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.     

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.     

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.     

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.     

The development of protein and oil bodies in the seed of Sinapis alba L.

Summary The cotyledons of Sinapis alba L. seed are the storage organs and first photosynthetic organs. The development of the cotyledon cell contents was studied using electron and light microscopy. From the heart shaped embryo (11 days from petal fall) to the mature seed, nine stages were examined.Both types of protein grains (designated aleurone grains and myrosin grains) were found to form within vacuoles, but the mode of protein accumulation differed with each type of grain.Oil bodies were apparent with the EM from 18 days onwards, but could not be seen to arise from the ER. They were granular in appearance at early stages, but later became electron transparent.     

Some studies on the composition and surface properties of oil bodies from the seed cotyledons of safflower (Carthamus tinctorius) and linseed (Linum ustatissimum).

1. The average oil-body diameter in intact cells of developing linseed (Linum usitatissimum) and safflower (Carthamus tinctorius) cotyledons was similar (about 1.4 micrometer), and there was little change in size after oil bodies were isolated and repeatedly washed. 2. The glycerolipid composition of washed oil bodies from both developing and mature cotyledons of the two species was similar; oil bodies from ten different batches of cotyledons contained 4.3 +/- 0.16 mumol of 3-sn-phosphatidylcholine and 25.2 +/- 1.7 mumol of diacylglycerol per 1000 mumol of triacylglycerol. During four successive washings of a once-washed oil-body preparation, the proportion of diacylglycerol to triacylglycerol remained constant and that of 3-sn-phosphatidylcholine to triacylglycerol decreased by only 20%. 3. The protein content of thrice-washed oil bodies from the two species was similar, about 2.4% of the weight of glycerolipids, and appeared to be independent of the stage of cotyledon maturity. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the protein of purified oil bodies from the two species consisted mainly of only four polypeptides and that two of the polypeptides from each species had apparent mol.wts. of 17500 and 15500. Similar patterns of polypeptides were obtained after the hydrolysis of the 15500-mol.wt. polypeptides from linseed and safflower oil bodies by Staphylococcus aureus V8 proteinase, whereas the proteolysis of the 17500-mol.wt. polypeptides from the two species produced different patterns of polypeptides. 4. The 3-sn-phosphatidylcholine in oil-body preparations was hydrolysed about 85% by bee-venom phospholipase A2 without any apparent coalescence of the oil bodies. Incubation with lipase from Rhizopus arrhizus caused rapid coalescence of the oil bodies, and this lipase appeared to initially hydrolyse diacylglycerols in preference to triacylglycerol. 5. Oil bodies from both species were almost completely dispersed in suspensions of pH between 7.1 and 8.3, but formed large aggregates at pH values between 6.7 and 3.9; pH-induced aggregation caused no coalescence. Aggregates formed under acidic conditions were dispersed by re-adjusting the pH of suspensions to 8.3. 6. A freeze-etch electron-microscopic examination of isolated oil bodies indicated that these organelles were bounded by some form of membrane with a particle-free outer surface.     

Animal-specific membrane components visualised on the surface of animal/plant heterokaryons

Summary Immunofluorescence was used to demonstrate the presence and distribution of animalspecific membrane components within the plasma membranes ofXenopus/carrot heterokaryons. The inhibitor of cellulose synthesis, 2,6-dichlorobenzonitrile, was used to impair cell wall regeneration so that the plasma membranes of cultured heterokaryons would remain accessible to antibodies.Xenopus-Specific surface components were observed in heterokaryons after 14 days of culture.     

Characterization and potential uses of Copaifera langsdorfii seeds and seed oil

Copaifera langsdorfii (Desf.) Kuntze (copaiba) seeds are abundantly produced and have not yet been characterized. The seed oil presents a characteristic odor of coumarin (250.1+/-6.57 mg/g determined through LC). The fatty acid composition of the oil was determined through CG/FID, being 45.3% linoleic acid, 32.3% monounsaturated, and 22.4% saturated fat. For the lipid-free seeds, the total carbohydrate, protein and moisture were 75.4%, 6.8% and 14.8%, respectively. The C. langsdorfii xyloglucan had an intrinsic viscosity of 804 mL/g, and the average molar mass (Mw) was 7.82 x 10(5)g/mol and Rg of 65 nm. The degree of polydispersion was 1.7, indicating the polydisperse family of polysaccharides. Its homogeneity, low degree of polymer contaminants and high intrinsic viscosity and molecular mass, represent good potential as a thickening agent. The presence of coumarin and xyloglucan as major components of C. langsdorfii seeds denotes its potential for use in the cosmetic or pharmaceutical industries.     

Lipid components of olive oil from Tunisian Cv. Sayali: Characterization and authenticity

The analysis of the total lipid fraction from the Sayali variety of olive oil was accomplished in the present investigation. Glyceridic, unsaponifiable and flavour fractions of the oil were isolated and identified using several analytical methods. Chromatographic techniques have proven to be suitable for these determinations, especially capillary gas chromatography. Gas chromatography coupled to mass spectrometry was successfully used to identify sterols, triterpenes alcohols, 4-monomethylsterols, aliphatic alcohols and aroma compounds in our samples. Furthermore, solid phase microextraction was used to isolate volatiles from the total lipid fraction. Results from the quantitative characterization of Sayali olive oil showed that oleic acid (77.4%) and triolein (47.4%) were the dominant glyceridic components. However, the main compounds of the unsaponifiable fraction were β-sitosterol (147.5 mg/100 g oil), 24-methylene cycloartenol (146.4 mg/100 g oil) and hexacosanol (49.3 mg/100 g oil). Moreover, results showed that the aldehydic compounds were the major flavours present in Sayali olive oil.     

Oleosin KD 18 on the surface of oil bodies in maize. Genomic and cDNA sequences and the deduced protein structure

Oleosins are newly discovered, abundant, and small Mr hydrophobic proteins localized on the surface of oil bodies in diverse seeds. So far, most of the studies have been on the general characteristics of the proteins, and only one protein (maize KD 16) has been studied using a cDNA clone containing an incomplete coding sequence. Here, we report the sequences of a genomic clone and a cDNA clone of a new maize oleosin (KD 18). There is no intron in the gene. The 5'-flanking region contains potential regulatory elements including RY repeats, CACA consensus, and CATC boxes, which are presumably involved in the specific expression of the proteins in maturing seeds. The deduced amino acid sequence was analyzed for secondary structures. We suggest that KD 18 of 187-amino acid residues contains three major structural domains: a largely hydrophilic domain at the N terminus, a hydrophobic hairpin alpha-helical domain at the center, and an amphipathic alpha-helix domain at the C terminus. These structural domains are very similar to those of oleosin KD 16. However, the KD 18 and KD 16 amino acid sequences as well as nucleotide sequences are highly similar only at the central domain (72 and 71%, respectively). The similarities are highest at the loop region of the alpha-helical hairpin. These results suggest that KD 18 and KD 16 are isoforms, encoded by genes derived from a common ancestor gene. We propose that the hairpin domain acts as an indispensible internal signal for intracellular trafficking of oleosins during protein synthesis as well as an anchor for oleosins on the oil bodies. The other two domains can undergo relatively massive amino acid substitutions without impairing the structure/function of the oleosins or have evolved to generate oleosins having different functions.     

油菜种子发育早期的油体发生与调控

以甘蓝型油菜( Brassica napus L.)品种‘Westar’和‘Topas’为材料,通过超微结构观察和荧光定量PCR技术对油菜胚胎发育早期油体的发生、油体蛋白及脂肪酸合成转录因子基因的表达情况进行分析。结果显示:油体出现在油菜胚胎发育早期,在授粉9 ~ 11 d后(球形胚时期)的胚体和胚柄中均存在直径小于0. 5 μm的油体;荧光定量实验结果表明,除 BnCLO3 的表达量在整个胚胎发育阶段无明显变化外,其他油体蛋白基因 Oleosins 、 Steroleosins 和 BnCLO1 的表达量在心形胚时期就明显增多并持续增长;脂肪酸合成转录因子 BnLEC1 、 BnL1L 、 BnWRI1 和 BnFUS3 在胚胎发育阶段,基因表达规律均呈先上升再下降的趋势,但达到最高值的时间存在差异,其中 BnLEC1 最早, BnL1L 其次, BnWRI1 和 BnFUS3 较晚。研究结果表明甘蓝型油菜在球形胚时期出现油体,其结构蛋白和转录调控因子基因的表达自心形胚开始明显增多。     

油菜种子发育早期的油体发生与调控

以甘蓝型油菜（Brassica napus L.）品种‘Westar’和‘Topas’为材料,通过超微结构观察和荧光定量PCR技术对油菜胚胎发育早期油体的发生、油体蛋白及脂肪酸合成转录因子基因的表达情况进行分析。结果显示：油体出现在油菜胚胎发育早期,在授粉9~11 d后（球形胚时期）的胚体和胚柄中均存在直径小于0.5 μm的油体;荧光定量实验结果表明,除BnCLO3的表达量在整个胚胎发育阶段无明显变化外,其他油体蛋白基因Oleosins、Steroleosins和BnCLO1的表达量在心形胚时期就明显增多并持续增长;脂肪酸合成转录因子BnLEC1、BnL1L、BnWRI1和BnFUS3在胚胎发育阶段,基因表达规律均呈先上升再下降的趋势,但达到最高值的时间存在差异,其中BnLEC1最早,BnL1L其次,BnWRI1和BnFUS3较晚。研究结果表明甘蓝型油菜在球形胚时期出现油体,其结构蛋白和转录调控因子基因的表达自心形胚开始明显增多。     

五味子科植种子表面微形态及其系统学意义

首次报道用扫描电镜观察的五味子科24种7变种1变型的92个居群117份种子样品的表面微形态特征。将该科种子表面微形态归纳为4大类型,其中第Ⅰ型又分为3个亚型。将种子表面微形态与该科的花粉微形态,以及花、果、茎和叶的宏观形态结合起来进行对比分析。结果表明,五味子科种子表面微形态特征几乎不受其植株生长地环境条件的影响,相当稳定,因而作为分类的鉴别特征完全可靠,已观察到的种子表面微形态特征不支持林祁和Saunders对五味子科所做的大部分分类学修订结果。种子表面微形态特征所揭示的Schisandra属与Kadsura属之间的关系,与花粉形态所揭示的两属关系极其相似,即两属可能起源于共同的祖先,分别沿两条不同路线演化,但亦不能排除两属间有更复杂的关系的可能性。Kadsura属的总体演化水平高于Schisandra属,因而不支持认为Kadsura属比Schisan-dra属原始的观点。     

Micromorphological features of the seed surface of Schisandraceae and their systematic significance

Micromorphological features of the seed surface of the Schisandraceae are reported for the first time. One hundred and seventeen seed samples from 92 populations, representing 24 species, seven varieties and one form of the Schisandraceae, were examined and photographed under scanning electron microscope (SEM). Micromorphological features of seed surfaces of the Schisandraceae are little affected by the habitats in which plants grow, and are quite constant within species, therefore they can be used as reliable diagnostic characters to distinguish species. The following taxonomic treatments are not supported by micromorphological features observed: ( 1 ) To reduce Schisandra wilsoniana A. C. Smith to S. henryi Clarke; (2) To reduce S. neglecta A. C. Smith, S. arisanensis Hayata, S. viridis A. C. Smith, S. sphenanthena Rehd. & Wils., S. gracilis A. C. Smith, S. micrantha A. C. Smith and S. lancifolia var. polycarpa Z. He to S. elongata ( Bl. ) Baill.; (3) To reduce S. henryi var. longipes (Merr. & Chun) A. C. Smith, S. tomentella A. C. Smith and S. pubescens var. pubinervis (Rehd. & Wils.)A. C. Smith to S. pubescens Hemsl. & Wils.; (4) To reduce S. rubriflora(Franch.)Rehd. & Wils., S. flaccidiramosa C. R. Sun, S. incarnata Stapf, S. sphaerandra Stapf, S. sphaerandra f. pallida A. C. Smith and S. glaucescens Diels to S. grandiflora ( Wall. ) Hook. f. & Thoms.; (5) To reduce Schisandra wilsoniana to S. bicolor Cheng; (6) To reduce S. lancifolia var. polycarpa to S. neglecta; (7) To raise S. henryi var. longipes to S. longipes(Merr. & Chun)R. M. K. Saunders; (8) To reduce Kadsura polysperma Yang to K. heteroclita(Roxb. )Craib. The relationship between Schisan-dra Michx. and Kadsura Kaempf. ex Juss. revealed by the micromorphological features of seed surface is very similar to that revealed by pollen morphology, namely these two genera might have originated from a common ancestor and then evolved along two different routes. However the probability of a more complicated relationship between the two genera than we have known hitherto can not be excluded. The general evolutionary level of Kadsura seems to be higher than that of Schisandra. Therefore, the viewpoint that Kadsura is more primitive than Schisandra is not supported.     

五味子科植物种子表面微形态及其系统学意义

首次报道用扫描电镜观察的五味子科24种7变种1变型的92个居群117份种子样品的表面微形态特征。将该科种子表面微形态归纳为4大类型,其中第Ⅰ型又分为3个亚型。将种子表面微形态与该科的花粉微形态,以及花、果、茎和叶的宏观形态结合起来进行对比分析,结果表明,五味子科种子表面微形态特征几乎不受其植株生长地环境条件的影响,相当稳定,因而作为分类的鉴别特征完全可靠。已观察到的种子表面微形态特征不支持林祁和Saunders对五味子科所做的大部分分类学修订结果。种子表面微形态特征所揭示的Schisandra属与Kadsura属之间的关系,与花粉形态所揭示的两属关系极其相似,即两属可能起源于共同的祖先,分别沿两条不同路线演化,但亦不能排除两属间有更复杂的关系的可能性。Kadsura属的总体演化水平高于Schisandra属,因而不支持认为Kadsura属比Schisandra属原始的观点。