Determination and analyses of the N-termini of oil-body proteins, steroleosin, caleosin and oleosin.

Seed oil bodies comprise a triacylglycerol matrix shielded by a monolayer of phospholipids and proteins. These surface proteins include an abundant structural protein, oleosin, and at least two minor protein classes termed caleosin and steroleosin. Two steroleosin isoforms (41 and 39 kDa), one caleosin (27 kDa), and two oleosin isoforms (17 and 15 kDa) have been identified in oil bodies isolated from sesame seeds. The signal peptides responsible for targeting of these proteins to oil bodies have not been experimentally determined. Hydropathy analyses indicate that the hydrophobic domain putatively responsible for oil-body anchoring is located in the N-terminal region of steroleosin, but in the central region of caleosin or oleosin. Direct amino acid sequencing showed that both steroleosin isoforms possessed a free methionine residue at their N-termini while caleosin and oleosin isoforms were N-terminally blocked. Mass spectrometry analyses revealed that N-termini of both caleosin and 17 kDa oleosin were acetylated after the removal of the first methionine. In addition, deamidation was observed at a glutamine residue in the N-terminal region of 17 kDa oleosin.     

Comparative Genomics of the Lipid-body-membrane Proteins Oleosin,Caleosin and Steroleosin in Magnoliophyte, Lycophyte and Bryophyte

Lipid bodies store oils in the form of triacylglycerols. Oleosin, caleosin and steroleosin are unique proteins localized on the surface of lipid bodies in seed plants. This study has identified genes encoding lipid body proteins oleosin, caleosin and steroleosin in the genomes of five plants: Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Selaginella moellendorffii and Physcomitrella patens. The protein sequence alignment indicated that each oleosin protein contains a highly-conserved proline knot motif, and proline knob motif is well conserved in steroleosin proteins, while caleosin proteins possess the Dx[D/N]xDG-containing calcium-binding motifs. The identification of motifs (proline knot and knob) and conserved amino acids at active site was further supported by the sequence logos. The phylogenetic analysis revealed the presence of magnoliophyte-and bryophyte-specific subgroups. We analyzed the public microarray data for expression of oleosin, caleosin and steroleosin in Arabidopsis and rice during the vegetative and reproductive stages, or under abiotic stresses. Our results indicated that genes encoding oleosin, caleosin and steroleosin proteins were expressed predominantly in plant seeds. This work may facilitate better understanding of the members of lipid-body-membrane proteins in diverse organisms and their gene expression in model plants Arabidopsis and rice.     

Steroleosin, a sterol-binding dehydrogenase in seed oil bodies

Besides abundant oleosin, three minor proteins, Sop 1, 2, and 3, are present in sesame (Sesamum indicum) oil bodies. The gene encoding Sop1, named caleosin for its calcium-binding capacity, has recently been cloned. In this study, Sop2 gene was obtained by immunoscreening, and it was subsequently confirmed by amino acid partial sequencing and immunological recognition of its overexpressed protein in Escherichia coli. Immunological cross recognition implies that Sop2 exists in seed oil bodies of diverse species. Along with oleosin and caleosin genes, Sop2 gene was transcribed in maturing seeds where oil bodies are actively assembled. Sequence analysis reveals that Sop2, tentatively named steroleosin, possesses a hydrophobic anchoring segment preceding a soluble domain homologous to sterol-binding dehydrogenases/reductases involved in signal transduction in diverse organisms. Three-dimensional structure of the soluble domain was predicted via homology modeling. The structure forms a seven-stranded parallel beta-sheet with the active site, S-(12X)-Y-(3X)-K, between an NADPH and a sterol-binding subdomain. Sterol-coupling dehydrogenase activity was demonstrated in the overexpressed soluble domain of steroleosin as well as in purified oil bodies. Southern hybridization suggests that one steroleosin gene and certain homologous genes may be present in the sesame genome. Comparably, eight hypothetical steroleosin-like proteins are present in the Arabidopsis genome with a conserved NADPH-binding subdomain, but a divergent sterol-binding subdomain. It is indicated that steroleosin-like proteins may represent a class of dehydrogenases/reductases that are involved in plant signal transduction regulated by various sterols.     

Membrane topology and sequence requirements for oil body targeting of oleosin

Oleosin protein is targeted to oil bodies via the endoplasmic reticulum (ER) and consists of a lipid-submerged hydrophobic (H) domain that is flanked by cytosolic hydrophilic domains. We investigated the relationship between oleosin ER topology and its subsequent ability to target to oil bodies. Oleosin variants were created to yield differing ER membrane topologies and tagged with a reporter enzyme. Localisation was assessed by fractionation after transient expression in embryonic cells. Membrane-straddled topologies with N-terminal sequence in the ER lumen and C-terminal sequence in the cytosol were unable to target to oil bodies efficiently. Similarly, a translocated topology with only ER membrane and lumenal sequence was unable to target to oil bodies efficiently. Both topology variants accumulated proportionately higher in ER microsomal fractions, demonstrating a block in transferring from ER to oil bodies. The residual oil body accumulation for the inverted topology was shown to be because of partial adoption of native ER membrane topology, using a reporter variant, which becomes inactivated by ER-mediated glycosylation. In addition, the importance of H domain sequence for oil body targeting was assessed using variants that maintain native ER topology. The central proline knot motif (PKM) has previously been shown to be critical for oil body targeting, but here the arms of the H domain flanking this motif were shown to be interchangeable with only a moderate reduction in oil body targeting. We conclude that oil body targeting of oleosin depends on a specific ER membrane topology but does not require a specific sequence in the H domain flanking arms.     

Protein composition analysis of oil bodies from maize embryos during germination

Seed oil bodies (OBs) are intracellular particles that store lipids. In maize embryos, the oil bodies are accumulated mainly in the scutellum. Oil bodies were purified from the scutellum of germinating maize seeds and the associated proteins were extracted and subjected to 2-DE analysis followed by LC-MS/MS for protein identification. In addition to the previously known oil body proteins oleosin, caleosin and steroleosin, new proteins were identified.     

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.     

An in vitro system to examine the effective phospholipids and structural domain for protein targeting to seed oil bodies.

An in vitro system was established to examine the targeting of proteins to maturing seed oil bodies. Oleosin, the most abundant structural protein, and caleosin, a newly identified minor constituent in seed oil bodies, were translated in a reticulocyte lysate system and simultaneously incubated with artificial oil emulsions composed of triacylglycerol and phospholipid. The results suggest that oil body proteins could spontaneously target to artificial oil emulsions in a co-translational mode. Incorporation of oleosin to artificial oil emulsions extensively protected a fragment of approximately 8 kDa from proteinase K digestion. In a competition experiment, in vitro translated caleosin and oleosin preferentially target to artificial oil emulsions instead of microsomal membranes. In oil emulsions with neutral phospholipids, relatively low protein targeting efficiency was observed. The targeting efficiency was substantially elevated when negatively charged phospholipids were supplemented to oil emulsions to mimic the native phospholipid composition of oil bodies. Mutated caleosin lacking various structural domains or subdomains was examined for its in vitro targeting efficiency. The results indicate that the subdomain comprising the proline knot motif is crucial for caleosin targeting to oil bodies. A model of direct targeting of oil-body proteins to maturing oil bodies is proposed.     

An aquatic perspective on the concepts of Ingestad relating plant nutrition to plant growth

Oil bodies are lipid storage organelles which have been analyzed biochemically due to the economic importance of oil seeds. Although oil bodies are structurally simple, the mechanisms involved in their formation and degradation remain controversial. At present, only two proteins associated with oil bodies have been described, oleosin and caleosin. Oleosin is thought to be important for oil body stabilization in the cytosol, although neither the structure nor the function of oleosin has been fully elucidated. Even less is known about caleosin, which has only recently been described [Chen et al. (1999) Plant Cell Physiol 40: 1079–1086; Næsted et al. (2000) Plant Mol Biol 44: 463–476]. Caleosin and caleosin-like proteins are not unique to oil bodies and are associated with an endoplasmatic reticulum subdomain in some cell types. Here we review the synthesis and degradation of oil bodies as they relate to structural and functional aspects of oleosin and caleosin.     

Protein and lipid composition analysis of oil bodies from two Brassica napus cultivars

Oil bodies were purified from mature seed of two Brassica napus crop cultivars, Reston and Westar. Purified oil body proteins were subjected to both 2-DE followed by LC-MS/MS and multidimensional protein identification technology. Besides previously known oil body proteins oleosin, putative embryo specific protein ATS1, (similar to caleosin), and 11-beta-hydroxysteroid dehydrogenase-like protein (steroleosin), several new proteins were identified in this study. One of the identified proteins, a short chain dehydrogenase/reductase, is similar to a triacylglycerol-associated factor from narrow-leafed lupin while the other, a protein annotated as a myrosinase associated protein, shows high similarity to the lipase/hydrolase family of enzymes with GDSL-motifs. These similarities suggest these two proteins could be involved in oil body degradation. Detailed analysis of the two other oil body components, polar lipids (lipid monolayer) and neutral lipids (triacylglycerol matrix) was also performed. Major differences were observed in the fatty acid composition of polar lipid fractions between the two B. napus cultivars. Neutral lipid composition confirmed erucic acid and oleic acid accumulation in Reston and Westar seed oil, respectively.     

Role of the proline knot motif in oleosin endoplasmic reticulum topology and oil body targeting.

An Arabidopsis oleosin was used as a model to study oleosin topology and targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteinase K mapping of the membrane topology. Oleosin maintains a conformation with a membrane-integrated hydrophobic domain flanked by N- and C-terminal domains located on the outer microsome surface. This is a unique membrane topology on the endoplasmic reticulum (ER). Three universally conserved proline residues within the "proline knot" motif of the oleosin hydrophobic domain were substituted by leucine residues. After in vitro translation, only minor differences in proteinase K protection could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on the ER was observed between the two oleosin forms. However, as an oleosin-beta-glucuronidase translational fusion, the proline knot variant failed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expressing oleosin-beta-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native form. Therefore, the proline knot motif is not important for ER integration and the determination of topology but is required for oil body targeting. The loss of the proline knot results in an intrinsic instability in the oleosin polypeptide during trafficking.     

Characterization of oil bodies in jelly fig achenes.

Thin-layer chromatography analysis revealed that the contents stored in oil bodies isolated from jelly fig (Ficus awkeotsang Makino) achenes were mainly neutral lipids (>90% triacylglycerols and approximately 5% diacylglycerols). Fatty acids released from the neutral lipids of achene oil bodies were highly unsaturated (62.65% alpha-linolenic acid, 18.24% linoleic acid, and 10.62% oleic acid). The integrity of isolated oil bodies was presumably maintained via electronegative repulsion and steric hindrance provided by their surface proteins. Immunological cross-recognition using antibodies against sesame oil-body proteins indicated that two oleosin isoforms and one caleosin were present in these oil bodies. MALDI-MS analyses confirmed that the three full-length cDNA fragments obtained by PCR cloning from maturing achenes encoded the two jelly fig oleosin isoforms and one caleosin identified by immunological screening.     

Two distinct steroleosins are present in seed oil bodies.

In addition to oleosin isoforms, three minor proteins, Sop1, 2 and 3 are present in sesame oil bodies. Genes encoding Sop1 and Sop2, named caleosin and steroleosin for their calcium and sterol-binding capacity, respectively, have been cloned recently. Blast sequence analysis of the first 32 N-terminal residues revealed that Sop3 was presumably a steroleosin-like protein homologous to Sop2. A putative cDNA clone of Sop3 was obtained by PCR, and subsequently confirmed by immunological recognition with antibodies against its over-expressed protein in Escherichia coli. Although Sop2 and Sop3, tentatively named steroleosin-A and -B, were found homologous, they could not be cross-recognized immunologically. Sequence comparison showed that these two steroleosins possessed a conserved NADP+ binding subdomain but a diverse sterol-binding subdomain of different size. Both steroleosins were progressively accumulated in maturing seeds but with different cumulating patterns. Dehydrogenase activity detected in their expressed proteins indicated that steroleosin-B might comparably possess a broader sterol selectivity and higher NADP+ specificity than steroleosin-A. Immunological cross-recognition implies that steroleosin-B is present in seed oil bodies of diverse species. A structural model of an oil-body was drawn with all its known essential constituents, and secondary structure organizations of the three classes of oil-body proteins were compared.     

A unique caleosin in oil bodies of lily pollen

In view of the recent isolation of stable oil bodies as well as a unique oleosin from lily pollen, this study examined whether other minor proteins were present in this lipid-storage organelle. Immunological cross-recognition using antibodies against three minor oil-body proteins from sesame suggested that a putative caleosin was specifically detected in the oil-body fraction of pollen extract. A cDNA fragment encoding this putative pollen caleosin, obtained by PCR cloning, was confirmed by immunodetection and MALDI-MS analyses of the recombinant protein over-expressed in Escherichia coli and the native form. Caleosin in lily pollen oil bodies seemed to be a unique isoform distinct from that in lily seed oil bodies.     

Identification of an oleosin-like gene in seagrass seeds

Objective

To investigate the oil body protein and function in seeds of mature seagrass, Thalassia hemprichii.

Results

Seeds of mature seagrass T. hemprichii when stained with a fluorescent probe BODIPY showed the presence of oil bodies in intracellular cells. Triacylglycerol was the major lipid class in the seeds. Protein extracted from seagrass seeds was subjected to immunological cross-recognition with land plant seed oil body proteins, such as oleosin and caleosin, resulting in no cross-reactivity. An oleosin-like gene was found in seagrass seeds. Next generation sequencing and sequence alignment indicated that the deduced seagrass seed oleosin-like protein has a central hydrophobic domain responsible for their anchoring onto the surface of oil bodies. Phylogenetic analysis showed that the oleosin-like protein was evolutionarily closer to pollen oleosin than to seed oleosins.

Conclusion

Oil body protein found in seagrass seeds represent a distinct class of land seed oil body proteins.

     

Computational identification and phylogenetic analysis of the oil-body structural proteins,oleosin and caleosin,in castor bean and flax

Oil bodies (OBs) are the intracellular particles derived from oilseeds. These OBs store lipids as a carbon resource, and have been exploited for a variety of industrial applications including biofuels. Oleosin and caleosin are the common OB structural proteins which are enabling biotechnological enhancement of oil content and OB-based pharmaceutical formations via stabilizing OBs. Although the draft whole genome sequence information for Ricinus communis L. (castor bean) and Linum usitatissimum L. (flax), important oil seed plants, is available in public database, OB-structural proteins in these plants are poorly indentified. Therefore, in this study, we performed a comprehensive bioinformatic analysis including analysis of the genome sequence, conserved domains and phylogenetic relationships to identify OB structural proteins in castor bean and flax genomes. Using comprehensive analysis, we have identified 6 and 15 OB-structural proteins from castor bean and flax, respectively. A complete overview of this gene family in castor bean and flax is presented, including the gene structures, phylogeny and conserved motifs, resulting in the presence of central hydrophobic regions with proline knot motif, providing an evolutionary proof that this central hydrophobic region had evolved from duplications in the primitive eukaryotes. In addition, expression analysis of L-oleosin and caleosin genes using quantitative real-time PCR demonstrated that seed contained their maximum expression, except that RcCLO-1 expressed maximum in cotyledon. Thus, our comparative genomics analysis of oleosin and caleosin genes and their putatively encoded proteins in two non-model plant species provides insights into the prospective usage of gene resources for improving OB-stability.     

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.     

Stable oil bodies sheltered by a unique caleosin in cycad megagametophytes

Stable oil bodies of smaller sizes and higher thermostability were isolated from mature cycad (Cycas revoluta) megagametophytes compared with those isolated from sesame seeds. Immunological cross-recognition revealed that cycad oil bodies contained a major protein of 27 kDa, tentatively identified as caleosin, while oleosin, the well-known structural protein, was apparently absent. Mass spectrometric analysis showed that the putative cycad caleosin possessed a tryptic fragment of 15 residues matching to that of a theoretical moss caleosin. A complete cDNA fragment encoding this putative caleosin was obtained by PCR cloning using a primer designed according to the tryptic peptide and another one designed according to a highly conservative region among diverse caleosins. The identification of this clone was subsequently confirmed by immunodetection and MALDI-MS analyses of its recombinant fusion protein over-expressed in Escherichia coli and the native form from cycad oil bodies. Stable artificial oil bodies were successfully constituted with triacylglycerol, phospholipid and the recombinant fusion protein containing the cycad caleosin. These results suggest that stable oil bodies in cycad megagametophytes are mainly sheltered by a unique structural protein caleosin.     

Plant lipid bodies and cell-cell signaling: A new role for an old organelle?

Plant lipid droplets are found in seeds and in post-embryonic tissues. Lipid droplets in seeds have been intensively studied, but those in post-embryonic tissues are less well characterised. Although known by a variety of names, here we will refer to all of them as lipid bodies (LBs). LBs are unique spherical organelles which bud off from the endoplasmic reticulum, and are composed of a single phospholipid (PL) layer enclosing a core of triacylglycerides. The PL monolayer is coated with oleosin, a structural protein that stabilizes the LB, restricts its size, and prevents fusion with adjacent LBs. Oleosin is uniquely present at LBs and is regarded as a LB marker. Although initially viewed as simple stores for energy and carbon, the emerging view is that LBs also function in cytoplasmic signalling, with the minor LB proteins caleosin and steroleosin in a prominent role. Apart from seeds, a variety of vegetative and floral structures contain LBs. Recently, it was found that numerous LBs emerge in the shoot apex of perennial plants during seasonal growth arrest and bud formation. They appear to function in dormancy release by reconstituting cell-cell signalling paths in the apex. As apices and orthodox seeds proceed through comparable cycles of dormancy and dehydration, the question arises to what degree LBs in apices share functions with those in seeds. We here review what is known about LBs, particularly in seeds, and speculate about possible unique functions of LBs in post-embryonic tissues in general and in apices in particular.     

Serine/threonine/tyrosine protein kinase phosphorylates oleosin, a regulator of lipid metabolic functions

Plant oils are stored in oleosomes or oil bodies, which are surrounded by a monolayer of phospholipids embedded with oleosin proteins that stabilize the structure. Recently, a structural protein, Oleosin3 (OLE3), was shown to exhibit both monoacylglycerol acyltransferase and phospholipase A(2) activities. The regulation of these distinct dual activities in a single protein is unclear. Here, we report that a serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using bimolecular fluorescence complementation analysis, we demonstrate that this kinase interacts with OLE3 and that the fluorescence was associated with chloroplasts. Oleosin-green fluorescent protein fusion protein was exclusively associated with the chloroplasts. Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase and increased phospholipase A(2) activities. Moreover, phosphatidylcholine and diacylglycerol activated oleosin phosphorylation, whereas lysophosphatidylcholine, oleic acid, and Ca(2+) inhibited phosphorylation. In addition, recombinant peanut (Arachis hypogaea) kinase was determined to predominantly phosphorylate serine residues, specifically serine-18 in OLE3. Phosphorylation levels of OLE3 during seed germination were determined to be higher than in developing peanut seeds. These findings provide direct evidence for the in vivo substrate selectivity of the dual-specificity kinase and demonstrate that the bifunctional activities of oleosin are regulated by phosphorylation.     

Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of oil-body formation in seeds

Two genomic clones, encoding isoforms A and B of the 24 kDa soybean oleosin and containing 5 kbp and 1 kbp, respectively, of promoter sequence, were inserted separately into rapeseed plants. T₂ seeds from five independent transgenic lines, three expressing isoform A and two expressing isoform B, each containing one or two copies of the transgene, were analysed in detail. In all five lines, the soybean transgenes exhibited the same patterns of mRNA and protein accumulation as the resident rapeseed oleosins, i.e. their expression was absolutely seed-specific and peaked at the mid-late stages of cotyledon development. The 24 kDa soybean oleosin was targeted to and stably integrated into oil bodies, despite the absence of a soybean partner isoform. The soybean protein accumulated in young embryos mainly as a 23 kDa polypeptide, whereas a 24 kDa protein predominated later in development. The ratio of rapeseed:soybean oleosin in the transgenic plants was about 5:1 to 6:1, as determined by SDS-PAGE and densitometry. Accumulation of these relatively high levels of soybean oleosin protein did not affect the amount of endogenous rapeseed oleosin. Immunoblotting studies showed that about 95% of the recombinant soybean 24 kDa oleosin (and the endogenous 19 kDa rapeseed oleosin) was targeted to oil bodies, with the remainder associated with the microsomal fraction. Sucrose density-gradient centrifugation showed that the oleosins were associated with a membrane fraction of buoyant density 1.10–1.14 g ml^?1, which partially overlapped with several endoplasmic reticulum (ER) markers. Unlike oleosins associated with oil bodies, none of the membrane-associated oleosins could be immunoprecipitated in the presence of protein A-Sepharose, indicating a possible conformational difference between the two pools of oleosin. Complementary electron microscopy-immunocytochemical studies of transgenic rapeseed revealed that all oil bodies examined could be labelled with both the soybean or rapeseed anti-oleosin antibodies, indicating that each oil body contained a mixed population of soybean and rapeseed oleosins. A small but significant proportion of both soybean and rapeseed oleosins was located on ER membranes in the vicinity of oil bodies, but none were detected on the bulk ER cisternae. This is the first report of apparent targeting of oleosins via ER to oil bodies in vivo and of possible associated conformational/ processing changes in the protein. Although oil-body formation per se can occur independently of oleosins, it is proposed that the relative net amounts of oleosin and oil accumulated during the course of seed development are a major determinant of oil-body size in desiccation-tolerant seeds.