Triacylglycerol Bioassembly in Microspore-Derived Embryos of Brassica napus L. cv Reston

Erucic acid (22:1) was chosen as a marker to study triacylglycerol (TAG) biosynthesis in a Brassica napus L. cv Reston microspore-derived (MD) embryo culture system. TAGs accumulating during embryo development exhibited changes in acyl composition similar to those observed in developing zygotic embryos of the same cv, particularly with respect to erucic and eicosenoic acids. However, MD embryos showed a much higher rate of incorporation of ¹⁴C-erucoyl moieties into TAGs in vitro than zygotic embryos. Homogenates of early-late cotyledonary stage MD embryos (14-29 days in culture) were assessed for the ability to incorporate 22:1 and 18:1 (oleoyl) moieties into glycerolipids. In the presence of [1-¹⁴C]22:1-coenzyme A (CoA) and various acyl acceptors, including glycerol-3-phosphate (G-3-P), radiolabeled erucoyl moieties were rapidly incorporated into the TAG fraction, but virtually excluded from other Kennedy Pathway intermediates as well as complex polar lipids. This pattern of erucoyl incorporation was unchanged during time course experiments or upon incubation of homogenates with chemicals known to inhibit Kennedy Pathway enzymes. In marked contrast, parallel experiments conducted using [1-¹⁴C]18:1-CoA and G-3-P indicated that ¹⁴C oleoyl moieties were incorporated into lyso-phosphatidic acids, phosphatidic acids, diacylglycerols, and TAGs of the Kennedy Pathway, as well as other complex polar lipids, such as phosphatidylcholines and phosphatidylethanolamines. When supplied with l-[2-³H(N)]G-3-P and [1-¹⁴C]22:1-CoA, the radiolabeled TAG pool contained both isotopes, indicating G-3-P to be a true acceptor of erucoyl moieties. Radio-high-performance liquid chromatography, argentation thin-layer chromatography/gas chromatography-mass spectrometry, and stereospecific analyses of radiolabeled TAGs indicated that 22:1 was selectively incorporated into the sn-3 position by a highly active diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), while oleoyl moieties were inserted into the sn-1 and sn-2 positions. In the presence of sn-1,2-dierucin and [1-¹⁴C]22:1-CoA, homogenates and microsomal preparations were able to produce radiolabeled trierucin, a TAG not found endogenously in this species. A 105,000g pellet fraction contained 22:1-CoA:DGAT exhibiting the highest specific activity. The rate of 22:1-CoA:DGAT activity in vitro could more than account for the maximal rate of TAG biosynthesis observed in vivo during embryo development. In double label experiments, G-3-P was shown to stimulate the conversion of [³H]phosphatidylcholines to [³H]diacylglycerols, which subsequently acted as acceptors for ¹⁴C erucoyl moieties. In vitro, 22:1 moieties did not enter the sn-1 position of TAGs by a postsynthetic modification or transacylation of preformed TAGs.     

Expression of Polarity during early Development of Microspore-derived and Zygotic Embryos of Brassica napus L. cv. Topas

Microspore derived (MS-)embryogenesis and zygotic embryogenesis of Brassica napus L. cv. Topas were investigated by light and scanning electron microscopy to reveal the expression of polarity during the transition phase from globular to heart and torpedo shape. During the first 5 days of MS-embryo formation, the cell wall of the former microspores remained intact and a globular mass of cells developed within. Pollen walls ruptured after 5 days of culture; embryos proceeded through heart-shape and torpedo-shape stages within 15 days in a way comparable to, but faster than observed during zygotic embryogenesis. Expression of polarity in globular and elongating MS-embryos was analyzed by detection of the distribution of activated calmodulin as well as of free cytosolic calcium by using confocal scanning laser microscopy, and by the detection of starch. Calmodulin was evenly distributed in globular embryos and only exhibited clear polar distribution in elongated embryos. Free cytosolic Ca²⁺ accumulated in the protoderm of globular embryos and in the central cylinder of torpedo shaped embryos, but never showed polar distribution. Accumulation of starch granules at the root poles of both sexual as well as MS-embryos, however, indicated polar distribution before the transition from globular to heart shape stage. Since the local rupture of the pollen wall of 6-day-old MS-embryos was never preceded by the decrease of starch at that site, it is likely that the rupture of the pollen wall plays an important role in the local activation of cell metabolism and thus in the determination of the polarity axis in MS-embryos.     

Zygotic embryo cloning in oilseed rape (Brassica napus L.)

When late torpedo- to early cotyledonary-stage zygotic embryos of Brassica napus were cultured on a solidified B5 basal medium with or without 0.05 mg l⁻¹ 6-benzylaminopurine (BAP), they proliferated by direct formation on the hypocotyl surface of structures resembling accessory cotyledons and root poles. When embryos bearing these structures were transferred to slowly rotating liquid culture on the same media, proliferation continued. As the underlying tissues senesced, meristematic structures became detached which produced cotyledonary and root tissues. A random sample of plants raised from these structures by direct transfer to sterilized potting mix all showed normal morphology.     

Oilbody Proteins in Microspore-Derived Embryos of Brassica napus: Hormonal, Osmotic, and Developmental Regulation of Synthesis

A number of treatments were tested for their ability to affect the synthesis of oilbody proteins in microspore-derived embryos of rapeseed (Brassica napus). Synthesis of the oilbody proteins was determined by [³⁵S]methionine incorporation in vivo and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of washed oilbody fractions. Oilbody proteins of approximately 19, 23, and 32 kilodaltons were found to be prominent. These proteins showed differential patterns of regulation. The 19 and 23 kilodalton proteins (oleosins) were greatly enhanced by treatments with abscisic acid, jasmonic acid, and osmotic stress imposed using sorbitol (12.5%). Synthesis of the 32 kilodalton protein was inhibited by abscisic acid and by sorbitol (12.5%), but unaffected by jasmonates. The strong promotion of synthesis of the 19 and 23 kilodalton oilbody proteins appeared to be specific as they are not seen with gibberellic acid treatment or with a stress such as heat shock. Time course experiments revealed that the abscisic acid stimulation of oleosin synthesis is quite rapid (less than 2 hours), reaching a maximum at 6 to 8 hours. The response of the oleosins to abscisic acid is found in all stages of embryogenesis, with a major increase in synthetic rates even in globular embryos on abscisic acid treatment. This suggests that these proteins may accumulate much earlier in embryogenesis than has previously been believed. The 32 kilodalton oilbody-associated protein appears different from the oleosins in several ways, including its distinct pattern of regulation and its unique property, among the oilbody proteins, of undergoing phosphorylation.     

The Effect of Temperature on the Level and Biosynthesis of Unsaturated Fatty Acids in Diacylglycerols of Brassica napus Leaves

Experiments on the effects of temperature on the levels of unsaturated fatty acids and their rates of desaturation in Brassica napus leaf lipids have shown that significant differences occur in the composition of all diacylglycerols in the leaf between plants grown at high and low temperatures. In the major thylakoid diacylglycerols, monogalactosyl-diacylglycerol and digalactosyldiacylglycerol, not only is there an increase in the level of unsaturation at low temperatures, but there is a change in the balance between molecular species of chloroplastic origin (16/18C) and cytosolic origin (18/18C). Radioactivity tracer data indicate that at low temperatures there are two distinct phases of desaturation in the fatty acids of the major diacylglycerols of these leaves. A rapid phase, which appears in plants grown at low temperatures and results in the desaturation of palmitic acid to hexadecadienoic acid and oleic acid to linoleic acid may explain the high levels of unsaturated fatty acids found in the leaf diacylglycerols from plants grown at low temperatures. The appearance of this rapid phase is controlled by the temperature at which the plant is grown and is not subject to rapid variations in environmental temperature.     

Influence of Meteorological Factors on Oil Content and Major Fatty Acids of Rapeseeds (Brassica napus L. )

1IntroductionOilseedrape(BrassicanapusL.)isthemostimportantsourceofedibleoil,andtheYangtzeRiverlowerbasinisthemaincultivationregioninChina.Duringlastdecade,winterrapeproduc-tionisincreasinginthecereal-growingregionsafterthericeharvest(Zhou,1994).Weathervari-ablessuchassolarradiation,temperature,precipitation,windandhumidityhavepronouncedef-fectsonrapegrowth,developmentandseedyield(Almondetal.1986).itisprobablethatahigh-ertemperatureduringvegetativegrowththanatmainfloweringandseedfillingprod…     

甘蓝型油菜芥酸和二十碳烯酸含量的基因效应

以甘蓝型油菜的4种纯合芥酸基因型之间所有可能的6个杂交组合的P_1、P_2、F_1、P_2、B_1和B_2世代为材料,用生统遗传学方法研究了芥酸和二十碳烯酸的基因作用形式及效应。发现无论亲本是单基因差异还是二基因差异,F_1和F_2代的芥酸含量都接近中亲值,F_1略大于中亲值和F_(20)世代均值分析表明,芥酸含量的遗传符合加性显性模型,加性效应占绝对优势,显性效应不显著。用数量遗传学方法估计的芥酸基因数与已知的结果相近。     

Fatty acid breakdown in developing embryos of Brassica napus L

Developing Brassica napus embryos are primarily concerned with the accumulation of storage products, namely oil, starch and protein. The presence of fatty acid catabolic pathways in the background of this biosynthetic activity was investigated. Enzymes involved in the process of lipid mobilization, such as malate synthase and isocitrate lyase, are detectable towards the late stages of embryo development. [(14)C]Acetate feeding experiments also reveal that fatty acid catabolism becomes increasingly functional as the embryo matures.     

植物合成长链多不饱和脂肪酸研究进展

长链不饱和脂肪酸（LC-PUFAs）对人类健康具有重要作用,通过转基因植物生产LC-PUFAs具有低成本、可持续、污染少等诸多优势。本文简要介绍了LC-PUFAs的作用、来源及其植物生物合成途径,综述了转基因植物合成LC-PUFAs的研究进展,并对如何进一步提高LC-PUFAs产量进行了探讨。     

Proline accumulation and sodium sulfate tolerance in callus cultures of Brassica napus L. cv. Westar

Callus cultures of Brassica napus L. cv. Westar were selected which contained 5 – 6 times more proline than unselected callus. Callus pieces from these cultures were able to survive much better after subculture to medium containing 105 mM Na₂SO₄ than unselected callus, or unselected callus cultured on exogenous proline before or during transfer to the salt. Exogenous proline was rapidly absorbed. In unselected callus there was a peak in proline accumulation ca. 2 days after transfer to Na₂SO₄, followed by a decline. In contrast proline accumulation in tolerant callus was linear with time, reaching maximum levels at 8 days. Proline levels induced by exposure to salt were maintained in the absence of stress.Abbreviations DW Dry weight - FW Fresh weight     

甘蓝型油菜‘沪油15'中两个CBF类转录因子的克隆与分析

以拟南芥CBF家族转录因子AP24保守域的氨基酸序列为信息探针,搜索油莱uniGerle数据库,得到2个uniGene：Bna．2193和Bna．2260,序列拼接得到2个cDNA序列（BnaCBF-1和BnaCBF-2）。通过PCR和RT—PCR方法分别从甘蓝型油莱‘沪油15’的DNA和cDNA中克隆了上述基因。序列分析显示,克隆的BnaCBF-1-Hy15与BnaCBF—1基因序列完全一样．而BnaCBF-2-Hy15与BnacBF-2的基因序列差异很小,只有4个氨基酸位点不同,且郝没有内含子。从cDNA序列、氨基酸序列的相似性、组成成分、理化性质、疏水性／亲水性、序列比对、进化树、功能域、二级结构、三级结构进行了预测。结果显示,‘沪油15’来源的BnaCBF-1-Hy15和BnaCBF-2-Hyl5转录因子属于AP2／ERF家族中的DREB—A1亚族,是亲水性蛋白,在蛋白质的三级结构上与atCBF1相似。     

Molecular Regulation of Biosynthesis of Long Chain Polyunsaturated Fatty Acids in Atlantic Salmon

Marine Biotechnology - Salmon is a rich source of health-promoting omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic...     

Allelic Variation of BnaC.TT2.a and Its Association with Seed Coat Color and Fatty Acids in Rapeseed (Brassica napus L.)

Efficient molecular markers for the selection of rapeseed genetic materials with high seed oil content and ideal fatty acid (FA) composition are preferred by rapeseed breeders. Recently, we reported the molecular mechanism of TRANSPARENT TESTA 2 (TT2) in inhibiting seed FA biosynthesis in Arabidopsis. However, evidence showing the association of rapeseed TT2 homologs and seed FA production are still insufficient. In this study, we collected 83 rapeseed (Brassica napus L.) landraces from different geographical backgrounds to conduct association mapping of BnaC.TT2.a in relation to seed coat color and FA biosynthesis. Population background was corrected by 84 pairs of SSR markers that were uniformly distributed among the linkage groups of the Tapidor-Ningyou-7 DH population. A single copy of BnaC.TT2.a for single nucleotide polymorphism (SNP) assay was cloned by a pair of previously reported specific primers. From the analysis of BnaC.TT2.a allelic variations using GLM+Q model, four SNPs on intron 1 of BnaC.TT2.a that were associated with seed FA were discovered. Moreover, an InDel at position 738 on exon 3 of BnaC.TT2.a indicated a change of protein function that was significantly associated with seed coat color, linoleic acid (C18:2), and total FA content. These findings revealed the role of BnaC.TT2.a in regulating the seed color formation and seed FA biosynthesis in rapeseed, thereby suggesting effective molecular markers for rapeseed breeding.     

Induction and Growth of 'Microspore-Derived' Embryos of Brassica napus ssp. oleifera

Three cultivars of spring rape (Brassica napus ssp. oleifera),Tower, Willi and Duplo, were used for a study of induction andgrowth of ‘microspore-derived’ embryos, Buds, 2.0mm in length, containing uninucleate microspores were harvestedand stored for 14 d at 4 ?C in darkness. Anthers were then removedand cultured on a liquid medium based upon that of Murashigeand Skoog and containing 8% sucrose, 0.5 mg l^–1 naphthylaceticacid and 0.05 mg l^–1 benzylaminopurine. Cultures werepre-incubated at 35 ?C for 0–3 d and then incubated at30 ?C. After a total of 42 d incubation, cultures were scoredfor the presence of macroscopic embryos (1–2 mm in length)and for the presence of anthers containing abortive embryoidswhich had not developed further. The cultivars differed greatly in terms both of the frequencyof anthers showing induced embryoids and of the final yieldof embryos. Tower showed the highest frequency of induction(maximum 38% of cultured anthers with induced embryoids) whereasthe highest yield (equivalent to 1.1 embryo per cultured anther)was obtained from anthers of the cv. Duplo after a 3 d treatmentat 35 ?C. Yields from the other cultivars were much lower andwere relatively unaffected by the 35 ?C treatment. Key words: Brassica napus, Rape, Anther culture, Pollen, Haploid     

Immunocytochemical localization of myrosinase in Brassica napus L.

The cytological and intracellular localization of myrosinase (EC 3.2.3.1) has been studied by immunochemical techniques using paraffin-embedded sections of radicles and cotyledons from seeds of Brassica napus L. cv. Niklas. For immunolabelling, sections were sequentially incubated with a monoclonal anti-myrosinase antibody and with peroxidase-and fluorescein-isothiocyanate-conjugated secondary antibodies. Enzyme and fluorescence label was present in typical myrosin cells both in radicles and in cotyledons. With higher magnification, fluorescence label revealed that the intracellular localization of myrosinase was associated with the tonoplast-like membrane surrounding the myrosin grains in the myrosin cells. The results also indicate that a large proportion of the positive myrosin cells are located in the second-outermost cell layer of the peripheral cortex region of the radicles.Abbreviations FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - PBS-T PBS with 0.5% (v/v) Tween-20 (polyoxyethylene sorbitane monolaurate) This work was supported by The Norwegian Research Council for Science and the Humanities. We wish to thank Professor Med. O.A. Haugen, Department of Pathology, University of Trondheim, Norway, for the skilful assistance provided regarding fixation and sectioning.     

Fatty acid inheritance in microspore-derived Populations of spring rapeseed (Brassica napus L.)

Summary Hard seededness in soybean [Glycine max (L.) Merr.] is a quantitative trait that affects the germination rate, viability, and quality of stored seeds. We have used 72 restriction fragment length polymorphisms (RFLPs) to identify genomic regions containing quantitative trait loci (QTL) affecting hard seededness in a segregating population from a G. max by a Glycine soja (Sieb. & Zucc.) cross. Five independent RFLP markers were found to be associated with variation in the hard-seeded trait. These markers and the epistatic interactions between them explain 71% of the variation for hard seededness. A genomic region associated with the i locus accounted for 32% of the variation in this segregating population. This study illustrates one approach to physiological genetic studies in plants.Journal Paper No. J-13557 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 2763     

Alterations in Membrane Lipids and Nutrient Uptake during Root Development of Rape-Seed (Brassica napus L.)

Diepenbrock, W. 1988. Alterations in membrane lipids and nutrientuptake during root development of rape-seed (Brassica napusL.). J. exp. Bot. 39: 193–198. Oil-seed rape was grown hydroponically in two experiments. Inthe first one, the effects of root development on phosphorusbound in lipids (lipid-P) and on nutrient uptake were tested.The content of lipid-P in root tissue decreased markedly withtime. This decline was accompanied by reduced rates of nitrogenaccumulation in the shoot. In the second experiment, plantswere grown in a split-root system with two chambers per pot.A part of the root system was harvested at 28 d after seedlingtransfer (DAS) referring to ‘continuous growth’.The other part was cut at 14 DAS and roots from the same chamberwere gathered at 14 d after root cutting (DAR) referring to‘re-growth’ Not only at 14 DAS but also at 14 DARhigh concentrations of lipid-P were detected whereas at 28 DASthe lipid-P content decreased dramatically. Changes in lipid-Pwere reflected in the fluctuation of fatty acid compositionof phosphatidyl choline, a major phospholipid in root tissue.It was clearly demonstrated that during re-growth, nutrientuptake was intensified as compared to continuous growth. Generally,membrane lipids and nutrient uptake were closely related. Key words: Root, lipids, nutrient uptake     

Effects of culture density,conditioned medium and feeder cultures on microspore embryogenesis in Brassica napus L. cv. Topas

Summary In microspore cultures of Brassica napus L. cv. Topas, embryo yield increases with culture density up to about 40,000 microspores per ml. A much higher density (100,000 per ml) appears inhibitory to embryogenesis. A relatively high culture density (30,000 or 40,000 per ml) for the first 2–4 days of culture is crucial for embryogenesis, after which cultures may be diluted to allow better embryo growth.Medium conditioned by culturing microspores at 30,000 or 40,000 per ml for 1 day improved microspore-embryo yield in low density cultures (3,000 or 4,000 per ml) more than 3-fold. In contrast, media conditioned with microspores from 1–4 days or 0–4 days of culture were inhibitory.Use of feeder cultures resulted in up to 10-fold increase of embryo yield in low density microspore cultures, depending on the method used. Filter papers and other membranes placed on top of feeders greatly inhibited embryogenesis in the feeder layer as well as microspores cultured on the feeder, possibly due to poorer gaseous exchange.