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.     

Oleosins prevent oil-body coalescence during seed imbibition as suggested by a low-temperature scanning electron microscope study of desiccation-tolerant and -sensitive oilseeds

In order to clarify further the physiological role of oleosins in seed development, we characterized the oil-body proteins of several oilseeds exhibiting a range of desiccation sensitivities from the recalcitrant (Theobroma cacao L., Quercus rubra L.), intermediate (Coffea arabica L., Azadirachta indica A. Juss.) and orthodox categories (Sterculia setigera Del., Brassica napus L.). The estimated ratio of putative oleosins to lipid in oil bodies of Q. rubra was less than 5% of the equivalent values for rapeseed oil bodies. No oleosin was detected in T. cacao oil bodies. In A. indica cotyledons, oil bodies contained very low amounts of putative oleosins. Oil bodies both from C. arabica and S. setigera exhibited a similar ratio of putative oleosins to lipid as found in rapeseed. In C. arabica seeds, the central domain of an oleosin was partially sequenced. Using a low temperature field-emission scanning electron microscope, the structural stability of oil bodies was investigated in seeds after drying, storage in cold conditions and rehydration. Despite the absence or relative dearth of oleosins in desiccation-sensitive, recalcitrant oilseeds, oil bodies remained relatively stable after slow or fast drying. In A. indica seeds exposed to a lethal cold storage treatment, no significant change in oil-body sizes was observed. In contrast, during imbibition of artificially dried seeds containing low amounts of putative oleosins, the oil bodies fused to form large droplets, resulting in the loss of cellular integrity. No damage to the oil bodies occurred in imbibed seeds of Q. rubra, C. arabica and S. setigera. Thus the rehydration phase appears to be detrimental to the stability of oil bodies when these are present in large amounts and are lacking oleosins. We therefore suggest that one of the functions of oleosins in oilseed development may be to stabilize oil bodies during seed imbibition prior to germination. Received: 22 April 1997 / Accepted: 5 June 1997     

A novel role for oleosins in freezing tolerance of oilseeds in Arabidopsis thaliana

Oil bodies in seeds of higher plants are surrounded with oleosins. Here we demonstrate a novel role for oleosins in protecting oilseeds against freeze/thaw-induced damage of their cells. We detected four oleosins in oil bodies isolated from seeds of Arabidopsis thaliana , and designated them OLE1, OLE2, OLE3 and OLE4 in decreasing order of abundance in the seeds. For reverse genetics, we isolated oleosin-deficient mutants ( ole1 , ole2 , ole3 and ole4 ) and generated three double mutants ( ole1 ole2 , ole1 ole3 and ole2 ole3 ). Electron microscopy showed an inverse relationship between oil body sizes and total oleosin levels. The double mutant ole1 ole2 , which had the lowest levels of oleosins, had irregular enlarged oil-containing structures throughout the seed cells. Germination rates were positively associated with oleosin levels, suggesting that defects in germination are related to the expansion of oil bodies due to oleosin deficiency. We found that freezing followed by imbibition at 4°C abolished seed germination of single mutants ( ole1 , ole2 and ole3 ), which germinated normally without freezing treatment. The treatment accelerated the fusion of oil bodies and the abnormal-positioning and deformation of nuclei in ole1 seeds, which caused seed mortality. In contrast, ole1 seeds that had undergone freezing treatment germinated normally when incubated at 22°C instead of 4°C, because degradation of oils abolished the acceleration of fusion of oil bodies during imbibition. Taken together, our findings suggest that oleosins increase the viability of over-wintering oilseeds by preventing abnormal fusion of oil bodies during imbibition in the spring.     

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.     

Oleosins in the gametophytes of Pinus and Brassica and their phylogenetic relationship with those in the sporophytes of various species

Oleosins, which are structural proteins on the surface of intracellular oil bodies, have been found in the sporophytic seeds of angiosperms. Here, we report an oleosin from the female gametophyte of gymnosperm Pinus ponderosa Laws, seed and another oleosin from the male gametophyte of Brassica napus L. With the pine seed gametophyte, we identified two putative oleosins of 15 and 10 kDa, which are similar to the oleosins in angiosperm seeds in terms of their presence in the oil bodies in massive quantity. The complete sequence of the cDNA encoding the gametophytic 15-kDa oleosin was obtained, and it has a predicted amino-acid sequence similar to those of oleosins in angiosperm sporophytic seeds. A Brassica napus pollen cDNA sequence, which was reported earlier, would encode an amino-acid sequence somewhat similar to those of seed oleosins. We tested if the dissimilarity signifies a substantially different oleosin in the Brassica male gametophyte or an analytic error. By direct sequencing of a polymerase chain reaction (PCR)-amplified fragment of genomic DNA, we obtained evidence showing that this reported dissimilarity is likely to have arisen from a sequencing error. Our predicted sequence of the Brassica pollen oleosin has all the structural characteristics of seed oleosins. A phylogenic tree of 20 oleosins, including those from sporophytic and gametophytic tissues of angiosperm and gymnosperm, was constructed based on their amino-acid sequences. We discuss the evolution of oleosins, and conclude that oleosins are ancient proteins with multiple lineages whose root cannot be determined at this time.Abbreviations PCR polymerase chain reaction - TAG triacylglycerols This work was supported by USDA grant 91-01439 (AHCH). We thank Dr. Mike Lassner of Calgene, Inc., (Davis, Calif., USA) for providing us with the unpublished jojoba oleosin amino acid sequence.     

Oleosin genes in maize kernels having diverse oil contents are constitutively expressed independent of oil contents

In seeds, the subcellular storage oil bodies have a matrix of oils (triacylglycerols) surrounded by a layer of phospholipids embedded with abundant structural proteins called oleosins. We used two maize (Zea mays L.) strains having diverse kernel (seed) oil contents to study the effects of varying the oil and oleosin contents on the structure of the oil bodies. Illinois High Oils (IHO, 15% w/w oils) and Illinois Low Oils (ILO, 0.5%) maize kernels were the products of breeding for diverse oil contents for about 100 generations. In both maize strains, although the genes for oil synthesis had apparently been modified drastically, the genes encoding oleosins appeared to be unaltered, as revealed by Southern blot analyses of the three oleosin genes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with immunoblotting of the oleosins. In addition, both strains contained the same three oleosin isoforms of a defined proportion, and both accumulated oils and oleosins coordinately. Oleosins in both strains were restricted to the oil bodies, as shown by analyses of the various subcellular fractions separated by sucrosedensity-gradient centrifugation. Electron microscopy of the embryos and the isolated organelles revealed that the oil bodies in IHO were larger and had a spherical shape, whereas those in ILO were smaller and had irregular shapes. We conclude that in seeds, oleosin genes are expressed independent of the oil contents, and the size and shape of the oil bodies are dictated by the ratio of oils to oleosins synthesized during seed maturation. The extensive breeding for diverse oil contents has not altered the apparent mechanism of oil-body synthesis and the occurrence of hetero-dimer or -multimer of oleosin isoforms on the oil bodies.Abbreviations IHO Illinois High Oils - ILO Illinois Low Oils This work was supported by a USDA NRICGP grant. We thank Dr. J.W. Dudley of the University of Illinois for the IHO and ILO maize kernels, and Dr. W. Thomson for discussion on the stereological method.     

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.     

Surface structure and properties of plant seed oil bodies

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

Oleosin fusion expression systems for the production of recombinant proteins

For the production of recombinant proteins, product purification is potentially difficult and expensive. Plant oleosins are capable of anchoring onto the surface of natural or artificial oil bodies. The oleosin fusion expression systems allow products to be extracted with oil bodies. In vivo, oleosin fusions are produced and directly localized to natural oil bodies in transgenic plant seeds. Via the oleosin fusion technology the thrombin inhibitor hirudin has been successfully produced and commercially used in Canada. In vitro, artificial oil bodies have been used as “carriers” for the recombinant proteins expressed in transformed microbes. In this article, plant oleosins, strategies and limitations of the oleosin fusion expression systems are summarized, alongside with progress and applications. The oleosin fusion expression systems reveal an available way to produce recombinant biopharmaceuticals at large scale.     

Light-enhanced oil body mobilization in sunflower seedlings accompanies faster protease action on oleosins

Germination of sunflower (Helianthus annuus L.) seeds in light is accompanied by greater susceptibility of oil bodies for lipolytic action following enhanced oleosin mobilization than in the dark. The 16- and 17.5-kDa oleosins are mobilized within the first 4 d of seedling growth in light, whereas 20-kDa oleosin remains detectable. Oleosin mobilization is slower in the dark and all three oleosins remain detectable until 7 d of seedling growth. Light-grown seedlings show higher activity of fatty acyl-ester hydrolase (EC 3.1.1.1) mainly due to greater expression of its major (40–50 kDa) isoforms. Increased susceptibility of oil bodies to lipolytic action in light-grown seedlings shows a correlation with higher activity of a cytosolic 65-kDa protease, oleosin mobilization and relative accumulation of 11-kDa protease-protected fragment. These observations support the view that the expression of 65-kDa protease is enhanced in light and it could be considered as a component of light-enhanced lipolysis.     

Protein composition of oil bodies in Arabidopsis thaliana ecotype WS

Till now, only scattered data are available in the literature, which describes the protein content of plant oil bodies. Especially, the proteins closely associated with the model plant Arabidopsis thaliana oil bodies have never been previously purified and characterized. Oil bodies have been purified using flotation techniques, combined with incubations under high salt concentration, in the presence of detergents and urea in order to remove non-specifically trapped proteins. The identity and integrity of the oil bodies have been characterized. Oil bodies exhibited hydrodynamic diameters close to 2.6 μm, and a ratio fatty acid-protein content near 20. The proteins composing these organelles were extracted, separated by SDS-PAGE, digested by trypsin, and their peptides were subsequently analyzed by nano-chromatography–mass spectrometry (nano-LC–MS/MS). This led to the identification of a limited number of proteins: four different oleosins, ATS1, a protein homologous to calcium binding protein, a 11-β-hydroxysteroid dehydrogenase-like protein, a probable aquaporin and a glycosylphosphatidylinositol-anchored protein with no known function. The two last proteins were till now never identified in plant oil bodies. Structural proteins (oleosins) represented up to 79% of oil body proteins and the 18.5 kDa oleosin was the most abundant among them.     

Stable oil bodies sheltered by a unique oleosin in lily pollen

Stable oil bodies were purified from mature lily (Lilium longiflorum Thunb.) pollen. The integrity of pollen oil bodies was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Immunodetection revealed that a major protein of 18 kDa was exclusively present in pollen oil bodies and massively accumulated in late stages of pollen maturation. According to mass spectrometric analyses, this oil body protein possessed a tryptic fragment of 13 residues matching that of a theoretical rice oleosin. A complete cDNA fragment encoding this putative oleosin was obtained by PCR cloning with primers derived from its known 13-residue sequence. Sequence analysis as well as immunological non-cross-reactivity suggests that this pollen oleosin represents a distinct class in comparison with oleosins found in seed oil bodies and tapetum. In pollen cells observed by electron microscopy, oil bodies were presumably surrounded by tubular membrane structures, and encapsulated in the vacuoles after germination. It seems that pollen oil bodies are mobilized via a different route from that of glyoxysomal mobilization of seed oil bodies after germination.     

Structural requirements of oleosin domains for subcellular targeting to the oil body.

We have investigated the protein domains responsible for the correct subcellular targeting of plant seed oleosins. We have attempted to study this targeting in vivo using "tagged" oleosins in transgenic plants. Different constructs were prepared lacking gene sequences encoding one of three structural domains of natural oleosins. Each was fused in frame to the Escherichia coli uid A gene encoding beta-glucuronidase (GUS). These constructs were introduced into Brassica napus using Agrobacterium-mediated transformation. GUS activity was measured in washed oil bodies and in the soluble protein fraction of the transgenic seeds. It was found that complete Arabidopsis oleosin-GUS fusions undergo correct subcellular targeting in transgenic Brassica seeds. Removal of the C-terminal domain of the Arabidopsis oleosin comprising the last 48 amino acids had no effect on overall subcellular targeting. In contrast, loss of the first 47 amino acids (N terminus) or amino acids 48 to 113 (which make up a lipophilic core) resulted in impaired targeting of the fusion protein to the oil bodies and greatly reduced accumulation of the fusion protein. Northern blotting revealed that this reduction is not due to differences in mRNA accumulation. Results from these measurements indicated that both the N-terminal and central oleosin domain are important for targeting to the oil body and show that there is a direct correlation between the inability to target to the oil body and protein stability.     

Use of oil bodies and oleosins in recombinant protein production and other biotechnological applications

Oil bodies obtained from oilseeds have been exploited for a variety of applications in biotechnology in the recent past. These applications are based on their non-coalescing nature, ease of extraction and presence of unique membrane proteins—oleosins. In suspension, oil bodies exist as separate entities and, hence, they can serve as emulsifying agent for a wide variety of products, ranging from vaccines, food, cosmetics and personal care products. Oil bodies have found significant uses in the production and purification of recombinant proteins with specific applications. The desired protein can be targeted to oil bodies in oilseeds by affinity tag or by fusing it directly to the N or C terminal of oleosins. Upon targeting, the hydrophobic domain of oleosin embeds into the TAG matrix of oil body, whereas the protein fused with N and/or C termini is exposed on the oil body surface, where it acquires correct confirmation spontaneously. Oil bodies with the attached foreign protein can be separated easily from other cellular components. They can be used directly or the protein can be cleaved from the fusion. The desired protein can be a pharmaceutically important polypeptide (e.g. hirudin, insulin and epidermal growth factor), a neutraceutical polypeptide (somatotropin), a commercially important enzyme (e.g. xylanase), a protein important for improvement of crops (e.g. chitinase) or a multimeric protein. These applications can further be widened as oil bodies can also be made artificially and oleosin gene can be expressed in bacterial systems. Thus, a protein fused to oleosin can be expressed in Escherichia coli and after cell lysis it can be incorporated into artificial oil bodies, thereby facilitating the extraction and purification of the desired protein. Artificial oil bodies can also be used for encapsulation of probiotics. The manipulation of oleosin gene for the expression of polyoleosins has further expanded the arena of the applications of oil bodies in biotechnology.     

A class of amphipathic proteins associated with lipid storage bodies in plants. Possible similarities with animal serum apolipoproteins

The lipid-storing tissues of plants contain many small (0.2-1 microns) lipid (normally triacylglycerol) droplets which are surrounded and stabilized by a mixed phospholipid and protein annulus. The proteinaceous components of the lipid storage bodies are termed oleosins and are not associated with any other cellular structures. The major oleosins of rapeseed and radish have been isolated by preparative SDS-PAGE and are respectively classes of 19 kDa and 20 kDa proteins. Both protein classes were N-terminally blocked for direct sequencing, but were partially sequenced following limited proteolytic digestion. The major rapeseed oleosin was made up of at least two 19 kDa polypeptides, termed nap-I and nap-II, which have closely related but different amino acid sequences. A single 20 kDa oleosin, termed rad-I, was found in radish. A near full length cDNA clone for a major rapeseed oleosin was sequenced and found to correspond almost exactly to the sequence of nap-II. The sequences of nap-I and rad-I show very close similarity to one another, as do the sequences of nap-II and the previously determined sequence for the major oleosin from maize. All four oleosins have a large central hydrophobic domain flanked by polar N- and C-terminal domains. Secondary structure predictions for the four oleosins are similar and a novel model is proposed based on a central hydrophobic beta-strand region flanked by an N-terminal polar alpha-helix and a C-terminal amphipathic alpha-helix. The possibility that oleosins exhibit structural and functional similarities with some animal apolipoproteins is discussed.     

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

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

Identification, subcellular localization, and developmental studies of oleosins in the anther of Brassica napus

mRNAs encoding putative oleosins have been detected in the tapetum of developing anthers in Brassica and Arabidopsis, but the authentic proteins have not been previously documented. Antibodies against a synthetic 15-residue polypeptide that represents a portion of the putative tapetum oleosins encoded by two cloned Brassica napus genes were raised. Using these antibodies for immunoblotting after SDS-PAGE of the sporophytic extracts of B. napus developing anthers, two oleosins of ～ 48 and 45 kDa were detected. These two oleosins were judged to be the putative oleosins encoded by cloned Brassica genes because of their identical N-terminal sequences. The two oleosins were present in the anthers only during the developmental stage when the tapetum cells were packed with organelles. A fraction of low-density organelles was isolated from the developing anthers by flotation centrifugation. The fraction contained plastoglobule-filled plastids and lipid-containing particles. The structures of these two isolated organelles were similar to those in situ in the tapetum cells. Of subcellular fractions of the anther homogenate, the two oleosins were present exclusively in the low-density organelle fraction. They were absent in the surface fractions of the developing microspores and the mature pollen, although fragmented oleosin molecules were earlier reported to be present on the pollen. By immunocytochemistry, immunogold particles were found largely on the periphery of the plastoglobuli inside the plastids in the tapetum cells. The antibodies also detected oleosins on the surface of storage oil bodies inside the maturing microspores. Apparently, the gametophytic microspore oil-body oleosins share common epitopes at the generally non-conserved C-terminal domain with the sporophytic tapetum oleosins.