The effect of different temperatures on fatty-acid synthesis and polyunsaturation in cell suspension cultures

Cell suspension cultures of Catharanthus roseus G. Don, Glycine max (L.) Merr. and Nicotiana tabacum L. were incubated with [¹⁴C]acetate, [¹⁴C]oleic acid and [¹⁴C]linoleic acid at five different temperatures ranging from 15 to 35° C. When the incubation temperature was increased, [¹⁴C]acetate was incorporated preferentially into [¹⁴C]palmitate, with a concomitant drop in [¹⁴C]oleate formation. Between 15 and 20° C, [¹⁴C]oleic acid accumulated in C. roseus cells. In all cultures, optimum desaturation of [¹⁴C]oleic acid to [¹⁴C]linoleic acid occurred between 20 and 25° C, and in G. max this was also the optimal range for desaturation of [¹⁴C]linoleic acid to [¹⁴C]linolenic acid. Elongation of [¹⁴C]palmitic acid was inhibited when cultures grown at 15° C for 25 h were subsequently incubated with [¹⁴C]acetate at 25° C. [¹⁴C]oleic acid accumulated in G. max and C. roseus cultures grown at 35° C for 25 h and subsequently incubated at 25° C. Desaturation of [¹⁴C]oleic acid increased up to 25° C, but then decreased or leveled off depending on the cell line and on the temperature prior to incubation.     

Fatty-acid composition and biosynthesis in cell suspension cultures of Glycine max (L.) Merr., Catharanthus roseus G. Don and Nicotiana tabacum L.

The fatty-acid composition of C. roseus and N. tabacum cell suspension cultures was unaffected by subculture on Wood and Braun, Murashige and Skoog, or Gamborg B5C media. However, placing the cultures — which were normally grown at 25° C — at 15° C reduced growth but resulted in enhanced formation of oleic and linolenic acids in C. roseus cultures and increased levels of linoleic and linolenic acids in cultures of G. max and N. tabacum, respectively. The incorporation of [¹⁴C]acetate into [¹⁴C]linoleic acid was more rapid in N. tabacum cells than in G. max cells, but was very poor in C. roseus where the [¹⁴C] label was distributed mainly between palmitic and oleic acids.     

Unusual fatty acids in the lipids from organs and cell cultures ofPetroselinum crispum

The lipids of seeds, leaves, and roots of parsley,Petroselinum crispum, and of heterotrophic as well as photomixotrophic cell cultures of this plant were characterized with the aim of finding a system for studying the biosynthesis of unusual fatty acids. It was found that (Z)-6-octadecenoic acid, petroselinic acid, which is the typical constituent fatty acid of triacylglycerols in seeds, occurs only in small proportions, if at all, in leaves, roots, and cell cultures of parsley. In all lipid classes studied petroselinic acid is accompanied by its (Z)-9- and (Z)-11-isomers, oleic and vaccenic acid, respectively. The phosphatidylcholines, phosphatidylethanolamines, and triacylglycerols of both heterotrophic and photomixotrophic callus cultures contain no petroselinic acid but rather oleic and vaccenic acids in equal ratios. Thus, cell cultures of parsley appear to be suitable for studying the biosynthesis of vaccenic acid. The constituent octadecadienoic acids in the lipids of various tissues and cell cultures of parsley consist almost exclusively of the (Z),(Z)-9,12-isomer, linoleic acid, which is derived from oleic acid. (Z),(Z)-6,9- and (Z),(Z)-11,14-Octadecadienoic acids, which could be expected as products of desaturation of petroselinic and vaccenic acids, were not found in any of the lipids of organs and cell cultures investigated.Abbreviations TLC thin-layer chromatography - GLC gas-liquid chromatography     

In Vivo Pathway of Oleate and Linoleate Desaturation in Developing Cotyledons of Cucumis sativus L. Seedlings

Exogenous [1-¹⁴C]oleic acid and [1-¹⁴C]linoleic acid were taken up and esterified to complex lipids by greening cucumber (Cucumis sativus L.) cotyledons. Both ¹⁴C-labeled fatty acids were initially esterified to phosphatidylcholine prior to eventual accumulation in triacylglycerols and galactolipids. Kinetic data suggest that esterification occurs prior to desaturation and that phosphatidylcholine is the initial site of both [¹⁴C]-oleate and [1-¹⁴C]linoleate esterification and of [1-¹⁴C]oleate desaturation to [1-¹⁴C]linoleate. [1-¹⁴C]Linoleic acid was esterified more rapidly than [¹⁴C]oleic acid and its desaturation product, [1-¹⁴C]α-linolenate, occurred mainly on monogalactosyl diacylglycerol, although some was also observed on the other major acyl lipids, including phosphatidylcholine.     

Inhibitory effect of ethanol on growth and solute accumulation by Saccharomyces cerevisiae as affected by plasma-membrane lipid composition

Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rate, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing d-[6-³H] glucose, d-[1-¹⁴C] glucosamine, l-[U-¹⁴C] lysine or arginine, or KH₂ ³²PO₄ lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5–7 times greater than that in the cell-free medium.     

Particulate fractions from Chloroflexus aurantiacus and distribution of lipids and polyprenoid forming activities

Two new particulate fractions, wax oleosomes and chlorophyll-free spindle-shaped particles in addition to chlorosomes and plasma membrane vesicles, were isolated and characterized from homogenates of the photosynthetic bacterium Chloroflexus aurantiacus. The wax oleosomes contained the bulk of cellular wax esters and carotenoids. Wax esters were also present in the spindle-shaped particles, chlorosomes, and plasma membrane vesicles. The spindleshaped particles were the only particulate fraction active in polyprenol and carotene synthesis from [1-¹⁴C]isopentenyl diphosphate. The significance of wax esters and spindle-shaped particles is discussed.     

Metabolism of alkyl glyceryl ethers and their noninvolvement in alkane biosynthesis in plants

[1-¹⁴C]Octadecyl glyceryl ether did not label alkanes in the leaves of Brassica oleracea and Pisum sativum while [1-¹⁴C]octadecanol and [1-¹⁴C]octadecanoic acid readily labeled the alkanes. About 40% of the exogenous-labeled glyceryl ether was incorporated intact into choline phosphatide while 10–20% was converted into fatty acids and alcohols. [1-¹⁴C]octadecanol was not converted into alkyl glyceryl ether, but it was oxidized to the corresponding acid and then incorporated into alkanes. These results show that alkyl ether is not an intermediate in alkane biosynthesis. When [1-¹⁴C-1-³H]-octadecanol was fed to the leaves of B. oleracea and P. sativum, only the ¹⁴C and no ³H was incorporated into alkanes, ketones, and secondary alcohols. These results show that fatty alcohols are first oxidized to the acid before being incorporated into alkanes, ruling out fatty alcohol, alkyl ether, and alk-1-enyl ether as intermediates in alkane biosynthesis. The exogenous alcohols were also readily esterified into wax esters in both tissues.     

Regioselective synthesis of kojic acid esters by Bacillus subtilis protease

The lipophilicity of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one] was improved by esterifying kojic acid with either divinyl adipate, vinyl hexanoate, vinyl octanoate or vinyl decanoate using protease from Bacillus subtilis for 7 d. ¹H-NMR and ¹³C-NMR showed that the primary hydroxyl group at the C-7 position of kojic acid was regioselectively esterified to afford 7-O-vinyl adipoyl kojic acid, 7-O-hexanoyl kojic acid, 7-O-octanoyl kojic acid and 7-O-decanoyl kojic acid (13–27% yield). The kojic acid esters had radical scavenging activities, inhibited tyrosinase activity and was biodegradable.     

Rapid recovery of photosynthetic cell suspension cultures following heterotrophic bleaching

Summary Seven suspension-cultured lines of five different species (Amaranthus powellii Datura innoxia, Glycine max, Gossypium hirsutum, andNicotiana tabacum × Nicotiana glutinosa fusion hybrid), which had been grown under photomixotrophic conditions, were placed under heterotrophic conditions (darkness and media with 3% sucrose or starch) where the chlorophyll levels declined to near zero. After three transfers over a 70-d period, the cells were placed back into photomixotrophic or photoautotrophic conditions where regreening occurred rapidly and continued growth was observed. This rapid adaptation to photosynthetic conditions contrasts with the original initiation process for these cultures, which required many months and an apparent selection since many of the original cells died. Thus, these seven photosynthetic cell suspension cultures appear to be different from the original cultures due possible to genetic or adaptive changes.     

Semi-synthetic preparation of 1-O-[1'-14C]hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) using plant cell cultures

Incubation of photomixotrophic cell suspension cultures of rape (Brassica napus) and heterotrophic cell suspension cultures of soya (Glycine max) with 1-O-[1'-14C]hexadecyl-sn-glycerol or rac-1-O-[1'-14C]hexadecylglycerol leads in high yield (up to 78%) to labeled 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines. Alkaline hydrolysis of the choline glycerophospholipids yields pure 1-O-[1'-14C]hexadecyl-sn-glycero-3-phosphocholine. 1-O-[1'-14C]Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) is obtained by acetylating the lyso compound. The semi-synthetic preparation described leads to labeled platelet activating factor in an overall yield of 50-60% without loss of specific activity.     

Fatty acid synthesis by slices from developing leaves

Fatty acid synthesis was studied in successive leaf sections from the base to the tip of developing barley (Hordeum vulgare L.), maize (Zea mays L.), rye grass (Lolium perenne L.) and wheat (Triticum aestivium L.) leaves. The basal regions of the leaves had the lowest rates of fatty acid synthesis and accumulated small amounts of very long chain fatty acids. Fatty acid synthesis was highest in the middle leaf sections in all four plants. Linolenic acid synthesis from [1-¹⁴C]acetate was highest in the distal leaf sections of rye grass. The labelling of the fatty acids of individual lipids of rye grass was examined and it was found that [¹⁴C]linolenic acid was highest in the galactolipids. Synthesis of this acid in the galactolipids was most active in leaf segment C. Only traces of [¹⁴C]linolenic acid were ever found in phosphatidylcholine and it is concluded that this phospholipid cannot serve as a substrate for linoleic acid desaturation in rye grass. The synthesis of fatty acids was sensitive to arsenite, fluoride and the herbicide EPTC. The latter was only inhibitory towards those leaf segments which made very long chain fatty acids. Formation of fatty acids from [1-¹⁴C]acetate was also studied in chloroplasts prepared from successive leaf sections of rye grass. Chloroplasts isolated from the middle leaf sections had the highest activity. Palmitic and oleic acids were the main fatty acid products in all chloroplast preparations. Linolenic acid synthesis was highest in chlorplasts isolated from the distal leaf sections of rye grass.     

Biosynthesis of myo-inositol and its role as a precursor of cell-wall polysaccharides in suspension cultures of wild-carrot cells

The biosynthesis of myo-inositol (MI) and its role as a precursor of cell-wall polysaccharides was studied in supension cultures of wild carrot (Daucus carota L.) cells. Suspension cultures, grown in the presence or absence of 2,4-dichlorophenoxyacetic acid for 7 and 14d were incubated with [U-¹⁴C]glucose and [2-³H]MI in the presence of different concentrations of unlabeled MI. Synthesis of [¹⁴C]MI from [U-¹⁴C]glucose occurred under all conditions. The amount of MI synthesized from glucose was sharply reduced when 10 mM MI was provided in the medium. Substantial quantities of ³H were incorporated in arabinose, xylose and galacturonic acid isolated and purified from the cell-wall polysaccharides of the cell cultures in various stages of growth or embryogenesis. No ³H was present in the glucose or galactose units of cell-wall polysaccharides. At the four stages of growth and states of development of the carrot cultures used, the MI oxidation pathway contributed to the synthesis of pentosyl and galacturonosyl units of the cell wall. However, the data indicate that the contribution of the MI oxidation pathway to pentosyl and galacturonosyl units is small.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MI myo-inositol     

The biosynthesis of indole-3-acetic acid byFrankia

Summary High perfomance liquid chromatography (HPLC) of the products of [5-³H] tryptophan metabolism byFrankia sp. Avc I1 indicates that small amounts of [³H] indole-3-acetic acid (IAA) are excreted into the growth medium.Frankia has a limited capacity for the catabolism of [2-¹⁴C]IAA and the product that accumulates is different from that detected inRhizobium japonicum cultures following inoculation with [2-¹⁴C]IAA. The data imply that the rate of turnover of IAA is much more rapid inRhizobium thanFrankia and that the two organisms employ different routes for the catabolism of IAA.     

Carbohydrate oxidation by leucoplasts from suspension cultures of soybean (Glycine max L.)

The aim of this work was to discover how leucoplasts from suspension cultures of soybean (Glycine max L.) oxidize hexose monophosphates. Leucoplasts were isolated from protoplast lysates on a continuous gradient of Nycodenz with a yield of 28% and an intactness of 80%. Incubation of the leucoplasts with ¹⁴C-labelled substrates led to ¹⁴CO₂ production, that was dependent upon leucoplast intactness, from [U-¹⁴C]glucose 6-phosphate, [U-¹⁴C]glucose 1-phosphate, [U-¹⁴C] fructose 6-phosphate and [U-¹⁴C]glucose+ATP, but not from [U-¹⁴C]fructose-1,6-bisphosphate or [U-¹⁴C]triose phosphate. The yield from [U-¹⁴C]glucose 6-phosphate was at least four times greater than that from any of the other substrates. When [1-¹⁴C]-, [2-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose 6-phosphate were supplied to leucoplasts significant ¹⁴CO₂ production that was dependent upon leucoplast intactness was found only for [1-¹⁴C]glucose 6-phosphate. It is argued that soybean cell leucoplasts oxidize glucose 6-phosphate via the oxidative pentose phosphate pathway with very little recycling, and that in these plastids glycolysis to acetyl CoA is negligible.S.A.C. thanks the Science and Engineering Research Council for a research studentship.     

Comparison of secondary product accumulation in photoautotrophic,photomixotrophic and heterotrophic Nicotiana tabacum cell suspension cultures

Summary Photoautotrophic, photomixotrophic and heterotrophic Nicotiana tabacum cell suspension cultures were compared for the constitutive accumulation of secondary metabolites and the elicitor-induced formation of the phytoalexin capsidiol. Nicotine and chlorogenic acid were found in high amounts in the heterotrophic cultures and in moderate concentrations in photomixotrophic but not in photoautotrophic cells. Nicotinic acid-N-glucoside occured in all culture types; in photoautotrophic and photomixotrophic cells the formation of N-methylnicotinic acid (trigonelline) was also observed. Treatment with a fungal elicitor led to substantial accumulation of capsidiol in heterotrophic and photomixotrophic cells and in only low levels in photoautotrophic cultures. Elicitor-treated photomixotrophic cells showed a pronounced increase in cell wall-bound phenolics. The levels of nicotine, nicotinic acid-N-glucoside and trigonelline were not affected by elicitation.Abbreviations hcc heterotrophic cell culture - mcc photomixotrophic cell culture - pcc photoautotrophic cell culture - fr.wt. freshweight - nic-N-glc nicotinic acid-N-glucoside - PMG Phytophthora megasperma f. sp. glycínea - HPLC high performance liquid chromatography - GC gas chromatography - TLC thin layer chromatography - 2,4D 2,4-dichlorophenoxyacetic acid - Kin kinetin - BAP 6-benzylaminopurine - NAA -naphthylacetic acid     

The lipids in photoautotrophic and heterotrophic cell suspension cultures of Chenopodium rubrum

Resembling the lipids in the leaves and other green organs of intact plants, the lipids in photoautotrophic cell cultures of Chenopodium rubrum were found to contain high proportions of monogalactosyldiacylglycerols and digalactosyldiacylglycerols, as well as fair amounts of sulfoquinovosyldiacylglycerols and diacylglycerophosphoglycerols. Conversely, the heterotrophic cell cultures, from which the photoautotrophic cultures had been derived, contained only traces of these compounds. The heterotrophic cultures were rich in sterols, sterol esters, sterol glycosides, and esterified sterol glycosides. The lipids of photoautotrophic cell cultures contained higher proportions of constituent linolenic acid, but lower concentrations of linoleic acid than those of heterotrophic cultures. In the photoautotrophic cultures, as in green leaves, linolenic acid was predominantly estrified in monogalactosyldiacylglycerols and digalactosyldiacylglycerols. This investigation shows that it is possible to select strains of cell cultures, which are capable of grosing photoautotrophically, with the aim of activating the biosynthesis of specific metabolites.     

Single step thin-layer chromatographic method for quantitation of enzymatic formation of fatty acid anilides

The activity of the enzyme involved in catalyzing the formation of fatty acid anilides can be measured by quantitating the fatty acid anilides formed. We have shown earlier that oleic acid is the most preferred substrate among other fatty acids studied for the conjugation with aniline. The reaction product (oleyl anilide) could be separated by thin-layer chromatography (TLC) and then quantified by reversed-phase high-performance liquid chromatography (HPLC). Using [1-¹⁴C]oleic acid as substrate, the fatty acid anilide forming activity can be determined in a single step by TLC analysis. The conventional TLC methods used for the separation of the fatty acid esters, however, could not resolve oleyl anilide from the residual [1-¹⁴C]oleic acid. Therefore, a simple and reliable TLC method was developed for the separation of oleyl anilide from oleic acid using a freshly prepared solvent consisting of petroleum ether–ethyl acetate–ammonium hydroxide (80:20:1, v/v). Using this solvent system the relative flow (R_f) values were found to be 0.54 for oleyl anilide and 0.34 for aniline, whereas oleic acid remained at the origin. The TLC procedure developed in the present study could be used to determine the fatty acid anilide forming activity using [1-¹⁴C]oleic or other fatty acids as substrate and was also found suitable for the analysis of fatty acid anilides from the biological samples.     

Intraprotoplasmic feruloylation of arabinoxylans in Festuca arundinacea cell cultures

Graminaceous primary cell walls contain polysaccharides to which are esterified feruloyl residues. Ester biosynthesis is highly specific and the present experiments were performed to ascertain the likely site of feruloylation in living grass cell cultures. Cell cultures of tall fescue grass (Festuca arundinacea Schreber) incorporated exogenous l-[1-³H]arabinose into polymers at a linear rate after a short lag of approx. 1–3 min. Radiolabelled polymers did not start to accumulate in the culture medium until 20–35 min after [³H]arabinose was supplied. However, polymer-bound feruloyl-arabinose residues began to accumulate ³H after a lag of 1–3 min. Assuming that the onset of secretion of radiolabelled polymers into the medium indicates the time before which essentially all the radiolabel was internal to the plasma membrane, the results show that the polysaccharide-bound [³H]arabinose residues must have been feruloylated within the protoplast.Abbreviations AIR alcohol-insoluble residue - BAW butan1-ol/acetic acid/water (12:3:5 by volume) - BEW butan-1-ol/ ethanol/water (20:5:11 by volume) - EPW ethyl acetate/pyridine/ water (8:2:1 by volume) - R_Ara Chromatographic mobility relative to that of l-arabinose We are very grateful to Mr. Gundolf Wende for assistance with the characterisation of the feruloyl esters. K.E.M. is funded by a studentship from the Science and Engineering Research Council in collaboration with Zeneca Agrochemicals.     

Elicitor-mediated induction of enzymes of lignin biosynthesis and formation of lignin-like material in a cell suspension culture of spruce (Picea abies)

Incubations of photomixotrophic suspension culture cells of spruce (Picea abies) (L.) (Karst) with an autoclaved cell wall preparation of Rhizosphaera kalkhoffii as elicitor led to a rapid increase of the activity of a number of enzymes involved in lignin biosynthesis. l-phenylalanine ammonia-lyase (EC 4.3.1.5) was induced about 10-fold, feruloyl-Coenzyme A reductase (ED 1.2.1.44) 4-fold, cinnamyl alcohol dehydrogenase (NADP⁺) (EC 1.1.1.195) 2-fold and peroxidase (EC 1.11.1.7) about 1.5-fold. The induction of the enzymes, with the exception of the peroxidase, was transient, showing maximal activity within 3 days after elicitation. Extracellular peroxidase activity, determined in the culture medium, rapidly decreased on initiation of elicitation.Concomitant with the increase of activity of the enzymes of lignin synthesis was a rapid clouding of the culture medium. Phloroglucinol-HCl staining revealed the presence of lignin-like material in the medium and also in the cells. The IR-spectrum of this material was identical with the IR-spectrum of authentic spruce lignin.Abbreviations PAL l-phenylalanine ammonia-lyase - FCR feruloyl-Coenzyme A reductase - CAD cinnamyl alcohol dehydrogenase - POD peroxidase