 945–956, 1996.

C. C. Peng and J. T. C. Tzen, “Analysis of the three essential constituents of oil bodies in developing sesame seeds,” Plant and Cell Physiology, vol. 39, no. 1, pp. 35–42, 1998.

J. T. C. Tzen, Y. Z. Cao, P. Laurent, C. Ratnayake, and A. H. C. Huang, “Lipids, proteins, and structure of seed oil bodies from diverse species,” Plant Physiology, vol. 101, no. 1, pp. 267–276, 1993.

C. R. Slack, W. S. Bertaud, B. D. Shaw, R. Holland, J. Browse, and H. Wright, “Some studies on the composition and surface properties of oil bodies from the seed cotyledons of safflower (Carthamus tinctorius) and linseed (Linum ustatissimum),” Biochemical Journal, vol. 190, no. 3, pp. 551–561, 1980.

R. Qu, S. M. Wang, Y. H. Lin, V. B. Vance, and A. H. Huang, “Characteristics and biosynthesis of membrane proteins of lipid bodies in the scutella of maize (Zea mays L.),” The Biochemical Journal, vol. 235, no. 1, pp. 57–65, 1986.

E. M. Herman, “Immunogold-localization and synthesis of an oil-body membrane protein in developing soybean seeds,” Planta, vol. 172, no. 3, pp. 336–345, 1987.

D. J. Murphy, “Storage lipid bodies in plants and other organisms,” Progress in Lipid Research, vol. 29, no. 4, pp. 299–324, 1990.

L. Y. Yatsu and T. J. Jacks, “Spherosome membranes: half unit-membranes,” Plant Physiology, vol. 49, no. 6, pp. 937–943, 1972.

A. H. C. Huang, “Oil bodies and oleosins in seeds,” Annual Review of Plant Physiology and Plant Molecular Biology, vol. 43, no. 1, pp. 177–200, 1992.

D. J. Murphy, “Structure, function and biogenesis of storage lipid bodies and oleosins in plants,” Progress in Lipid Research, vol. 32, no. 3, pp. 247–280, 1993.

G. I. Frandsen, J. Mundy, and J. T. C. Tzen, “Oil bodies and their associated proteins, oleosin and caleosin,” Physiologia Plantarum, vol. 112, no. 3, pp. 301–307, 2001.

Z. Purkrtova, P. Jolivet, M. Miquel, and T. Chardot, “Structure and function of seed lipid body-associated proteins,” Comptes Rendus Biologies, vol. 331, no. 10, pp. 746–754, 2008.

V. B. Vance and A. H. Huang, “The major protein from lipid bodies of maize. Characterization and structure based on cDNA cloning,” Journal of Biological Chemistry, vol. 262, no. 23, pp. 11275–11279, 1987.

R. Qu and A. H. C. Huang, “Oleosin KD 18 on the surface of oil bodies in maize. Genomic and cDNA sequences and the deduced protein structure,” Journal of Biological Chemistry, vol. 265, no. 4, pp. 2238–2243, 1990.

D. J. Murphy and D. M. Y. Au, “A new class of highly abundant apolipoproteins involved in lipid storage in oilseeds,” Biochemical Society Transactions, vol. 117, no. 4, pp. 682–683, 1989.

D. J. Murphy, J. N. Keen, J. N. O'Sullivan et al., “A class of amphipathic proteins associated with lipid storage bodies in plants. Possible similarities with animal serum apolipoproteins,” Biochimica et Biophysica Acta, vol. 1088, no. 1, pp. 86–94, 1991.

J. S. Keddie, G. Hübner, S. P. Slocombe et al., “Cloning and characterisation of an oleosin gene from Brassica napus,” Plant Molecular Biology, vol. 19, no. 3, pp. 443–453, 1992.

A. Kalinski, D. S. Loer, J. M. Weisemann, B. F. Matthews, and E. M. Herman, “Isoforms of soybean seed oil body membrane protein 24 kDa oleosin are encoded by closely related cDNAs,” Plant Molecular Biology, vol. 17, no. 5, pp. 1095–1098, 1991.

P. Hatzopoulos, G. Franz, L. Choy, and R. Z. Sung, “Interaction of nuclear factors with upstream sequences of a lipid body membrane protein gene from carrot,” Plant Cell, vol. 2, no. 5, pp. 457–467, 1990.

I. Cummins and D. J. Murphy, “cDNA sequence of a sunflower oleosin and transcript tissue specificity,” Plant Molecular Biology, vol. 19, no. 5, pp. 873–876, 1992.

G. J. H. van Rooijen, L. I. Terning, and M. M. Moloney, “Nucleotide sequence of an Arabidopsis thaliana oleosin gene,” Plant Molecular Biology, vol. 18, no. 6, pp. 1177–1179, 1992.

D. W. Hughes, H. Y. Wang, and G. A. Galau, “Cotton (Gossypium hirsutum) MatP6 and MatP7 oleosin genes,” Plant Physiology, vol. 101, no. 2, pp. 697–698, 1993.

Q. Liu, Y. Sun, W. Su, et al., “Species-specific size expansion and molecular evolution of the oleosins in angiosperms,” Gene, vol. 509, no. 2, pp. 247–257, 2012.

R. B. Aalen, “The transcripts encoding two oleosin isoforms are both present in the aleurone and in the embryo of barley (Hordeum vulgare L.) seeds,” Plant Molecular Biology, vol. 28, no. 3, pp. 583–588, 1995.

R. L. C. Chuang, J. C. F. Chen, J. Chu, and J. T. C. Tzen, “Characterization of seed oil bodies and their surface oleosin isoforms from rice embryos,” Journal of Biochemistry, vol. 120, no. 1, pp. 74–81, 1996.

J. C. F. Chen, R. H. Lin, H. C. Huang, and J. T. C. Tzen, “Cloning, expression and isoform classification of a minor oleosin in sesame oil bodies,” Journal of Biochemistry, vol. 122, no. 4, pp. 819–824, 1997.

L. S. H. Wu, L. D. Wang, P. W. Chen, L. J. Chen, and J. T. C. Tzen, “Genomic cloning of 18 kDa oleosin and detection of triacylglycerols and oleosin isoforms in maturing rice and postgerminative seedlings,” Journal of Biochemistry, vol. 123, no. 3, pp. 386–391, 1998.

J. T. C. Tzen and A. H. C. Huang, “Surface structure and properties of plant seed oil bodies,” Journal of Cell Biology, vol. 117, no. 2, pp. 327–335, 1992.

J. T. C. Tzen, G. C. Lie, and A. H. C. Huang, “Characterization of the charged components and their topology on the surface of plant seed oil bodies,” Journal of Biological Chemistry, vol. 267, no. 22, pp. 15626–15634, 1992.

J. T. C. Tzen, C. C. Peng, D. J. Cheng, E. C. F. Chen, and J. M. H. Chiu, “A new method for seed oil body purification and examination of oil body integrity following germination,” Journal of Biochemistry, vol. 121, no. 4, pp. 762–768, 1997.

E. C. F. Chen, S. S. K. Tai, C. C. Peng, and J. T. C. Tzen, “Identification of three novel unique proteins in seed oil bodies of sesame,” Plant and Cell Physiology, vol. 39, no. 9, pp. 935–941, 1998.

L. J. Lin and J. T. C. Tzen, “Two distinct steroleosins are present in seed oil bodies,” Plant Physiology and Biochemistry, vol. 42, no. 7-8, pp. 601–608, 2004.

J. T. C. Tzen, M. M. Wang, J. C. F. Chen, L. J. Lin, and M. C. M. Chen, “Seed oil body proteins: oleosin, caleosin, and steroleosin,” Current Topic in Biochemical Reseaech, vol. 5, pp. 133–139, 2003.

J. C. F. Chen, C. C. Y. Tsai, and J. T. C. Tzen, “Cloning and secondary structure analysis of caleosin, a unique calcium-binding protein in oil bodies of plant seeds,” Plant and Cell Physiology, vol. 40, no. 10, pp. 1079–1086, 1999.

L. J. Lin, S. S. K. Tai, C. C. Peng, and J. T. C. Tzen, “Steroleosin, a sterol-binding dehydrogenase in seed oil bodies,” Plant Physiology, vol. 128, no. 4, pp. 1200–1211, 2002.

H. Tnani, I. López, T. Jouenne, and C. M. Vicient, “Quantitative subproteomic analysis of germinating related changes in the scutellum oil bodies of Zea mays,” Plant Science, vol. 191-192, pp. 1–7, 2012.

H. Tnani, I. López, T. Jouenne, and C. M. Vicient, “Protein composition analysis of oil bodies from maize embryos during germination,” Journal of Plant Physiology, vol. 168, no. 5, pp. 510–513, 2011.

S. Popluechai, M. Froissard, P. Jolivet et al., “Jatropha curcas oil body proteome and oleosins: L-form JcOle3 as a potential phylogenetic marker,” Plant Physiology and Biochemistry, vol. 49, no. 3, pp. 352–356, 2011.

F. Capuano, N. J. Bond, L. Gatto, et al., “LC-MS/MS methods for absolute quantification and identification of proteins associated with chimeric plant oil bodies,” Analytical Chemistry, vol. 83, no. 24, pp. 9267–9272, 2011.

P. Jolivet, C. Boulard, A. Bellamy et al., “Protein composition of oil bodies from mature Brassica napus seeds,” Proteomics, vol. 9, no. 12, pp. 3268–3284, 2009.

V. Katavic, G. K. Agrawal, M. Hajduch, S. L. Harris, and J. J. Thelen, “Protein and lipid composition analysis of oil bodies from two Brassica napus cultivars,” Proteomics, vol. 6, no. 16, pp. 4586–4598, 2006.

P. Jolivet, E. Roux, S. D'Andrea et al., “Protein composition of oil bodies in Arabidopsis thaliana ecotype WS,” Plant Physiology and Biochemistry, vol. 42, no. 6, pp. 501–509, 2004.

J. T. C. Tzen, M. M. C. Wang, S. S. K. Tai, T. T. T. Lee, and C. C. Peng, “The abundant proteins in sesame seed: storage proteins in protein bodies and oleosins in oil bodies,” Advances in Plant Physiolology, vol. 6, pp. 93–105, 2003.

L. J. Lin, P. C. Liao, H. H. Yang, and J. T. C. Tzen, “Determination and analyses of the N-termini of oil-body proteins, steroleosin, caleosin and oleosin,” Plant Physiology and Biochemistry, vol. 43, no. 8, pp. 770–776, 2005.

D. J. Murphy, “The biogenesis and functions of lipid bodies in animals, plants and microorganisms,” Progress in Lipid Research, vol. 40, no. 5, pp. 325–438, 2001.

F. Capuano, F. Beaudoin, J. A. Napier, and P. R. Shewry, “Properties and exploitation of oleosins,” Biotechnology Advances, vol. 25, no. 2, pp. 203–206, 2007.

T. L. Shimada and I. Hara-Nishimura, “Oil-body-membrane proteins and their physiological functions in plants,” Biological and Pharmaceutical Bulletin, vol. 33, no. 3, pp. 360–363, 2010.

C. C. Peng, V. S. Y. Lee, M. Y. Lin, H. Y. Huang, and J. T. C. Tzen, “Minimizing the central hydrophobic domain in oleosin for the constitution of artificial oil bodies,” Journal of Agricultural and Food Chemistry, vol. 55, no. 14, pp. 5604–5610, 2007.

B. M. Abell, M. Hahn, L. A. Holbrook, and M. M. Moloney, “Membrane topology and sequence requirements for oil body targeting of oleosin,” Plant Journal, vol. 37, no. 4, pp. 461–470, 2004.

K. Giannoulia, G. Banilas, and P. Hatzopoulos, “Oleosin gene expression in olive,” Journal of Plant Physiology, vol. 164, no. 1, pp. 104–107, 2007.

B. M. Abell, L. A. Holbrook, M. Abenes, D. J. Murphy, M. J. Hills, and M. M. Moloney, “Role of the proline knot motif in oleosin endoplasmic reticulum topology and oil body targeting,” Plant Cell, vol. 9, no. 8, pp. 1481–1493, 1997.

K. B. Vargo, R. Parthasarathy, and D. A. Hammer, “Self-assembly of tunable protein suprastructures from recombinant oleosin,” Proceedings of the National Academy of Sciences of the United States of America, vol. 109, no. 29, pp. 11657–11662, 2012.

Y. Gohon, J. D. Vindigni, A. Pallier et al., “High water solubility and fold in amphipols of proteins with large hydrophobic regions: oleosins and caleosin from seed lipid bodies,” Biochimica et Biophysica Acta, vol. 1808, no. 3, pp. 706–716, 2011.

L. G. Alexander, R. B. Sessions, A. R. Clarke, A. S. Tatham, P. R. Shewry, and J. A. Napier, “Characterization and modelling of the hydrophobic domain of a sunflower oleosin,” Planta, vol. 214, no. 4, pp. 546–551, 2002.

M. Li, D. J. Murphy, K. H. K. Lee et al., “Purification and structural characterization of the central hydrophobic domain of oleosin,” Journal of Biological Chemistry, vol. 277, no. 40, pp. 37888–37895, 2002.

B. M. Abell, S. High, and M. M. Moloney, “Membrane protein topology of oleosin is constrained by its long hydrophobic domain,” Journal of Biological Chemistry, vol. 277, no. 10, pp. 8602–8610, 2002.

D. J. Lacey, N. Wellner, F. Beaudoin, J. A. Napier, and P. R. Shewry, “Secondary structure of oleosins in oil bodies isolated from seeds of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.),” Biochemical Journal, vol. 334, no. 2, pp. 469–477, 1998.

M. Millichip, A. S. Tatham, F. Jackson, G. Griffiths, P. R. Shewry, and A. K. Stobart, “Purification and characterization of oil-bodies (oleosomes) and oil-body boundary proteins (oleosins) from the developing cotyledons of sunflower (Helianthus annuus L.),” Biochemical Journal, vol. 314, no. 1, pp. 333–337, 1996.

M. Li, J. S. Keddie, L. J. Smith, D. C. Clark, and D. J. Murphy, “Expression and characterization of the N-terminal domain of an oleosin protein from sunflower,” Journal of Biological Chemistry, vol. 268, no. 23, pp. 17504–17512, 1993.

M. Li, L. J. Smith, D. C. Clark, R. Wilson, and D. J. Murphy, “Secondary structures of a new class of lipid body proteins from oilseeds,” Journal of Biological Chemistry, vol. 267, no. 12, pp. 8245–8253, 1992.

S. Takahashi, T. Katagiri, K. Yamaguchi-Shinozaki, and K. Shinozaki, “An Arabidopsis gene encoding a Ca²⁺ -binding protein is induced by abscisic acid during dehydration,” Plant and Cell Physiology, vol. 41, no. 7, pp. 898–903, 2000.

Z. Purkrtova, C. Le Bon, B. Kralova, M. H. Ropers, M. Anton, and T. Chardot, “Caleosin of Arabidopsis thaliana: effect of calcium on functional and structural properties,” Journal of Agricultural and Food Chemistry, vol. 56, no. 23, pp. 11217–11224, 2008.

M. B. Busch, K. H. Kortje, H. Rahmann, and A. Sievers, “Characteristic and differential calcium signals from cell structures of the root cap detected by energy-filtering electron microscopy (EELS/ESI),” European Journal of Cell Biology, vol. 60, no. 1, pp. 88–100, 1993.

P. L. Jiang and J. T. C. Tzen, “Caleosin serves as the major structural protein as efficient as oleosin on the surface of seed oil bodies,” Plant Signaling and Behavior, vol. 5, no. 4, pp. 447–449, 2010.

T. H. Liu, C. L. Chyan, F. Y. Li, and J. T. C. Tzen, “Stability of artificial oil bodies constituted with recombinant caleosins,” Journal of Agricultural and Food Chemistry, vol. 57, no. 6, pp. 2308–2313, 2009.

J. C. F. Chen and J. T. C. Tzen, “An in vitro system to examine the effective phospholipids and structural domain for protein targeting to seed oil bodies,” Plant and Cell Physiology, vol. 42, no. 11, pp. 1245–1252, 2001.

Z. Purkrtova, S. d'Andrea, P. Jolivet et al., “Structural properties of caleosin: a MS and CD study,” Archives of Biochemistry and Biophysics, vol. 464, no. 2, pp. 335–343, 2007.

C. I. Brändeén, “Relation between structure and function of alpha/beta-proteins,” Quarterly Reviews of Biophysics, vol. 13, no. 3, pp. 317–338, 1980.

C. van der Schoot, L. K. Paul, S. B. Paul, and P. L. Rinne, “Plant lipid bodies and cell-cell signaling: a new role for an old organelle?” Plant Signaling and Behavior, vol. 6, no. 11, pp. 1732–1738, 2011.

T. L. Shimada, T. Shimada, H. Takahashi, Y. Fukao, and I. Hara-Nishimura, “A novel role for oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana,” Plant Journal, vol. 55, no. 5, pp. 798–809, 2008.

Y. Y. Wu, Y. R. Chou, C. S. Wang, T. H. Tseng, L. J. Chen, and J. T. C. Tzen, “Different effects on triacylglycerol packaging to oil bodies in transgenic rice seeds by specifically eliminating one of their two oleosin isoforms,” Plant Physiology and Biochemistry, vol. 48, no. 2-3, pp. 81–89, 2010.

M. A. Schmidt and E. M. Herman, “Suppression of soybean oleosin produces micro-oil bodies that aggregate into oil body/ER complexes,” Molecular Plant, vol. 1, no. 6, pp. 910–924, 2008.

R. M. P. Siloto, K. Findlay, A. Lopez-Villalobos, E. C. Yeung, C. L. Nykiforuk, and M. M. Moloney, “The accumulation of oleosins determines the size of seed oilbodies in Arabidopsis,” Plant Cell, vol. 18, no. 8, pp. 1961–1974, 2006.

E. Roux, S. Baumberger, M. A. V. Axelos, and T. Chardot, “Oleosins of Arabidopsis thaliana: expression in Escherichia coli, purification, and functional properties,” Journal of Agricultural and Food Chemistry, vol. 52, no. 16, pp. 5245–5249, 2004.

C. C. Peng, I. P. Lin, C. K. Lin, and J. T. C. Tzen, “Size and stability of reconstituted sesame oil bodies,” Biotechnology Progress, vol. 19, no. 5, pp. 1623–1626, 2003.

J. T. L. Ting, K. Lee, C. Ratnayake, K. A. Platt, R. A. Balsamo, and A. H. C. Huang, “Oleosin genes in maize kernels having diverse oil contents are constitutively expressed independent of oil contents: size and shape of intracellular oil bodies are determined by the oleosins/oils ratio,” Planta, vol. 199, no. 1, pp. 158–165, 1996.

N. Babazadeh, M. Poursaadat, H. R. Sadeghipour, and A. Hossein Zadeh Colagar, “Oil body mobilization in sunflower seedlings is potentially regulated by thioredoxin h,” Plant Physiology and Biochemistry, vol. 57, pp. 134–142, 2012.

M. Rudolph, A. Schlereth, M. Körner et al., “The lipoxygenase-dependent oxygenation of lipid body membranes is promoted by a patatin-type phospholipase in cucumber cotyledons,” Journal of Experimental Botany, vol. 62, no. 2, pp. 749–760, 2011.

E. S. L. Hsiao and J. T. C. Tzen, “Ubiquitination of oleosin-H and caleosin in sesame oil bodies after seed germination,” Plant Physiology and Biochemistry, vol. 49, no. 1, pp. 77–81, 2011.

S. Vandana and S. C. Bhatla, “Evidence for the probable oil body association of a thiol-protease, leading to oleosin degradation in sunflower seedling cotyledons,” Plant Physiology and Biochemistry, vol. 44, no. 11-12, pp. 714–723, 2006.

V. Parthibane, S. Rajakumari, V. Venkateshwari, R. Iyappan, and R. Rajasekharan, “Oleosin is bifunctional enzyme that has both monoacylglycerol acyltransferase and phospholipase activities,” The Journal of Biological Chemistry, vol. 287, no. 3, pp. 1946–1954, 2012.

V. Parthibane, R. Iyappan, A. Vijayakumar, V. Venkateshwari, and R. Rajasekharan, “Serine/threonine/tyrosine protein kinase phosphorylates oleosin, a regulator of lipid metabolic functions,” Plant Physiology, vol. 159, no. 1, pp. 95–104, 2012.

P. L. Jiang, J. C. F. Chen, S. T. Chiu, and J. T. C. Tzen, “Stable oil bodies sheltered by a unique caleosin in cycad megagametophytes,” Plant Physiology and Biochemistry, vol. 47, no. 11-12, pp. 1009–1016, 2009.

M. Poxleitner, S. W. Rogers, A. Lacey Samuels, J. Browse, and J. C. Rogers, “A role for caleosin in degradation of oil-body storage lipid during seed germination,” Plant Journal, vol. 47, no. 6, pp. 917–933, 2006.

P. L. Jiang, C. S. Wang, C. M. Hsu, G. Y. Jauh, and J. T. C. Tzen, “Stable oil bodies sheltered by a unique oleosin in lily pollen,” Plant and Cell Physiology, vol. 48, no. 6, pp. 812–821, 2007.

P. L. Jiang, G. Y. Jauh, C. S. Wang, and J. T. C. Tzen, “A unique caleosin in oil bodies of lily pollen,” Plant and Cell Physiology, vol. 49, no. 9, pp. 1390–1395, 2008.

K. Zienkiewicz, A. J. Castro, J. D. D. Alché, A. Zienkiewicz, C. Suárez, and M. I. Rodríguez-García, “Identification and localization of a caleosin in olive (Olea europaea L.) pollen during in vitro germination,” Journal of Experimental Botany, vol. 61, no. 5, pp. 1537–1546, 2010.

A. Hanano, M. Burcklen, M. Flenet et al., “Plant seed peroxygenase is an original heme-oxygenase with an EF-hand calcium binding motif,” Journal of Biological Chemistry, vol. 281, no. 44, pp. 33140–33151, 2006.

M. Partridge and D. J. Murphy, “Roles of a membrane-bound caleosin and putative peroxygenase in biotic and abiotic stress responses in Arabidopsis,” Plant Physiology and Biochemistry, vol. 47, no. 9, pp. 796–806, 2009.

H. Feng, X. Wang, Y. Sun et al., “Cloning and characterization of a calcium binding EF-hand protein gene TaCab1 from wheat and its expression in response to Puccinia striiformis f. sp. tritici and abiotic stresses,” Molecular Biology Reports, vol. 38, no. 6, pp. 3857–3866, 2011.

Y. Y. Kim, K. W. Jung, K. S. Yoo, J. U. Jeung, and J. S. Shin, “A stress-responsive caleosin-like protein, AtCLO4, Acts as a Negative Regulator of ABA Responses in Arabidopsis,” Plant and Cell Physiology, vol. 52, no. 5, pp. 874–884, 2011.

Y. Aubert, L. Leba, C. Cheval et al., “Involvement of RD20, a member of caleosin family, in ABA-mediated regulation of germination in Arabidopsis thaliana,” Plant Signaling and Behavior, vol. 6, no. 4, pp. 538–540, 2011.

H. B. Khalil, Z. Wang, J. A. Wright, et al., “Heterotrimeric Gα subunit from wheat (Triticum aestivum), GA3, interacts with the calcium-binding protein, Clo3, and the phosphoinositide-specific phospholipase C, PI-PLC1,” Plant Molecular Biology, vol. 77, pp. 145–158, 2011.

E. Blée, M. Flenet, B. Boachon, and M. L. Fauconnier, “A non-canonical caleosin from Arabidopsis efficiently epoxidizes physiological unsaturated fatty acids with complete stereoselectivity,” The FEBS Journal, vol. 279, no. 20, pp. 3981–3995, 2012.

S. De Domenico, S. Bonsegna, M. S. Lenucci, et al., “Localization of seed oil body proteins in tobacco protoplasts reveals specific mechanisms of protein targeting to leaf lipid droplets,” Journal of Integrative Plant Biology, vol. 53, no. 11, pp. 858–868, 2011.

J. Hänisch, M. Wältermann, H. Robenek, and A. Steinbüchel, “Eukaryotic lipid body proteins in oleogenous actinomycetes and their targeting to intracellular triacylglycerol inclusions: impact on models of lipid body biogenesis,” Applied and Environmental Microbiology, vol. 72, no. 10, pp. 6743–6750, 2006.

W. Li, L. G. Li, X. F. Sun, and K. X. Tang, “An oleosin-fusion protein driven by the CaMV35S promoter is accumulated in Arabidopsis (Brassicaceae) seeds and correctly targeted to oil bodies,” Genetics and Molecular Research, vol. 11, no. 3, pp. 2138–2146, 2012.

G. J. Van Rooijen and M. M. Moloney, “Structural requirements of oleosin domains for subcellular targeting to the oil body,” Plant Physiology, vol. 109, no. 4, pp. 1353–1361, 1995.

I. Cummins, M. J. Hills, J. H. E. Ross, D. H. Hobbs, M. D. Watson, and D. J. Murphy, “Differential, temporal and spatial expression of genes involved in storage oil and oleosin accumulation in developing rapeseed embryos: implications for the role of oleosins and the mechanisms of oil-body formation,” Plant Molecular Biology, vol. 23, no. 5, pp. 1015–1027, 1993.

W. S. Lee, J. T. C. Tzen, J. C. Kridl, S. E. Radke, and A. H. C. Huang, “Maize oleosin is correctly targeted to seed oil bodies in Brassica napus transformed with the maize oleosin gene,” Proceedings of the National Academy of Sciences of the United States of America, vol. 88, no. 14, pp. 6181–6185, 1991.

T. Wahlroos, J. Soukka, A. Denesyuk, R. Wahlroos, T. Korpela, and N. J. Kilby, “Oleosin expression and trafficking during oil body biogenesis in tobacco leaf cells,” Genesis, vol. 35, no. 2, pp. 125–132, 2003.

J. T. L. Ting, R. A. Balsamo, C. Ratnayake, and A. H. C. Huang, “Oleosin of plant seed oil bodies is correctly targeted to the lipid bodies in transformed yeast,” Journal of Biological Chemistry, vol. 272, no. 6, pp. 3699–3706, 1997.

F. Beaudoin, B. M. Wilkinson, C. J. Stirling, and J. A. Napier, “In vivo targeting of a sunflower oil body protein in yeast secretory (sec) mutants,” Plant Journal, vol. 23, no. 2, pp. 159–170, 2000.

F. Beaudoin and J. A. Napier, “The targeting and accumulation of ectopically expressed oleosin in non-seed tissues of Arabidopsis thaliana,” Planta, vol. 210, no. 3, pp. 439–445, 2000.

C. Sarmiento, J. H. E. Ross, E. Herman, and D. J. Murphy, “Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of oil-body formation in seeds,” Plant Journal, vol. 11, no. 4, pp. 783–796, 1997.

P. J. Thoyts, M. I. Millichip, A. K. Stobart, W. T. Griffiths, P. R. Shewry, and J. A. Napier, “Expression and in vitro targeting of a sunflower oleosin,” Plant Molecular Biology, vol. 29, no. 2, pp. 403–410, 1995.

D. S. Loer and E. M. Herman, “Cotranslational integration of soybean (Glycine max) oil body membrane protein oleosin into microsomal membranes,” Plant Physiology, vol. 101, no. 3, pp. 993–998, 1993.

M. J. Hills, M. D. Watson, and D. J. Murphy, “Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.),” Planta, vol. 189, no. 1, pp. 24–29, 1993.

M. Froissard, S. D'Andréa, C. Boulard, and T. Chardot, “Heterologous expression of AtClo1, a plant oil body protein, induces lipid accumulation in yeast,” FEMS Yeast Research, vol. 9, no. 3, pp. 428–438, 2009.

F. Beaudoin and J. A. Napier, “Targeting and membrane-insertion of a sunflower oleosin in vitro and in Saccharomyces cerevisiae: the central hydrophobic domain contains more than one signal sequence, and directs oleosin insertion into the endoplasmic reticulum membrane using a signal anchor sequence mechanism,” Planta, vol. 215, no. 2, pp. 293–303, 2002.

J. T. C. Tzen, Y. K. Lai, K. L. Chan, and A. H. C. Huang, “Oleosin isoforms of high and low molecular weights are present in the oil bodies of diverse seed species,” Plant Physiology, vol. 94, no. 3, pp. 1282–1289, 1990.

J. T. C. Tzen, R. L. C. Chuang, J. C. F. Chen, and L. S. H. Wu, “Coexistence of both oleosin isoforms on the surface of seed oil bodies and their individual stabilization to the organelles,” Journal of Biochemistry, vol. 123, no. 2, pp. 318–323, 1998.

S. S. K. Tai, M. C. M. Chen, C. C. Peng, and J. T. C. Tzen, “Gene family of oleosin isoforms and their structural stabilization in sesame seed oil bodies,” Bioscience, Biotechnology and Biochemistry, vol. 66, no. 10, pp. 2146–2153, 2002.

A. C. N. Chua, P. L. Jiang, L. S. Shi, W. M. Chou, and J. T. C. Tzen, “Characterization of oil bodies in jelly fig achenes,” Plant Physiology and Biochemistry, vol. 46, no. 5-6, pp. 525–532, 2008.

A. J. Simkin, T. Qian, V. Caillet et al., “Oleosin gene family of Coffea canephora: quantitative expression analysis of five oleosin genes in developing and germinating coffee grain,” Journal of Plant Physiology, vol. 163, no. 7, pp. 691–708, 2006.

L. S. H. Wu, G. H. H. Hong, R. F. Hou, and J. T. C. Tzen, “Classification of the single oleosin isoform and characterization of seed oil bodies in gymnosperms,” Plant and Cell Physiology, vol. 40, no. 3, pp. 326–334, 1999.

M. R. Roberts, R. Hodge, and R. Scott, “Brassica napus pollen oleosins possess a characteristic C-terminal domain,” Planta, vol. 195, no. 3, pp. 469–470, 1995.

D. J. Murphy and J. H. E. Ross, “Biosynthesis, targeting and processing of oleosin-like proteins, which are major pollen coat components in Brassica napus,” Plant Journal, vol. 13, no. 1, pp. 1–16, 1998.

C. Y. Huang, C. I. Chung, Y. C. Lin, Y. I. C. Hsing, and A. H. C. Huang, “Oil bodies and oleosins in Physcomitrella possess characteristics representative of early trends in evolution,” Plant Physiology, vol. 150, no. 3, pp. 1192–1203, 2009.

L. S. Robert, J. Gerster, S. Allard, L. Cass, and J. Simmonds, “Molecular characterization of two Brassica napus genes related to oleosins which are highly expressed in the tapetum,” Plant Journal, vol. 6, no. 6, pp. 927–933, 1994.

J. H. E. Ross and D. J. Murphy, “Characterization of anther-expressed genes encoding a major class of extracellular oleosin-like proteins in the pollen coat of Brassicaceae,” Plant Journal, vol. 9, no. 5, pp. 625–637, 1996.

L. O. Franco, C. L. De, S. Hamdi, G. Sachetto-Martins, and D. E. De Oliveira, “Distal regulatory regions restrict the expression of cis-linked genes to the tapetal cells,” The FEBS Letters, vol. 517, no. 1–3, pp. 13–18, 2002.

D. H. Chen, C. L. Chyan, P. L. Jiang, C. S. Chen, and J. T. C. Tzen, “The same oleosin isoforms are present in oil bodies of rice embryo and aleurone layer while caleosin exists only in those of the embryo,” Plant Physiology and Biochemistry, vol. 60, pp. 18–24, 2012.

H. C. Lu, P. L. Jiang, L. R. C. Hsu, C. L. Chyan, and J. T. C. Tzen, “Characterization of Oil bodies in adlay (Coix lachryma-jobi L),” Bioscience, Biotechnology and Biochemistry, vol. 74, no. 9, pp. 1841–1847, 2010.

H. Liu, P. Hedley, L. Cardle et al., “Characterisation and functional analysis of two barley caleosins expressed during barley caryopsis development,” Planta, vol. 221, no. 4, pp. 513–522, 2005.

K. Zienkiewicz, A. Zienkiewicz, M. I. Rodríguez-García, and A. J. Castro, “Characterization of a caleosin expressed during olive (Olea europaea L.) pollen ontogeny,” BMC Plant Biology, vol. 11, article 122, 2011.

I. P. Lin, P. L. Jiang, C. S. Chen, and J. T. C. Tzen, “A unique caleosin serving as the major integral protein in oil bodies isolated from Chlorella sp. cells cultured with limited nitrogen,” Plant Physiology and Biochemistry, vol. 44, no. 61, pp. 80–87, 2012.

H. Næsted, G. I. Frandsen, G. Y. Jauh et al., “Caleosins: Ca²⁺-binding proteins associated with lipid bodies,” Plant Molecular Biology, vol. 44, no. 4, pp. 463–476, 2000.

C. C. Peng, J. C. F. Chen, D. J. H. Shyu, M. J. Chen, and J. T. C. Tzen, “A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides,” Journal of Biotechnology, vol. 111, no. 1, pp. 51–57, 2004.

C. C. Peng, D. J. H. Shyu, W. M. Chou, M. J. Chen, and J. T. C. Tzen, “Method for bacterial expression and purification of sesame cystatin via artificial oil bodies,” Journal of Agricultural and Food Chemistry, vol. 52, no. 10, pp. 3115–3119, 2004.

M. C. M. Chen, C. L. Chyan, T. T. T. Lee, S. H. Huang, and J. T. C. Tzen, “Constitution of stable artificial oil bodies with triacylglycerol, phospholipid, and caleosin,” Journal of Agricultural and Food Chemistry, vol. 52, no. 12, pp. 3982–3987, 2004.

G. J. H. Van Rooijen and M. M. Moloney, “Plant seed oil-bodies as carriers for foreign proteins,” Bio/Technology, vol. 13, no. 1, pp. 72–77, 1995.

J. G. Boothe, J. A. Saponja, and D. L. Parmenter, “Molecular farming in plants: oilseeds as vehicles for the production of pharmaceutical proteins,” Drug Development Research, vol. 42, no. 3-4, pp. 172–181, 1997.

N. Markley, C. Nykiforuk, J. Boothe, and M. Moloney, “Producing proteins using transgenic oilbody-oleosin technology,” BioPharm International, vol. 19, no. 6, pp. 34–57, 2006.

S. C. Bhatla, V. Kaushik, and M. K. Yadav, “Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications,” Biotechnology Advances, vol. 28, no. 3, pp. 293–300, 2010.

M. D. McLean, R. Chen, D. Yu, et al., “Purification of the therapeutic antibody trastuzumab from genetically modified plants using safflower Protein A-oleosin oilbody technology,” Transgenic Research. In press

G. Banilas, G. Daras, S. Rigas, M. M. Moloney, and P. Hatzopoulos, “Oleosin di-or tri-meric fusions with GFP undergo correct targeting and provide advantages for recombinant protein production,” Plant Physiology and Biochemistry, vol. 49, no. 2, pp. 216–222, 2011.

C. Y. Yang, S. Y. Chen, and G. C. Duan, “Transgenic peanut (Arachis hypogaea L.) expressing the urease subunit B gene of Helicobacter pylori,” Current Microbiology, vol. 63, no. 4, pp. 387–391, 2011.

W. Li, L. Li, K. Li, J. Lin, X. Sun, and K. Tang, “Expression of biologically active human insulin-like growth factor 1 in Arabidopsis thaliana seeds via oleosin fusion technology,” Biotechnology and Applied Biochemistry, vol. 58, no. 3, pp. 139–146, 2011.

A. Ahmad, E. O. Pereira, A. J. Conley, A. S. Richman, and R. Menassa, “Green biofactories: recombinant protein production in plants,” Recent Patents on Biotechnology, vol. 4, no. 3, pp. 242–259, 2010.

C. E. Orozco-Barrios, S. F. Battaglia-Hsu, M. L. Arango-Rodriguez et al., “Vitamin B12-impaired metabolism produces apoptosis and Parkinson phenotype in rats expressing the transcobalamin-oleosin chimera in substantia nigra,” PLoS ONE, vol. 4, no. 12, Article ID e8268, 2009.

L. Pons, S. F. Battaglia-Hsu, C. E. Orozco-Barrios et al., “Anchoring secreted proteins in endoplasmic reticulum by plant oleosin: the example of vitamin B12 cellular sequestration by transcobalamin,” PLoS ONE, vol. 4, no. 7, Article ID e6325, 2009.

M. M. Moloney, “Oil-body proteins as carriers of high-value peptides in plants,” Patent US, 659835, 1991

M. M. Moloney and G. van Rooijen Sembiosys, “Expression of epidermal growth factor in plant seeds,” Patent US, 7091401, 2006

M. M. Moloney, “Oil-body proteins as carriers of high-value peptides in plants,” Patent US, 5650554, 1997

M. M. Moloney, J. Boothe, and G. van Rooijen, “Oil bodies and associated proteins as affinity matrices,” Patent US, 6509453, 2003

S. Szarka, G. van Rooijen, and M. M. Moloney, “Methods for the production of multimeric immunoglobulins, and related compositions,” Patent US, 7098383, 2006

G. van Rooijen, S. Zaplachinski, P. B. Heifetz, et al., “Methods for the production of multimeric protein complexes, and related compositions,” Patent US, 2006/0179514, 2006

J. McCarthy and S. A. Nestec, “Recombinant oleosins from cacao and their use as flavoring or emulsifying agents,” Patent US, 7126042, 2006

T. Harada, K. Kashihara, and N. Nio, “Oleosin/phospholipid complex and process for producing the same,” Patent WO, 2002/026788, 2002

H. M. Deckers, G. van Rooijen, J. Boothe, et al., “Uses of oil bodies,” Patent US, 6210742, 2001

C. J. Chiang, H. C. Chen, Y. P. Chad, and J. T. C. Tzen, “Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli,” Journal of Agricultural and Food Chemistry, vol. 53, no. 12, pp. 4799–4804, 2005.

J. R. Liu, C. H. Duan, X. Zhao, J. T. C. Tzen, K. J. Cheng, and C. K. Pai, “Cloning of a rumen fungal xylanase gene and purification of the recombinant enzyme via artificial oil bodies,” Applied Microbiology and Biotechnology, vol. 79, no. 2, pp. 225–233, 2008.

Y. J. Hung, C. C. Peng, J. T. C. Tzen, M. J. Chen, and J. R. Liu, “Immobilization of Neocallimastix patriciarum xylanase on artificial oil bodies and statistical optimization of enzyme activity,” Bioresource Technology, vol. 99, no. 18, pp. 8662–8666, 2008.

R. W. Scott, S. Winichayakul, M. Roldan et al., “Elevation of oil body integrity and emulsion stability by polyoleosins, multiple oleosin units joined in tandem head-to-tail fusions,” Plant Biotechnology Journal, vol. 8, no. 8, pp. 912–927, 2010.

J. M. Tseng, J. R. Huang, H. C. Huang, J. T. C. Tzen, W. M. Chou, and C. C. Peng, “Facilitative production of an antimicrobial peptide royalisin and its antibody via an artificial oil-body system,” Biotechnology Progress, vol. 27, no. 1, pp. 153–161, 2011.

S. Winichayakul, A. Pernthaner, S. Livingston, et al., “Production of active single-chain antibodies in seeds using trimeric polyoleosin fusion,” Journal of Biotechnology, vol. 161, no. 4, pp. 407–413, 2012.

C. J. Chiang, H. C. Chen, H. F. Kuo, Y. P. Chao, and J. T. C. Tzen, “A simple and effective method to prepare immobilized enzymes using artificial oil bodies,” Enzyme and Microbial Technology, vol. 39, no. 5, pp. 1152–1158, 2006.

C. J. Chiang, H. C. Chen, Y. P. Chao, and J. T. C. Tzen, “One-step purification of insoluble hydantoinase overproduced in Escherichia coli,” Protein Expression and Purification, vol. 52, no. 1, pp. 14–18, 2007.

R. C. W. Hou, M. Y. Lin, M. M. C. Wang, and J. T. C. Tzen, “Increase of viability of entrapped cells of Lactobacillus delbrueckii ssp. bulgaricus in artificial sesame oil emulsions,” Journal of Dairy Science, vol. 86, no. 2, pp. 424–428, 2003.

M. C. M. Chen, J. L. Wang, and J. T. C. Tzen, “Elevating bioavailability of cyclosporine A via encapsulation in artificial oil bodies stabilized by caleosin,” Biotechnology Progress, vol. 21, no. 4, pp. 1297–1301, 2005.

C. J. Chiang, C. C. Lin, T. L. Lu, and H. F. Wang, “Functionalized nanoscale oil bodies for targeted delivery of a hydrophobic drug,” Nanotechnology, vol. 22, no. 41, Article ID 415102, 2011.

C. J. Chiang, C. J. Chen, L. J. Lin, C. H. Chang, and Y. P. Chao, “Selective delivery of cargo entities to tumor cells by nanoscale artificial oil bodies,” Journal of Agricultural and Food Chemistry, vol. 58, no. 22, pp. 11695–11702, 2010.

C. J. Chiang, L. J. Lin, C. C. Lin, C. H. Chang, and Y. P. Chao, “Selective internalization of self-assembled artificial oil bodies by HER2/neu-positive cells,” Nanotechnology, vol. 22, no. 1, Article ID 015102, 2011.

M. T. Chang, C. R. Chen, T. H. Liu, C. P. Lee, and J. T. C. Tzen, “Development of a protocol to solidify native and artificial oil bodies for long-term storage at room temperature,” Journal of the Science of Food and Agriculture. In press

T. H. Liu, C. L. Chyan, F. Y. Li, Y. J. Chen, and J. T. C. Tzen, “Engineering lysine-rich caleosins as carrier proteins torenderbiotin as a hapten on artificial oil bodies for antibody production,” Biotechnology Progress, vol. 27, no. 6, pp. 1760–1767, 2011.