Lipid biosynthesis in cultures of oilseed rape

Summary This review focuses on how microspore-derived (MD) embros and cell suspension cultures of oilseed rape have been used to advance our understanding of the biochemistry and molecular biology of lipid biosynthesis in plants. Both types of cultures are easily maintained and circumvent the difficulties associated with using developing seeds for investigations of lipid biosynthesis. Developing MD embryos exhibit a similar storage lipid accumulation profile and fatty acid composition to developing seed. The use of dihaploids derived from plantlets of MD embryos have accelerated breeding programs and have proven useful in the detection of recessive mutations. MD embryos and MD cell suspension cultures have been particularly useful in investigating the properties of key enzymes involved in triacylglycerol (TG) bioassembly. MD cell suspension cultures, however, offer the advantage of being able to study lipid metabolism in the absence of cellular differentiation. TG accumulation can be induced in MD cell suspension cultures by increasing the sucrose concentration of the growth medium thereby providing a useful system to investigate gene expression and the proteomics of lipid biosynthesis.     

Metabolism of long-chain alcohols in cell suspension cultures of soya and rape

Heterotrophic cell suspension cultures of soya (Glycine max) and photomixotrophic cell suspension cultures of rape (Brassica napus) were incubated with cis-9-[1-¹⁴C]octadecenol for 3–48 h. It was found that under aerobic conditions large proportions of the alcohol are oxidized to oleic acid, which is incorporated predominantly into phospholipids, whereas up to 30% of the substrate is esterified to wax esters. This is true for both the heterotrophic and the photomixotrophic cell suspension cultures, but the metabolic rates are much higher in the latter. Under anaerobic conditions only small proportions of the radioactively labeled alcohol are oxidized to oleic acid, whereas a major portion of the alcohol is esterified to wax esters both in heterotrophic and photomixotrophic cultures. Incubations of homogenates of photomixotrophic rape cells with labeled cis-9-octadecenol showed that pH 6 is optimum for the formation of wax esters. This monounsaturated alcohol is preferred as a substrate over saturated longchain alcohols, whereas short-chain alcohols, cholesterol, and glycerol are not acylated. Incubations of an enzyme concentrate from a homogenate of rape cells with unlabeled cis-9-octadecenol and [1-¹⁴C]oleic acid, or [1-¹⁴C]stearoyl-CoA, or di[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine showed that acylation of the longchain alcohol proceeds predominantly through acyl-CoA. Direct esterification of the alcohol with fatty acid as well as acyl transfer from diacylglycerophosphocholine could be demonstrated to occur to a much smaller extent.     

High frequency production of embryos from liquid flask cultures of oilseed rape

As part of the program to scale-up the production of artificial seeds of winter oilseed rape, Brassica napus ssp. oleifera, we established a liquid flask culture system that enables the high frequency production of freely suspended embryos. As many as 4000 embryos could be obtained from 1 mL packed-cell-volume of cells. For initiation of liquid flask cultures, four different types of callus tissues were used. Among them, the most embryogenic cell suspension cultures were obtained from spontaneous callus developed on the surface of secondary embryos precultured in medium supplemented with 2,4-dichlorophenoxyacetic acid (4.52 muM) and kinetin (0.46 muM) (type B callus). Growth curves of the cell suspension were determined and the cell suspension was able to grow in medium without plant growth regulators. Embryos were observed to developed directly from the cells without going through an obvious callus phase. When subcultured to agar medium containing 44.38 muM benzylaminopurine, about 43% of the embryos developed into plants. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 231-238, 1997.     

Formation of complex ether lipids from 1-O-alkylglycerols in cell suspension cultures of rape

Photomixotrophic cell suspension cultures of rape, Brassica napus, were incubated with rac-1-O-[1′-¹⁴C]hexadecylglycerol. Radioactivity was incorporated predominantly into choline glycerophospholipids. Prolonged incubation led also to considerable proportions of labeled ethanolamine glycerophospholipids. In addition to these ionic lipids,isomeric hexadecylacylglycerols as well as hexadecyldiacylglycerols were formed. About a third of the hexadecylglycerol supplied as substrate was cleaved within 48 h incubation. The palmitic acid formed by oxidative cleavage of the substrate was incorporated predominantly into choline glycerophospholipids, ethanolamine glycerophospholipids, and triacylglycerols. Incubation of an equimolar mixture of homologous saturated rac-1-O-[1′¹⁴C]alkylglycerols (C₁₄, C₁₆, C₁₈, C₂₀) with rape cells showed that alkylglycerols with alkyl moieties having 16 and 18 carbon atoms were incorporated preferentially. Incubation of labeled hexadecyglycerol with a homogenate of rape cells led also predominantly to choline glycerophospholipids; highest yields were obtained at pH 7. Neither the 1-O-alkyl moieties in choline glycerophospholipis nor those in ethanolamine glycerophospholipids were desaturated to 1-O-alk-1′-enylmoieties. The results of these experiments led to the following conclusions: (1) The acylation of 1-O-alkylglycerols to isomeric alkylacylglycerols is catalyzed by two acyltransferases differing in their specificity with regard to the chain length of the alkyl moiety in the substrate. (2) CDP-Choline: diacylglycerol cholinephosphotransferase and CDP-ethanolamine: diacylglycerol ethanolaminephosphotransferase are two enzymes differing in various respects. Cholinephosphotransferase exhibits a much higher affinity for 1-O-alkyl-2-O-acylglycerols than ethanolaminephosphotransferase. The two enzymes show marked differences with regard to their specificity for 1-O-alkyl-2-O-acylglycerols differing in the chain lengths of their alkyl moieties.     

Microspore-derived cell suspension cultures of oilseed rape as a system for studying gene expression

Abiotic stress, such as extreme temperature, drought, or excessive salinity, is one of the leading causes of crop loss worldwide. Microspore-derived (MD) cell suspension cultures of Brassica napus L. cv. Jet Neuf have been shown to be a useful system for studying the biochemistry of developing oilseeds. In the present study, we describe the application of MD cell suspension cultures of B. napus as a system for studying gene expression in response to abiotic stress, and demonstrate emybryogenic competence in cultures that have been continuously subcultured for more than 20 years. MD cell suspension cultures of B. napus L. cv Jet Neuf were exposed to low temperature or osmotic stress and the expression profile of known stress responsive genes was evaluated. The gene expression profile of BN115, a known cold-responsive gene in B. napus, was similar to that described for intact cold-acclimated plants. Likewise, two late embryogenesis abundant (Lea) genes were shown to be up-regulated in response to low temperature or osmotic stress. The results demonstrate that B. napus MD cell suspension cultures are a useful system for the investigation of changes in gene expression in plants brought about by abiotic stress.     

Production of complex ether glycerolipids from exogenous alkylglycerols by cell suspension cultures of rape

Summary Neutral and ionic ether glycerolipids, especially alkylacylglycerophosphocholines and 1-alkenylacylglycerophosphocholines, are formed from exogenous 1-O-alkylglycerols, 1-O-(1-alkenyl)glycerols or 2-O-alkylglycerols by photomixotrophic cell suspension cultures of rape (Brassica napus). Best yields of ether glycerolipids were obtained by incubating rape cells with optically active 1-O-alkyl-sn-glycerols. Racemic or symmetric alkylglycerols are also utilized by rape cell suspension cultures for the biosynthesis of optically active ionic ether glycerolipids. In contrast, 3-O-hexadecyl-sn-glycerol is not incorporated into ether glycerophospholipids of rape cells. Incorporation of the substrates into ionic ether lipids is dependent on chain length (C₁₄>C₁₆>C₁₈) and degree of unsaturation (C_18:1C_18:0) of alkyl chains.Stereochemically uniform 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines and 2-O-alkyl-1-acyl-sn-glycero-3-phosphocholines with defined alkyl moieties can be prepared from exogenous alkylglycerols. This method recommends itself especially for the preparation of 1-O-(1-alkenyl)-2-acyl-sn-glycero-3-phosphocholines (choline plasmalogens) from 1-O-(1-alkenyl)-sn-glycerols. Ether glycerophospholipids with physiological activity, such as 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholines (platelet activating factor, PAF) and 1-O-alkyl-sn-glycero-3-phosphocholines (lyso PAF), were synthesized from 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines formed by cell suspension cultures of rape.     

外源四环素对土壤酶活性和油菜品质的影响

通过向土壤中添加四环素溶液研究种植油菜条件下,不同浓度（0、0.30、0.60、0.90 mg·kg^-1）的外源四环素对土壤酶活性和油菜品质的影响.结果表明：各处理土壤的脲酶与0.90 mg·kg^-1浓度处理的土壤转化酶活性在整个培养期均受到抑制,0.30、0.60 mg·kg^-1浓度处理的土壤转化酶活性则表现为激活-抑制-激活趋势,各处理土壤蛋白酶活性表现为抑制-激活趋势,且土壤酶活性受到外源四环素的影响程度及持续时间与处理浓度呈正相关（r=0.950**）.各处理土壤过氧化氢酶活性在前期被激活,后期差异不明显.四环素对土壤脲酶、过氧化氢酶、转化酶和蛋白酶活性的作用时间分别为：7周、6～8周、7周和6～7周.收获时0.30、0.60、0.90 mg·kg^-1处理的油菜叶片中可溶性糖含量与对照相比分别下降91.99%、87.92%和90.12%,而可溶性蛋白质含量分别上升26.47%、28.13%和23.22%.     

Cysteine synthase from rape leaves.

Cysteine synthase [O-Acetyl-L-serine acetate-lyase (adding hydrogen-sulfide) EC 4.2.99.8] has been highly purified from the extract of rape, Brassica chinensis var. Komatsuna. The purified preparation appeared to be homogeneous on Sephadex G-100 gel filtration and dodecylsulfate-polyacrylamide gel electrophoresis, showing a molecular weight of about 62,000. The latter method also suggested that this enzyme was composed of two identical subunits. The enzyme contained 2 moles of pyridoxal phosphate per mole of enzyme.     

Management of the rape victim.

A woman''s response to rape can be divided into three phases: an acute reaction, an intermediate stage and a period of resolution. Proper management of the physical and emotional problems of each phase, ideally by the woman''s family doctor or gynecologist, may prevent future problems. Treatment during the first phase includes responding to the emotional needs of the patient as well as doing a pelvic and general physical examination to detect any injuries; information for possible legal procedures may be obtained quickly and efficiently. Follow-up particularly psychological, is important in the second and third phases.     

Effect of copper on shoot and root regeneration in wheat,triticale, rape and tobacco tissue cultures

CuSO₄ (0.1–100 M) significantly enhanced shoot regeneration from calli of wheat and triticale and of tobacco leaf disc cultures. In cultures of wheat and triticale, CuSO₄ also stimulated root formation. When equal concentrations of CuSO₄ were applied in different media, it was found that the components of the basal media had only modifying effects. CuSO₄ pretreatment promoted plant survival when regenerated wheat plants were transferred directly to potting soil. In contrast with CuSO₄, AgNO₃, which also stimulated shoot regeneration, inhibited rooting in wheat and triticale. In Brassica napus callus cultures, AgNO₃ strongly increased morphogenesis, whereas CuSO₄ had no significant effect.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - KN kinetin - NAA naphthaleneacetic acid     

Light- and sucrose-dependent gene expression in photomixotrophic cell suspension cultures and protoplasts of rape (Brassica napus L.)

Light-dependent gene expression was analysed in photomixotrophic cell suspension cultures of rape (Brassica napus L.) growing in media containing either 2.0% or 0.6% sucrose. During growth in darkness phytochrome type I and NADPH-protochlorophyllide oxidoreductase (Pchlide reductase) accumulated in both cell culture lines to a similar extent. Illumination with continuous white, blue or red light, but not with far-red light, resulted in disappearance of both chromoproteins within 24 h in both cell cultures. Further analysis showed that the phytochrome system of rape cell cultures reacts in a similar way to that of re-etiolated dicotyledonous plants, showing rapid P_fr destruction and rapid P_fr dark reversion. In contrast, the light-dependent expression of genes encoding the major chlorophyll a- and b-binding protein (CAB) and the re-accumulation of chlorophyll were found to be strongly dependent on sucrose concentration in culture media. Whereas cells grown in darkness in medium containing 2.0% sucrose showed, after exposure to continuous white light, a very weak re-induction of CAB mRNA, CAB protein and chlorophyll accumulation, the cells in medium containing 0.6% sucrose reacted very strongly. It was also possible to demonstrate that phytochrome (by high irradiance response, HIR, and by low fluence response, LF) and the blue/UV-A receptor are involved in the light-dependent gene expression of CAB. Similar to complete cells, protoplasts derived from the two different cell cultures showed an almost identical sucrose concentration-dependent and light-quality-dependent regulation of CAB mRNA accumulation. As the dark-grown photomixotrophic cells and protoplasts reflect some typical photoregulatory characteristics known from dark-grown plants it is supposed that this system will be an excellent tool for studying biochemical and molecular biological aspects of light-dependent signal transduction in cells of higher plants.     

Organogenesis and plant formation from cotyledon explant cultures of wild turnip rape (Brassica tournefortii L.)

Cotyledon explants of Brassica tournefortii L. were excised from germinated seedlings and cultured on Murashige & Skoog's [6] basal medium supplemented with various combinations of cytokinins and auxins, Both cytokinin and auxin were required for induction of shoot organogenesis. Of the three cytokinins tested (in combination with a low concentration of IAA), kinetin was found to be the best for shoot regeneration. On this medium, cotyledonary explants invariably underwent callusing followed by multiple shoot formation. NAA in combination with any of the three cytokinins yielded a reduced number of shoots or none, but favoured good callus growth. Callus so produced also regenerated shoots when subcultured on media containing high concentration of KIN or ZEA and low concentration of IAA. Shoots were rooted during prolonged incubation on the same medium or on MS medium free of growth regulators. Mature plants were grown in the greenhouse.     

Lipids in plant tissue cultures. III. Very long-chain fatty acids in the lipids of callus cultures and suspension cultures

The lipids in plant tissue cultures contain in addition to the common saturated and unsaturated fatty acids even- and odd-numbered fatty acids having chain-lengths up to 26 carbon atoms.