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

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

Identification of three novel unique proteins in seed oil bodies of sesame

Plant seeds store triacylglycerols in discrete organelles called oil bodies. An oil body preserves a matrix of triacylglycerols surrounded by a monolayer of phospholipids embedded with abundant structural proteins termed oleosins and probably some uninvestigated minor proteins of higher molecular mass. Three polypeptides of 27, 37, and 39 kDa (temporarily denominated as Sop1, Sop2, and Sop3) were regularly co-purified with seed oil bodies of sesame. Comparison of amino acid composition indicated that they were substantially less hydrophobic than the known oleosins, and thus should not be aggregated multimers of oleosins. The results of immuno-recognition to sesame proteins extracted from subcellular fractions of mature seeds, various tissues, and oil bodies purified from different stages of seed formation revealed that these three polypeptides were unique proteins gathered in oil bodies, accompanying oleosins and triacylglycerols, during the active assembly of the organelles in maturing seeds. Both in vivo and in intro, immunofluorescence labeling using secondary antibodies conjugated with FITC (fluorescein isothiocyanate) confirmed the localization of these three polypeptides in oil bodies.     

A new method for seed oil body purification and examination of oil body integrity following germination

Plant seeds store triacylglycerols as energy sources for germination and postgerminative growth of seedlings. The triacylglycerols are preserved in small, discrete, intracellular organelles called oil bodies. A new method was developed to purify seed oil bodies. The method included extraction, flotation by centrifugation, detergent washing, ionic elution, treatment with a chaotropic agent, and integrity testing by use of hexane. These processes subsequently removed non-specifically associated or trapped proteins within the oil bodies. Oil bodies purified by this method maintained their integrity and displayed electrostatic repulsion and steric hindrance on their surface. Compared with the previous procedure, this method allowed higher purification of oil bodies, as demonstrated by SDS-PAGE using five species of oilseeds. Oil bodies purified from sesame were further analyzed by two-dimensional gel electrophoresis and revealed two potential oleosin isoforms. The integrity of oil bodies in germinating sesame seedlings was examined by hexane extraction. Our results indicated that consumption of triacylglycerols reduced gradually the total amount of oil bodies in seedlings, whereas no alteration was observed in the integrity of remaining oil bodies. This observation implies that oil bodies in germinating seeds are not degraded simultaneously. It is suggested that glyoxisomes, with the assistance of mitochondria, fuse and digest oil bodies one at a time, while the remaining oil bodies are preserved intact during the whole period of germination.     

Differential stability of baculovirus late transcription complexes during initiation and elongation

Oleosins are amphipathic proteins associated with oil bodies in seeds. We purified the major 16,500 peanut oleosin by preparative SDS-PAGE. Autoradiography after SDS-PAGE separation of the iodinated oleosin revealed covalently bound oligomers with Mr of 21,000, 33,000, 44,000 and 51,000. The strong capacity of these oligomers to form aggregates and to be incorporated into large-sized detergent micelles was demonstrated by gel permeation and isoelectric focusing. A 50% ethanol concentration was necessary to elute the 16,500 oleosin from octyl groups in hydrophobic interaction chromatography showing its natural tendency to interact with lipid acyl chains. This oligomerization behavior in aqueous solution is an indirect reflection of the interactions that occur in the oil body.     

Liquid chromatographic method for determination of triglycerides in vegetable oils in terms of their partition numbers: summary of collaborative study

The IUPAC Commission on Oils, Fats and Derivatives undertook development of a method and collaborative study for the determination of triglycerides in vegetable oils by liquid chromatography. Three collaborative studies were conducted from 1985 to 1987. Refinements were made in the method after the first collaborative study, and the second and third collaborative studies demonstrated that the method produces acceptable results. Materials studied were soybean oil, almond oil, sunflower oil, olive oil, rapeseed oil, and blends of palm and sunflower oils, and almond and sunflower oils. Six test samples were analyzed by 18 laboratories from 11 countries in the second study; 4 test samples were analyzed by 16 laboratories from 12 countries in the third study. The method for determination of triglycerides (by partition numbers) in vegetable oils by liquid chromatography was adopted first action by AOAC INTERNATIONAL as an IUPAC-AOCS-AOAC method.     

Fumonisin production and other traits of Fusarium moniliforme strains from maize in northeast Mexico

Strains of Fusarium moniliforme from maize seed collected in four fields in northeast Mexico were tested for fumonisin production in culture, for sexual compatibility, and for vegetative compatibility by using non-nitrate-utilizing mutants. The test results indicate that a diverse population of fumonisin-producing strains of F. moniliforme (Gibberella fujikuroi) mating population A predominates and that a potential exists for production of fumonisins in Mexican maize.     

Mapping of Tonoplast Intrinsic Proteins in Maturing and Germinating Arabidopsis Seeds Reveals Dual Localization of Embryonic TIPS to the Tonoplast and Plasma Membrane

We have mapped the expression of the tonoplast intrinsic protein (TIP) gene family members in Arabidopsis seeds by fluorescent protein tagging of their genomic sequences and confocal microscopy. Three isoforms (TIP1;1, TIP2;1,and TIP2;2) have distinct patterns of expression in maternal tissues (outer integument and placento-chalazal region). Two isoforms, TIP3;1 and the previously uncharacterized TIP3;2, are the only detectable TIPS in embryos during seed maturation and the early stages of seed germination. Throughout these developmental stages, both isoforms co-locate to the tonoplast of the protein storage vacuoles, but also appear to label the plasma membrane. Plasma membrane labeling is specific to TIP3;1 and TIP3;2, is independent of the position of the fluorescent protein tag, and appears to be specific to early seed maturation and early germination stages. We discuss these results in the context of the predicted distribution of aquaporins in Arabidopsis seeds.     

Eukaryotic lipid-biosynthetic enzymes: the same but not the same

The discovery that different eukaryotic enzymes catalyse the same lipid-biosynthetic reaction has paralleled the recognition that segregated pools of lipids have unique biological functions. This review considers enzymes involved in the synthesis of diverse classes of lipids--glycerolipid precursors, phospholipids, sterols and eicosanoids--and summarizes recent data that show that these duplicate enzymes are frequently encoded by different genes and have unique subcellular locations. Does this duality merely represent redundancy or do the different isoforms provide pools of lipids for specific biological purposes?     

Characterization of acyl-ACP thioesterases of mangosteen (Garcinia mangostana) seed and high levels of stearate production in transgenic canola

Acyl-acyl-carrier protein (ACP) thioesterases are, at least in part, responsible for the fatty acyl chain length composition of seed storage oils. Acyl-ACP thioesterases with specificity for each of the saturated acyl-ACP substrates from 8:0 through 16:0 have been cloned, with the exception of 18:0, and are members of the FatB class of thioesterases. The authors have determined that the tropical tree species mangosteen (Garcinia mangostana) stores 18:0 (stearate) in its seed oil in amounts of up to 56% by weight. Acyl-ACP thioesterase activity as measured in crude mangosteen seed extracts showed a preference for 18:1-ACP substrates, but had significant activity with 18:0 relative to that with 16:0-ACP, suggesting a thioesterase might be involved in the production of stearate. Three distinct acyl-ACP thioesterases were cloned from mangosteen seed cDNA; two representative of the FatA class and one representative of the FatB class. When expressed in vitro, the enzyme encoded by one of the FatAs (Garm FatA1) while preferring 18:1-ACP showed relatively low activity with 16:0-ACP as compared to 18:0-ACP, similar to the substrate preferences shown by the crude seed extract. Expression of Garm FatA1 in Brassica seeds led to the accumulation of stearate up to 22% in seed oil. These results suggest that Garm FatA1 is at least partially responsible for determining the high stearate composition of mangosteen seed oil and that FatA as well FatB thioesterases have evolved for specialized roles.     

Antibacterial activity of essential oils from Cymbopogon: inter- and intra-specific differences

The influence of the genetic background of a plant on the antibacterial activity of essential oil derived from it was investigated. Essential oils from six distinct strains of Cymbopogon were tested against eighteen bacteria. Interspecific and intra-specific differences were evident in the antibacterial activity of the essential oils derived from the the six Cymbopogon strains.     

Isolation and identification of bacteria from activated sludge purifying petroleum wastewaters

Several strains growing well in minimal media with 500 and 1000 mg/l of oil or phenol as a sole carbon source were isolated from activated sludge purifying petroleum waste waters and identified. Five of the best growing strains classified as Arthrobacter, Pseudomonas, Xanthomonas and Enterobacter were selected and their capacity to remove petroleum components and phenol (in the oil fraction of petrochemical waste waters) was studied. The enzymatic activity of the strains, including respiration intensity and dehydrogenase activity was also determined. All the examined strains were found to use oils as the sole source of carbon (percent age of the oils reduction during cultivation of the individual strains ranged from 58 to 78). Phenol was completely reduced by only one strain. The rest of the strain removed only from 7 to 24% of this compound. The activity of dehydrogenases and the respiration intensity in the presence of the studied substrates -- oil and phenol was low for all the examined strains.     

Newcastle disease oil emulsion vaccines prepared with animal, vegetable, and synthetic oils

Animal, vegetable, and synthetic oils were tested as potential replacements for mineral oil in Newcastle disease oil emulsion vaccines. Emulsifying surfactants of seed oil origin comprised 10% of the the oil phase that was used to prepare water-in-oil emulsion vaccines that contained a final concentration of 20% aqueous antigen. The hemagglutination inhibition responses of chickens inoculated with 46 of the newly formulated oil vaccines were, in most cases, not significantly different from those of control chickens inoculated with mineral oil vaccine. Tissue reactions associated with animal, vegetable, and synthetic oil vaccines were less severe than those associated with mineral oil vaccines. Viscosity of the mineral oil formulations ranged from 1/2 to 3 1/2 times that of the mineral oil control vaccines. These findings indicate that any of several oils may be more suitable than mineral oil for preparation of immune adjuvants for poultry vaccines.     

Fatty acids in 80 brands of edible vegetable oil and in 14 brands of mayonnaise (author's transl)

The fatty acid patterns have been defined in 80 commonly used table oils chosen at random and of 14 brands of mayonnaise commercially available in West Germany. In addition, sterols of the various mayonnaises were analysed. The oils could be grouped into 8 different categories: 12 oliver oils, 8 maize oils, 14 soya oils, 5 linseed oils, 22 sunflower oils, 3 safflower oils, 5 groundnut oils, and 8 rapeseed oils. As a result of the analyses, it is in almost every case a question of a pure variety of non-animal oils. The range of the values obtained for the composition of the fatty acids for each variety, which can be considerable, represents a modern complement to previous analyses. The fatty acid pattern of 3 additional commerical table oils suggest an amalgamation with fats of other types, among which are 2 cases of mixture with rapeseed oils. For mayonnaise, soya oil is used almost exclusively as a fat component. The presence of sunflower oil was only confirmed in one brand of mayonnaise. The concentrations of cholesterol which were determined lie between 17.0 and 72.3 mg (average 53 mg) per 100 g mayonnaise, so that even in the case of persons which present an elevated risk of atherosclerosis there can be no objection to the consumption of mayonnaise. The nutritive-physiological significance of these various oils for human nutrition is thoroughly discussed. A possible health hazard is the sale of vegetable oils rich in erucic acid permitted in the West German Republic (proportion of erucic acid in the 8 samples examined: 223-53%). These vegetable oils, as is shown by the additionally indicated trade names with the corresponding supplementary specifications are overwhelmingly labelled as vegetable oil (Pflanzen?l) or table oil (Speise?l). The purchase prices indicated and paid by us for the oils examined are subject to considerable fluctuations even for oils of the same quality.     

Effects of continuous medroxyprogesterone acetate on lipoprotein metabolism in postmenopausal women receiving estrogen

Seed storage globulins of the 7S and 11S type are synthesized in the seeds of angiosperms and gymnosperms. We have isolated and characterized a vicilin-like gene expressed in the cycad Zamia furfuraceae. Sequence comparisons reveal clear similarities to a sucrose-binding protein isolated from soybean. We suggest the existence of a superfamily of related genes including both vicilin-like and legumin-like seed globulin genes as well as genes coding for spherulins, germins and sucrose-binding-proteins.     

Mechanism of Effect of Lanthanum Nitrate on Vigor of Aged Rice Seeds

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Developmental control of H+/amino acid permease gene expression during seed development of Arabidopsis

Long distance transport of amino acids is mediated by several families of differentially expressed amino acid transporters. The two genes AAP1 and AAP2 encode broad specificity H(+)-amino acid co-transporters and are expressed to high levels in siliques of Arabidopsis, indicating a potential role in supplying the seeds with organic nitrogen. The expression of both genes is developmentally controlled and is strongly induced in siliques at heart stage of embryogenesis, shortly before induction of storage protein genes. Histochemical analysis of transgenic plants expressing promoter-GUS fusions shows that the genes have nonoverlapping expression patterns in siliques. AAP1 is expressed in the endosperm and the cotyledons whereas AAP2 is expressed in the vascular strands of siliques and in funiculi. The endosperm expression of AAP1 during early stages of seed development indicates that the endosperm serves as a transient storage tissue for organic nitrogen. Amino acids are transported in both xylem and phloem but during seed filling are imported only via the phloem. AAP2, which is expressed in the phloem of stems and in the veins supplying seeds, may function in uptake of amino acids assimilated in the green silique tissue, in the retrieval of amino acids leaking passively out of the phloem and in xylem-to-phloem transfer along the path. The promoters provide excellent tools to study developmental, hormonal and metabolic control of nitrogen nutrition during development and may help to manipulate the timing and composition of amino acid import into seeds.     

Cystine content of legume seed proteins: estimation by determination of cysteine with 2-vinylquinoline, and relation to protein content and activity of cysteine synthase

The cystine content of the protein of a number of different lines of legume seeds has been determined by the method of Krull et al. which selectively reacts cysteine residues of intact, reduced proteins with 2-vinylquinoline, giving an adduct with an absorption maximum at 318 nm. Some seed lines were found to have 3.5 times as much cysteine as the seed line with the lowest cysteine content, perhaps offering opportunities for improvement in the nutritional quality of bean seed proteins through breeding and selections. While no correlation between cysteine levels and protein content was observed, a positive correlation was found between the specific activity of the terminal enzyme of cysteine synthesis, cysteine synthase, and the cysteine content of seeds.     

Effect of Moisture Content on Melilotus suaveolens Seed Quality During Ultra-drying Storage

In order to enhance the seed storability and supply high-quality seeds for vegetation restoration in the arid and semi-arid regions in Northwest China, the effects of ultra-drying and accelerated aging on the physiological characterstics of Melilotus suaveolens seeds were studied. Melilotus suaveolens seeds were dried in a desiccator containing silica gel to 80, 53, 42, 33, 23 and 16g·kg<'-1> of moisture contents (MC), respectively. The parameters of the seed quality including germination energy (GE), germination percentage (GP), relative conductivity (RC), dehydrogenase activity (DA) and a-amylase activity (AA) were determined afier ultra-drying and accelerated aging. The results showed that ultra-dried seeds with 42 g,kg-1 of MC showed the minimum changes of GE and GP before and after seed aging. Moreover, ultra-dry seeds with 42 g·kg<'-1> of MC showed higher DA and AA, and lower RC than non-ultra-drying seeds. Therefore, ultra-drying to 42 g·kg<'-1> of MC was helpful for M. Suaveolens seed storage.