Evidence for the probable oil body association of a thiol-protease, leading to oleosin degradation in sunflower seedling cotyledons

The activity of a 65 kDa, cytosolic protease from sunflower seedling cotyledons coincides with the degradation of oleosins during seed germination. Further investigations carried out in this laboratory have demonstrated the probable association of a thiol-protease with oil bodies, leading to gradual degradation of oleosins during seedling growth. Evidence to this effect have been brought out through zymographic detection of protease activity from oil bodies, degradation of oleosins by electrophoretically eluted protease from the seedling cotyledons and inhibition of protease activity by thiol-protease inhibitor, such as N-ethylmaleimide (NEM). In addition to these biochemical evidence, visualization of thiol-protease activity has also been achieved by a novel fluorescence microscopic method and confocal imaging. It involves the uptake and binding of a fluorogenic thiol-protease inhibitor (fluorescein mercuric acetate, FMA) at the intracellular thiol-protease activity sites in protoplasts, leading to fluorescence emission at 523 nm following excitation at 499 nm. Maximum protease activity is observed in 4-d-old seedling cotyledons, coinciding with the phase of active triacylglycerol (TAGs) hydrolysis. All these observations provide evidence for the expression of the said thiol-protease activity on the oil body surface, leading to gradual proteolysis of oleosins during seed germination.     

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.     

Differential sensitivity of oleosins to proteolysis during oil body mobilization in sunflower seedlings

Until now, there has been no conclusive demonstration of any in vivo oleosin degradation at the early stages of oil body mobilization. The present work on sunflower (Helianthus annuus L.) has demonstrated limited oleosin degradation during seed germination. Seedling cotyledon homogenization in Tris-urea buffer, followed by SDS-PAGE, revealed three oleosins (16, 17.5 and 20 kDa). Incubation of oil bodies with total soluble protein from 4-day-old seedlings resulted in oleosin degradation. In vitro and in vivo degradation of the 17.5-kDa oleosin was faster than the other two, indicating its greater susceptibility to proteolysis. Oleosin degradation by the total soluble protein resulted in a transient 14.5-kDa polypeptide, followed by an 11-kDa protease-protected fragment, which appeared post-germinatively and accumulated corresponding to increased rate of lipid mobilization. A 65-kDa protease, active at pH 7.5-9.5, was zymographically detected in the total soluble protein. Its activity increased along with in vivo accumulation of the protease-protected fragment during seed germination and accompanying lipid mobilization. Protease-treated oil bodies were more susceptible to maize lipase action. Differential proteolytic sensitivity of different oleosins in the oil body membranes could be a determinant of oil body longevity during seed germination.     

Spatial and temporal changes in lipase activity sites during oil body mobilization in protoplasts from sunflower seedling cotyledons

Hydrolysis of triacylglycerols (TAGs) catalyzed by lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) action, is the principal biochemical event during oil body mobilization in germinating oilseeds. Employing a fluorescence microscopic technique developed in the author’s laboratory, a shift in the intracellular lipase activity has been demonstrated in the protoplasts of sunflower seedling cotyledons during seed germination. Lipase activity is primarily confined to protein storage vacuoles (PSVs) in 1 d old seedling cotyledons. At 2 d old stage, a relocalization of lipase activity begins and activity can be observed both on PSVs and oil bodies. At later stages of development (3–6 d), smaller PSVs coalesce into a large vegetative vacuole devoid of lipase activity. During this phase, lipase activity is confined to oil bodies only and maximum activity is detected in 4 d old seedlings, coinciding with maximum rate of lipolysis. Thus, present investigations on protoplasts from seedling cotyledons provide evidence for intracellular shift in lipase activity to sites of TAG hydrolysis (oil bodies) and also show a structural and functional reorganization of PSVs.     

Localization of seed oil body proteins in tobacco protoplasts reveals specific mechanisms of protein targeting to leaf lipid droplets

Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy. Our results indicated clear differences in the endocellular localization of the three proteins. Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets (LD), whereas steroleosin, characterized by an N-terminal oil body anchoring domain, was mainly retained in the endoplasmic reticulum (ER). Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosin-green fluorescent protein (GFP) peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein (BiP), were recovered. Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered. Finally, only oleosin- and caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids. Taken together, our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.     

Heterologous expression of AtClo1, a plant oil body protein, induces lipid accumulation in yeast

Proteomic approaches on lipid bodies have led to the identification of proteins associated with this compartment, showing that, rather than the inert fat depot, lipid droplets appear as complex dynamic organelles with roles in metabolism control and cell signaling. We focused our investigations on caleosin [ Arabidopsis thaliana caleosin 1 (AtClo1)], a minor protein of the Arabidopsis thaliana seed lipid body. AtClo1 shares an original triblock structure, which confers to the protein the capacity to insert at the lipid body surface. In addition, AtClo1 possesses a calcium-binding domain. The study of plants deficient in caleosin revealed its involvement in storage lipid degradation during seed germination. Using Saccharomyces cerevisiae as a heterologous expression system, we investigated the potential role of AtClo1 in lipid body biogenesis and filling. The green fluorescent protein-tagged protein was correctly targeted to lipid bodies. We observed an increase in the number and size of lipid bodies. Moreover, transformed yeasts accumulated more fatty acids (+46.6%). We confirmed that this excess of fatty acids was due to overaccumulation of lipid body neutral lipids, triacylglycerols and steryl esters. We showed that the original intrinsic properties of AtClo1 protein were sufficient to generate a functional lipid body membrane and to promote overaccumulation of storage lipids in yeast oil bodies.     

Protein mobilization in germinating mung bean seeds involves vacuolar sorting receptors and multivesicular bodies

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination.     

Cytolocalization of ion-channel antagonist binding sites in sunflower protoplasts during the early steps of culture

Summary A fluorescently labeled phenylalkylamine (PAA), DM-Bodipy PAA, was used as a probe for in vivo labeling of PAA binding sites in sunflower hypocotyl protoplasts in culture. Verapamil, a PAA known as a calcium channel antagonist in plants, lowers the division rate of sunflower protoplasts in culture. The binding specificity of DM-Bodipy PAA was established at various culture times by competition experiments with (–)bepridil. Studies on the Cytolocalization of DM-Bodipy PAA binding sites by confocal imaging showed that in freshly isolated protoplasts PAA receptors were organized into clusters uniformly distributed over the cell surface. During protoplast culture, the fluorescence labeling pattern evolved from peripheral to cytoplasmic. After a few days of culture, PAA binding sites were present inside the cell, along cytoplasmic strands, on the membrane of vesicles and vacuoles, and were highly concentrated around the nucleus. After protoplast division, the labeling was mainly restricted to a zone close to the new cell wall. On symmetrical division, binding sites were uniformly distributed on both sides of the new cell wall. With asymmetrical division, binding sites were concentrated in a ring surrounding the new cell plate.Abbreviations PAA phenylalkylamine - DHP dihydropyridine - FDA fluorescein diacetate     

Temporal and spatial analysis of lipid accumulation, oleosin expression and fatty acid partitioning during seed development in sunflower (Helianthus annuus L.)

Biochemical and fluorescence microscopic imaging approach has been adopted to investigate the accumulation of oil bodies at specific stages of seed development in Helianthus annuus L. cv. Morden. Seed filling in sunflower is marked with a rapid accumulation of proteins and lipids upto 30 DAA, after which protein accumulation declines whereas lipids continue to accumulate. Earliest signs of lipid accumulation are evident as early as during globular stage of embryo development. Spatially, a developing seed exhibits enhanced lipid deposition in peripheral cells. Oil body biogenesis is observed as early as 10 DAA, as is evident from the fluorescence microscopic detection of Nile red-positive entities in the protoplasts. To begin with, expression of one of the oleosin (the principal oil body membrane proteins) isoforms (16 kDa), is slower than the other two (17.5 and 20 kDa). Fatty acid composition of oil body lipids is quite similar to that of total seed lipids. An enhanced accumulation of linoleic acid is evident during later stages of seed filling. The proportion of major saturated fatty acids, palmitic (16:0) and stearic (18:0), however, do not alter much during the later phases of seed development. Present findings provide new information on oil body development, lipid accumulation and fatty acid composition, for a better understanding of the phasing of physiological and biochemical events associated with oilseed development.     

Confocal microscopy: a tool to visualise differentiation of stem cells into cardiomyocytes

Confocal microscopy offers important advantages compared to conventional epifluorescence microscopy. It works as an "optical microtome" leading to a accurate image resolution of a defined focal plane. Furthermore, the addition of a Nipkow disk on the confocal microscope greatly accelerates the image acquisition, up to 30 frames per second. Nevertheless, the software-assisted mathematical restoration of images acquired using a wide-field microscope allows to get images with a resolution similar to the one obtained in confocal microscopy. These imaging technologies allowed us to monitor on line cardiac differentiation of murine embryonic stem (ES) cells within 3D structures called embryoid bodies. The high rate acquisition of images using the confocal microscope equipped with a Nipkow disk allows to monitor calcium spiking in differentiating cardiomyocytes within embryoid bodies.     

甘蓝型油菜油体数量及面积之和与含油量的相关性

利用荧光染料尼罗红染色和激光扫描共聚焦显微观察技术, 建立了油菜油体观察或生物体内中性脂类物质定性鉴定的研究体系。对高油品种宁油14号、宁油18号、ZH-088和低油品种ZL-366、NjY008、Westar共6个甘蓝型油菜品种子叶 贮藏细胞内的油体进行了观察。研究发现: 油菜种子成熟过程中, 油体从着色不明显的小颗粒, 逐渐发育形成着色清晰的球状大油体。种子成熟干燥后, 油体间很少发生聚合。在成熟干燥的种子中, 油体集中分布于子叶贮藏细胞中央, 呈椭圆形或不规则形状, 较少为圆形。通过研究种子内油体与含油量的关系, 发现高油品种组与低油品种组之间在单个子叶贮藏细胞内油体数量和截面积之和存在明显差异, 而在高油品种组内或低油品种组内的差异不明显。结果显示, 油菜种子细胞中油体的数量和总面积与含油量之间存在正相关, 可作为高油分材料的选择依据。     

甘蓝型油菜油体数量及面积之和与含油量的相关性

利用荧光染料尼罗红染色和激光扫描共聚焦显微观察技术,建立了油菜油体观察或生物体内中性脂类物质定性鉴定的研究体系。对高油品种宁油14号、宁油18号、ZH-088和低油品种ZL-366、NjY008、Westar共6个甘蓝型油菜品种子叶贮藏细胞内的油体进行了观察。研究发现：油菜种子成熟过程中,油体从着色不明显的小颗粒,逐渐发育形成着色清晰的球状大油体。种子成熟干燥后,油体间很少发生聚合。在成熟干燥的种子中,油体集中分布于子叶贮藏细胞中央,呈椭圆形或不规则形状,较少为圆形。通过研究种子内油体与含油量的关系,发现高油品种组与低油品种组之间在单个子叶贮藏细胞内油体数量和截面积之和存在明显差异,而在高油品种组内或低油品种组内的差异不明显。结果显示,油菜种子细胞中油体的数量和总面积与含油量之间存在正相关,可作为高油分材料的选择依据。     

Interactions between proteins implicated in exocytosis and voltage-gated calcium channels

Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium influx through P/Q-type or N-type calcium channels. Purification of N-type channels from rat brain synaptosomes initially suggested molecular interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experiments were consistent with the hypothesis that both N- and P/Q-type calcium channels, but not L-type channels, are associated with the 7S complex containing syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immunofluorescence confocal microscopy at the frog neuromuscular junction confirmed that calcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experiments with recombinant proteins were performed to map synaptic protein interaction sites on the alpha 1A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the alpha 1A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium influx close to the channel mouth. Cysteine string proteins (CSPs) contain a J-domain characteristic of molecular chaperones that cooperate with Hsp70. They are located on synaptic vesicles and thought to be involved in modulating the activity of presynaptic calcium channels. CSPs were found to bind to the same domain of the calcium channel as synaptotagmin, and also to associate with VAMP. CSPs may act as molecular chaperones in association with Hsp70 to direct assembly or dissociation of multiprotein complexes at the calcium channel.     

Oleosin expression and trafficking during oil body biogenesis in tobacco leaf cells

We have established a versatile method for studying the interaction of the oleosin gene product with oil bodies during oil body biogenesis in plants. Our approach has been to transiently express a green fluorescent protein (GFP)-tagged Arabidopsis oleosin gene fusion in tobacco leaf cells containing bona fide oil bodies and then to monitor oleosin-GFP expression using real-time confocal laser scanning microscopy. We show that normally non-oil-storing tobacco leaf cells are able to synthesize and then transport oleosin-GFP fusion protein to leaf oil bodies. Synthesis and transport of oleosin-GFP fusion protein to oil bodies occurred within the first 6 h posttransformation. Oleosin-GFP fusion protein exclusively associated with the endoplasmic reticulum and was trafficked in a Golgi-independent manner at speeds approaching 0.5 microm sec(-1) along highly dynamic endoplasmic reticulum positioned over essentially static polygonal cortical endoplasmic reticulum. Our data indicate that oil body biogenesis can occur outside of the embryo and that oleosin-GFP can be used to monitor early events in oil body biogenesis in real-time.     

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.     

Application of YO-PRO-1 as an Epifluorescent Dye for in situ Detection of Small Amount DNA in Plant Cells

i.e. plastid and mitochondrial DNA in the plant cells such as the sperm cell of Jasminum nudiflorum, the generative cell of Pharbitis lim-bata, the cultured cell of Nicotiana tabacum and the root cell of Vicia faba with epifluorescence microscopy and laser confocal microscopy using YO-PRO-1 as a fluorescent dye. The excitation for YO-PRO-1 was blue light in epifluorescence microscopy and 488 nm Kr/Ar ion laser in confocal microscopy. Dimorphic epifluorescent spots that corresponded plastid DNA and mitochondrial DNA were distinctly detected in the cells of each species examined. In this report, we introduce YO-PRO-1 as a new epifluorescent dye for successful in situ detection of small amount DNA in plant live cells and cell sections with perticular emphasis on the importance of sample preparation. Received 10 November 1998/ Accepted in revised form 13 January 1999     

Effects of Sunflower Oil on Tomato Powdery Mildew Caused by Oidium neolycopersici

When tomato leaves were sprayed with 0.1% emulsified canola oil, corn oil, grape seed oil, peanut oil, safflower oil, soya bean oil or sunflower oil, the severity of powdery mildew caused by Oidium neolycopersici was greatly reduced. Among these edible oils tested, sunflower oil was the most effective in the control of powdery mildew. When sprayed with 0.5% sunflower oil, powdery mildew on tomato leaves was reduced to a negligible level. Sunflower oil applied to halves of upper leaf surface did not induce resistance against the pathogen in the non‐treated halves. When applied to halves of lower leaf surface, it also failed to reduce the severity of powdery mildew on the upper leaf surface right above the treated area indicating that control of the powdery mildew by sunflower oil did not result from activation of host defence mechanisms. Scanning electron microscopy showed that control of powdery mildew with sunflower oil resulted mainly from the inhibition of conidial germination and suppression of mycelial growth of the pathogen.     

Temporal association of Ca(2+)-dependent protein kinase with oil bodies during seed development in Santalum album L.: its biochemical characterization and significance

Calcium-dependent protein kinase (CDPK) is expressed in sandalwood (Santalum album L.) seeds under developmental regulation, and it is localized with spherical storage organelles in the endosperm [Anil et al. (2000) Plant Physiol. 122: 1035]. This study identifies these storage organelles as oil bodies. A 55 kDa protein associated with isolated oil bodies, showed Ca(2+)-dependent autophosphorylation and also cross-reacted with anti-soybean CDPK. The CDPK activity detected in the oil body-protein fraction was calmodulin-independent and sensitive to W7 (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide) inhibition. Differences in Michaelis Menton kinetics, rate of histone phosphorylation and sensitivity to W7 inhibition between a soluble CDPK from embryos and the oil body-associated CDPK of endosperm suggest that these are tissue-specific isozymes. The association of CDPK with oil bodies of endosperm was found to show a temporal pattern during seed development. CDPK protein and activity, and the in vivo phosphorylation of Ser and Thr residues were detected strongly in the oil bodies of endosperm from maturing seed. Since oil body formation occurs during seed maturation, the observations indicate that CDPK and Ca(2+) may have a regulatory role during oil accumulation/oil body biogenesis. The detection of CDPK-protein and activity in oil bodies of groundnut, sesame, cotton, sunflower, soybean and safflower suggests the ubiquity of the association of CDPKs with oil bodies.     

Sunflower (Helianthus annuus) long‐chain acyl‐coenzyme A synthetases expressed at high levels in developing seeds

Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl‐CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis.