Correlation of housefly sex pheromone production with ovarian development

Pheromone production in the housefly was monitored during oögenesis and in ovariectomized insects by gas-liquid chromatography (GLC) and radio-GLC. The presence of vitellogenic ovaries was required for the initiation of (Z)-9-tricosene (muscalure), (Z)-9,10-epoxytricosane and (Z)-14-tricosen-10-one synthesis. Methylalkane synthesis was enhanced by developing ovaries. Insects ovariectomized within 12 hr after emergence produced no detectable amounts of (Z)-9-tricosene, C₂₃ epoxide nor C₂₃ ketone and synthesized less methylalkanes than the controls. This effect was reversed by ovary implants. When flies were ovariectomized after oviposition, synthesis of (Z)-9-tricosene, C₂₃ epoxide and C₂₃ ketone continued. Thus, initiation of the synthesis of these C₂₃ pheromone compounds required a vitellogenic ovary, but the ovary was not required to maintain synthesis.     

Induction of female sex pheromone production in male houseflies by ovary implants or 20-hydroxyecdysone

Implanting ovaries or injecting 20-hydroxyecdysone into male houseflies induced sex pheromone production, including (Z)-9-tricosene (muscalure), 9,10-epoxytricosane and (Z)-14-tricosen-10-one, which normally occurs only in vitellogenic females. Control males did not produce detectable amounts of these compounds. Injection of 20-hydroxyecdysone (5 μg/insect per day) for 3 days resulted in the accumulation of 1.81 μg/insect of (Z)-9-tricosene, 0.97 μg/insect of 9,10-epoxytricosane and 0.12 μg/insect (Z)-14-tricosen-10-one. Multiple injections of 20-hydroxyecdysone at doses as low as 50 ng resulted in the accumulation of 23:1, C₂₃ epoxide and C₂₃ ketone; shifted the distribution of label within the alkenes from 27:1 to 23:1 and decreased the amount of label in the hydrocarbon fractions as alkenes. Structures of the C₂₃ alkene and epoxide produced by the males were verified by gas chromatography-mass spectrometry. Radioactivity from [1-¹⁴C] acetate was incorporated into the C₂₃ alkene, epoxide and ketone in male insects after ovaries were implanted or they were injected with 20-hydroxyecdysone. Synthesis of the C₂₃ pheromone components decreased rapidly within several days after the administration of 20-hydroxyecdysone ceased, indicating that the enzymes involved in sex pheromone production were not permanently induced by hormone treatment. Ecdysone was also effective in initianing pheromone production in males, whereas inokosterone and cholesterol were not effective. Data presented demonstrate that male houseflies possess the metabolic capability to produce the sex pheromone components, and this suggests that 20-hydroxyecdysone alters the production of cuticular hydrocarbons such that the C₂₃ sex pheromone components become major products.     

Evidence for a sex pheromone metabolizing cytochrome P-450 mono-oxygenase in the housefly

Direct evidence is presented for the role of a cytochrome P-450 monooxygenase (called mixed-function oxidase, or polysubstrate mono-oxygenase, PSMO) in the metabolism of the sex pheromone (Z)-9-tricosene to its corresponding epoxide and ketone in the housefly. A secondary alcohol, most likely an intermediate in the conversion of the alkene to the ketone, was also tentatively identified. The results of in vivo and in vitro experiments showed that the PSMO inhibitors, piperonyl butoxide (PB) and carbon monoxide, markedly inhibited the formation of epoxide and ketone from (9,10-³H) (Z)-9-tricosene. An examination of the relative rates of (Z)-9-tricosene metabolism showed that males exhibited a higher rate of metabolism than females with the antennae of males showing the highest activity of any tissue/organ examined. The major product from all tissues/organs was the epoxide. Data from experiments with subcellular fractions showed that the microsomal fraction had the majority of enzyme activity, which was strongly inhibited by PB and CO and required NADPH and O₂ for activity. A carbon monoxide difference spectrum with reduced cytochrome showed maximal absorbance at 450 nm and allowed quantification of the cytochrome P-450 in the microsomal fraction of 0.410-nmol cytochrome P-450 mg^?1 protein. Interaction of (Z)-9-tricosene with the cytochrome P-450 resulted in a type I spectrum, indicating that the pheromone binds to a hydrophobic site adjacent to the heme moiety of the oxidized cytochrome P-450.     

Effect of age and sex on the production of internal and external hydrocarbons and pheromones in the housefly, Musca domestica

The epicuticular and internal waxes of male and female houseflies were examined by capillary gas chromatography-mass spectrometry at closely timed intervals from emergence until day-6 of adulthood. New components identified included tricosan-10-one, 9,10-epoxyheptacosane, heptacosen-12-one, a series of odd-carbon numbered dienes from C31 to C39, several positional isomers of monoenes including (Z)-9- and 7-pentacosene and a number of methyl- and dimethylalkanes. (Z)-9-tricosene appears in internal lipids prior to appearing on the surface of the insect, suggesting that it is transported in the hemolymph to its site of deposition on the epicuticle. The large increases in the amount of (Z)-9-tricosene in females from day-2 until day-6 is compensated for by a concomitant decrease in (Z)-9-heptacosene. The C23 epoxide and ketone only appear in females after the production of (Z)-9-tricosene is induced, and are only abundant in epicuticular waxes, suggesting they are formed after (Z)-9-tricosene is transported to the cells which are involved in taking them to the surface of the insect. Mathematical analysis indicated that the time shift between internal production and external accumulation in females is more than 24 h. The divergence between male and female lipid production occurs at an early stage, when insects are less than one day old.     

The effects of laboratory culturing on (Z)-9-tricosene (muscalure) quantities on female houseflies

Using gas chromatography the relative amounts of (Z)-9-tricosene (muscalure) and some other hydrocarbons on the cuticle of 1- to 20-day-old houseflies (Musca domestica L.) from different strains were determined. Flies from a WHO strain, in culture since 1961, and first-generation laboratory-cultured flies from two wild-type strains from a poultry breeding and a cow-house with pigsty, respectively, were compared. On WHO females hydrocarbons with 23–25 C atoms constituted about 65% of the total hydrocarbons, whereas on wild-type females less than 2% of these compounds was present. (Z)-9-tricosene comprised up to 20–30% of the total hydrocarbons on 5- to 20-day-old WHO females, whereas less than 0.5% (Z)-9-tricosene was present on the wild-type females. We also compared the amounts of (Z)-9-tricosene and some other hydrocarbons on female houseflies, kept in culture in the laboratory for several generations. It appeared that whereas on first-generation wild-type females hardly or no (Z)-9-tricosene could be detected, the amounts of this substance had increased considerably after some tens of generations in the laboratory. It is suggested that this was due to selection in subsequent generations of high-density populations. Production of (Z)-9-tricosene and of tricosane was shown to be closely linked. Selection did not affect the production of other cuticular hydrocarbons by the females. It is suggested that in mixed populations (both sexes together in a cage) in the course of time (Z)-9-tricosene is transferred from females to males and (Z)-9-heptacosene from males to females. It is concluded that reproductive ability of houseflies does not primarily depend on the amounts of (Z)-9-tricosene on females, although higher amounts of this substance may increase contacts between males and females.     

Direct evidence for the involvement of epoxide intermediates in the biosynthesis of the C18 family of cutin acids

Cutin, the structural component of plant cuticle, is a polymer of C₁₆ and C₁₈ hydroxy fatty acids. Previous results have suggested that oleic acid undergoes ω-hydroxylation, epoxidation of the double bond, and, finally, hydration of the epoxide to give rise to the three major components of the C₁₈ family of cutin acids. 18-Hydroxy [18-³H]oleic acid and 18-hydroxy-9,10-epoxy[18-³H]stfaric acid have been synthesized and, with these synthetic substrates, the conversion of 18-hydroxyoleic acid to 18-hydroxy-9,10-epoxystearic acid and the hydrolysis of 18-hydroxy-9,10-epoxystearic acid to 9,10,18-trihydroxystearic acid were directly demonstrated in apple fruit skin and in the leaves of apple and Senecio odoris. Trichloropropene oxide, an inhibitor of microsomal epoxide hydrases of animals, specifically inhibited the conversion of [1-¹⁴C]oleic acid into 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid, while it had no effect on the conversion of [1-¹⁴C]palmitic acid into hydroxylated palmitic acid, a process which does not involve epoxy acid intermediates. Therefore, it appears that this inhibitor affects epoxidation and or epoxide hydration steps involved in cutin biosynthesis.     

Epicuticular hydrocarbons of Drosophila pseudoobscura (Diptera; Drosophilidae) Identification of unusual alkadiene and alkatriene positional isomers

Epicuticular hydrocarbons of Drosophila pseudoobscura were analyzed by gas chromatography and mass spectrometry. Methyl-branched alkanes and alkadienes were the predominant hydrocarbons, with lesser amounts of monoenes (14%) and trienes (9%) also present. Alkanes (49%) were mostly odd carbon number 2-methylalkanes (C₂₅–C₃₁). Alkadienes (27%) were odd carbon number components (C₂₅–C₃₃), with the (Z,Z)-5,9-isomer predominating. Monounsaturated hydrocarbons were a mixture of 5-, 7-, 9-, 11-, 13-, 14- and 15-isomers containing 25–33 carbon atoms. The major alkatriene components contained 29–31 carbon atoms and were either 5,19,17- or 5,9,19-isomers. Sodium[1-¹⁴C]acetate was incorporated into each class of hydrocarbon and into each of the major alkadienes.     

Adult house fly (Diptera: Muscidae) activity and age of females near varying levels of (Z)-9-tricosene on a southern California dairy

The number of adult male and female house flies, Musca domestica L. (Diptera: Muscidae), near varying levels of (Z)-9-tricosene alone (5, 50, or 100 micdrol) or combined (50 microl) with sugar was determined using conical screened traps on a dairy in southern California. Overall, significantly more males than females were collected in the traps. Significantly more flies (male and female) were collected in traps with (Z)-9-tricosene. There were no significant differences among doses of (Z)-9-tricosene alone, but numbers of both sexes were significantly higher in traps baited with (Z)-9-tricosene and sugar compared with the 5- and 50-microl doses without sugar. The age of female flies collected in traps was determined by pterin analysis. Mean female ages ranged from 94.7 to 99.6 degree-days (6.3-6.8 d of age) and did not differ significantly among treatments. Dissections of a subset of females from each treatment determined that collected females were primarily nongravid (86.3%). Proportions of gravid females that were collected did not differ among treatments.     

The incorporation of [1-14C]acetate into the methyl ketones that occur in steam-distillates of bovine milk fat

1. The ¹⁴C-labelling of the fatty acids and the methyl ketones in steam-distillates of milk fat from a lactating cow that had been injected intravenously with [1-¹⁴C]acetate was determined. 2. The labelling patterns of the C₆–C₁₆ fatty acids and the corresponding methyl ketones with one fewer carbon atoms were similar, particularly so for the C₅–C₁₀ compounds at 9 and 22hr. after the injection of [1-¹⁴C]acetate. The isolation of ¹⁴C-labelled methyl ketones in the range C₃–C₁₅ is evidence that the β-oxo acid precursors, which are glyceride-bound in the milk fat, are synthesized in the mammary gland from acetate. The absence of heptadecan-2-one in steam-distillates and the extremely low specific radioactivity of stearic acid are further evidence for this biosynthetic pathway. 3. The specific radioactivities of the C₅–C₁₅ methyl ketones were higher (with the exception of C₉ methyl ketone in the second milking) than the specific activities of the corresponding fatty acids with one more carbon atom. This is consistent with the methyl ketone precursors' being formed during the biosynthesis of fatty acids rather than being products of β-oxidation of fatty acids.     

Regulation of sex pheromone biosynthesis in the housefly, Musca domestica: relative contribution of the elongation and reductive steps.

The regulation of production of the sex pheromone (Z)-9-tricosene (Z9-23:Hy) in the housefly, Musca domestica, was studied by examining the chain length specificity of the fatty acyl-CoA elongation reactions and the reductive conversion of fatty acyl-CoAs to alkenes in 1- and 4-day-old male and female houseflies. Microsomal preparations from 4-day-old female insects produced as the predominant alkene Z9-23:Hy when incubated with malonyl-CoA, NADPH, and [9,10-3H2]oleoyl-CoA (18:1-CoA), whereas microsomal preparations from 4-day-old male insects produced predominantly (Z)-9-heptacosene (Z9-27:Hy). These are the major alkenes produced in vivo by Day 4 females and males, respectively. Microsomes prepared from both Day 1 males and Day 1 females produced Z9-27:Hy as the major alkene from labeled 18:1-CoA. This is the major alkene produced in vivo by both sexes at Day 1. An examination of the chain length specificity of the elongation reactions showed that microsomes prepared from Day 4 male insects readily elongated both 18:1-CoA and 15-[15,16-3H2]tetracosenoyl-CoA (24:1-CoA) to 28-carbon moieties, whereas microsomes from Day 4 female insects did not efficiently elongate either substrate beyond 24 carbons. With high substrate concentrations, microsomes prepared from male insects converted 24:1-CoA to Z9-23:Hy more efficiently than did those from females, whereas under lower and presumably more physiological substrate concentrations, microsomes from females had slightly higher activity than did those from males. Taken together, these data show that the regulation of the chain length of the alkenes, and thus sex pheromone production, in the housefly resides predominantly in the elongation reactions and not in the step which converts the fatty acyl-CoA to hydrocarbon.     

Biochemistry of Suberization: Incorporation of [1-C]Oleic Acid and [1-C]Acetate into the Aliphatic Components of Suberin in Potato Tuber Disks (Solanum tuberosum)

Biosynthesis of the aliphatic components of suberin was studied in suberizing potato (Solanum tuberosum) slices with [1-¹⁴C]oleic acid and [1-¹⁴C]acetate as precursors. In 4-day aged tissue, [1-¹⁴C]oleic acid was incorporated into an insoluble residue, which, upon hydrogenolysis (LiA1H₄), released the label into chloroform-soluble products. Radio thin layer and gas chromatographic analyses of these products showed that ¹⁴C was contained exclusively in octadecenol and octadecene-1, 18-diol. OsO₄ treatment and periodate cleavage of the resulting tetraol showed that the labeled diol was octadec-9-ene-1, 18-diol, the product expected from the two major components of suberin, namely 18-hydroxyoleic acid and the corresponding dicarboxylic acid. Aged potato slices also incorporated [1-¹⁴C]acetate into an insoluble material. Hydrogenolysis followed by radio chromatographic analyses of the products showed that ¹⁴C was contained in alkanols and alkane-α,ω-diols. In the former fraction, a substantial proportion of the label was contained in aliphatic chains longer than C₂₀, which are known to be common constituents of suberin. In the labeled diol fraction, the major component was octadec-9-ene-1,18-diol, with smaller quantities of saturated C₁₆, C₁₈, C₂₀, C₂₂, and C₂₄-α,ω-diols. Soluble lipids derived from [1-¹⁴C]acetate in the aged tissue also contained labeled very long acids from C₂₀ to C₂₈, as well as C₂₂ and C₂₄ alcohols, but no labeled ω-hydroxy acids or dicarboxylic acids were detected. Label was also found in n-alkanes isolated from the soluble lipids, and the distribution of label among them was consistent with the composition of n-alkanes found in the wound periderm of this tissue; C₂₁ and C₂₃ were the major components with lesser amounts of C₁₉ and C₂₅. The amount of ¹⁴C incorporated into these bifunctional monomers in 0-, 2-, 4-, 6-, and 8-day aged tissue were 0, 1.5, 2.5, 0.8, and 0.3% of the applied [1-¹⁴C]oleic acid, respectively. Incorporation of [1-¹⁴C]acetate into the insoluble residue was low up to the 3rd day of aging, rapid during the next 4 days of aging, and subsequently the rate decreased. These changes in the rates of incorporation of exogenous oleic acid and acetate reflected the development of diffusion resistance of the tissue surface to water vapor. As the tissue aged, increasing amounts of the [1-¹⁴C]acetate were incorporated into longer aliphatic chains of the residue and the soluble lipids, but no changes in the distribution of radioactivity among the α-ω-diols were obvious. The above results demonstrated that aging potato slices constitute a convenient system with which to study the biochemistry of suberization.     

House fly (Diptera: Muscidae) activity near baits containing (Z)-9-tricosene and efficacy of commercial toxic fly baits on a southern California dairy

Sticky card captures of house flies, Musca domestica L. (Diptera: Muscidae), were used to compare efficacy of screen-covered baits containing sugar, sugar and 0.1% (Z)-9-tricosene, sugar and 1.0% (Z)-9-tricosene, Golden Malrin [1.1% methomyl and 0.049% (Z)-9-tricosene], and Quick-Bayt [0.5% imidacloprid and 0.1% (Z)-9-tricosene]. The QuickBayt treatment caught more flies per hour (mean = 116.5) than sugar alone (mean = 81.0), but the addition of (Z)-9-tricosene to sugar did not increase fly capture compared with sugar alone. More males (65% of total) than females were collected on the sticky cards for all treatments. Fly kill by plain sugar (control) and the commercial baits Golden Malrin, QuikStrike Fly Abatement strips (1.0% nithiazine), and QuickBayt was tested over a 90-min period. An average of 1.4, 5.6, 363.0, and 1,266.0 flies were killed using sugar, Golden Malrin, QuikStrike, and QuickBayt, respectively. The similarity between Golden Malrin and plain sugar reflects severe resistance to this once effective methomyl bait. A no-choice feeding assay using lab-reared methomyl-susceptible and methomyl-resistant house flies was conducted with and without (Z)-9-tricosene. Adult mortality was significantly higher in the methomyl-susceptible strain exposed to treatments containing methomyl. Lower consumption of the methomyl treatments by resistant flies suggested resistance was behavioral and mortality was not influenced by (Z)-9-tricosene for either fly strain.     

Amino acid conjugates as metabolites of the plant growth regulator dihydrojasmonic acid in barley (Hordeum vulgare)

The biotransformation of [2-¹⁴C](±)9, 10-dihydrojasmonic acid (DJA) was studied in excised shoots of 6-day-old barley seedlings after 72 h. From the ethyl acetate extract, some minor metabolites were isolated and purified by DEAE-Sephadex A-25 chromatography, thin-layer chromatography (TLC), C₁₈-cartridges, and high-performance liquid chromatography (HPLC). The structural identification of these metabolites was performed by gas chromatography-mass spectrometry (GC-MS), circular dichroism (CD), and amino acid analysis, and the following amino acid conjugates were found:N-[(–)9,10-dihydrojasmonoyl]valine,N-[(–)9,10-dihydrojasmonoyl]isoleucine,N-[9,10-dihydrojasmonoyl]leucine,N-[11-hydroxy-9,10-dihydrojasmonoyl]valine,N-[11-hydroxy-9,10-dihydrojasmonoyl]isoleucine,N-[12-hydroxy-9,10-dihydrojasmonoyl]isoleucine; and the cucurbic acid-related compoundsN-{[3-hydroxy-2(4-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine andN-{[3-hydroxy-2(5-hydroxypentyl)-cyclopent-1-yl]-acetyl}isoleucine. The results suggest conjugation with isoleucine and valine, as well as preferential hydroxylation at position C-11 or hydrogenation at position C-6, as being important steps in the metabolism of (±)DJA in barley shoots.     

Male-specific (Z)-9-tricosene stimulates female mating behaviour in the spider Pholcus beijingensis

Chemical signals play an important role in spider sexual communication, yet the chemistry of spider sex pheromones remains poorly understood. Chemical identification of male-produced pheromone-mediating sexual behaviour in spiders has also, to our knowledge, not been reported before. This study aimed to examine whether chemically mediated strategies are used by males of the spider Pholcus beijingensis for increasing the probability of copulation. Based on data from gas chromatography–mass spectrometry analysis, electroantennography assay and a series of behavioural tests, we verified that (Z)-9-tricosene is a male-specific compound in the spider P. beijingensis. This compound acts as an aphrodisiac: it increases the likelihood that a female will mate. Mate-searching males release (Z)-9-tricosene to stimulate sexual behaviour of conspecific females. In the two-choice assay, however, sexually receptive females show no preference to the chambers containing (Z)-9-tricosene. This indicates that the male pheromone of P. beijingensis is not an attractant per se to the conspecific females. This is, to our knowledge, the first identification of a male-produced aphrodisiac pheromone in spiders.     

New synthesis of the (3Z,6Z,9S,10R)-isomers of 9,10-epoxy-3,6-henicosadiene and 9,10-epoxy-1,3,6-henicosatriene, pheromone components of the female fall webworm moth, Hyphantria cunea

(3Z,6Z,9S,10R)-9,10-Epoxy-3,6-henicosadiene (1) and (3Z,6Z,9S,10R)-9,10-epoxy-1,3,6-henicosatriene (5), pheromone components of the female fall webworm moth (Hyphantria cunea Drury), were synthesized by starting from (2S,3R)-2,3-epoxy-4-t-butyldimethylsilyloxy-1-butanol (8). Epoxide 8 was prepared by employing lipase-catalyzed asymmetric acetylation of (+/-)-8 as the key optical resolution step.     

An evaluation of (Z)-9-tricosene and food odours for attracting house flies, Musca domestica, to baited targets in deep-pit poultry units

Field trials investigating the effect of food baits on catches of Musca domestica at toxic targets impregnated with the female sex pheromone, (Z)-9-tricosene, were conducted in a caged-layer deep-pit poultry unit in southern England. Targets treated with an Alfacron-sugar mixture and baited with 2.5 g of 40% (Z)-9-tricosene beads caught significantly greater numbers of both male and female M. domestica than control targets. Egg and milk-baited targets were less attractive than controls, while brewers yeast slightly increased the numbers of M. domestica attracted. However, the inclusion of brewers yeast in (Z)-9-tricosene-impregnated targets produced a significant reduction in the number of male M. domestica attracted. Increased female attraction was elicited by baiting the targets with 2-phenylethanol, at the quantities of 1 mg and 10 mg. However, 2-phenylethanol had no effect on female attraction when presented in conjunction with (Z)-9-tricosene. The implications of these results in relation to the control of M. domestica populations in poultry units are discussed.     

Metabolism of Monoterpenes : Metabolic Fate of (+)-Camphor in Sage (Salvia officinalis)

The bicyclic monoterpene ketone (+)-camphor undergoes lactonization to 1,2-campholide in mature sage (Salvia officinalis L.) leaves followed by conversion to the β-d-glucoside-6-O-glucose ester of the corresponding hydroxy acid (1-carboxymethyl-3-hydroxy-2,2,3-trimethyl cyclopentane). Analysis of the disposition of (+)-[G-³H]camphor applied to midstem leaves of intact flowering plants allowed the kinetics of synthesis of the bis-glucose derivative and its transport from leaf to root to be determined, and gave strong indication that the transport derivative was subsequently metabolized in the root. Root extracts were shown to possess β-glucosidase and acyl glucose esterase activities, and studies with (+)-1,2[U-¹⁴C]campholide as substrate, using excised root segments, revealed that the terpenoid was converted to lipid materials. Localization studies confirmed the radiolabeled lipids to reside in the membranous fractions of root extracts, and analysis of this material indicated the presence of labeled phytosterols and labeled fatty acids (C₁₄ to C₂₀) of acyl lipids. Although it was not possible to detail the metabolic steps between 1,2-campholide and the acyl lipids and phytosterols derived therefrom because of the lack of readily detectable intermediates, it seemed likely that the monoterpene lactone was degraded to acetyl CoA which was reincorporated into root membrane components via standard acyl lipid and isoprenoid biosynthetic pathways. Monoterpene catabolism thus appears to represent a salvage mechanism for recycling mobile carbon from senescing oil glands on the leaves to the roots.     

Solid-phase extraction of cytokinins from aqueous solutions with C18 cartridges and their use in a rapid purification procedure

Eleven cytokinins-including bases, ribosides, glucosides, and ribotides-were tested for their retention on C₁₈ cartridges that were washed with 40 mL of water or a dilute acid at pH 3. Cytokinins were then eluted with methanol and analyzed by high performance liquid chromatography (HPLC). All pure cytokinin were well retained when the cartridge was washed with water, but Z and (diH)Z were less well retained at pH 3. The ribotides required 80% methanol for elution. Cotton leaf tissue (500 mg dry wt) was spiked with cytokinins, extracted with 80% methanol, and the extract bulk purified with hexane, insoluble polyvinylpyrrolidone, and minicolumns (strong anion exchange, amino, and C₁₈ cartridges). Ribotides, added to leaf tissue, could not be recovered as ribotides; it was necessary to hydrolyze and purify them as ribosides. The cytokinins were separated and analyzed by HPLC on strong cation exchange and C₁₈ columns. Recoveries through the entire procedure averaged 70%.Cytokinin abbreviations (diH)Z Dihydrozeatin - (diH)Z dihydrozeatin riboside - (diH)[9R]Z trans-zeatin - Z t-zeatin riboside - [9R]Z t-zeatin-O-glucoside - (OG)Z t-zeatin riboside-O-glucoside - (OG)[9R]Z t-zeatin riboside-5-monophosphate - [9R-5P]Z N6(^2-isopentenyl)adenine - iP N6(^2-isopentenyl)adenosine - [9R]iP N6(^2-isopentenyl)adenosine-5-monophosphate-[9R-5P]iP     

Structure and Biosynthesis of Cuticular Lipids: Hydroxylation of Palmitic Acid and Decarboxylation of C(28), C(30), and C(32) Acids in Vicia faba Flowers

The structure and composition of the cutin monomers from the flower petals of Vicia faba were determined by hydrogenolysis (LiAlH₄) or deuterolysis (LiAlD₄) followed by thin layer chromatography and combined gas-liquid chromatography and mass spectrometry. The major components were 10, 16-dihydroxyhexadecanoic acid (79.8%), 9, 16-dihydroxyhexadecanoic acid (4.2%), 16-hydroxyhexadecanoic acid (4.2%), 18-hydroxyoctadecanoic acid (1.6%), and hexadecanoic acid (2.4%). These results show that flower petal cutin is very similar to leaf cutin of V. faba. Developing petals readily incorporated exogenous [1-¹⁴C]palmitic acid into cutin. Direct conversion of the exogeneous acid into 16-hydroxyhexadecanoic acid, 10, 16-dihydroxy-, and 9, 16-dihydroxyhexadecanoic acid was demonstrated by radio gas-liquid chromatography of their chemical degradation products. About 1% of the exogenous [1-¹⁴C]palmitic acid was incorporated into C₂₇, C₂₉, and C₃₁n-alkanes, which were identified by combined gas-liquid chromatography and mass spectrometry as the major components of the hydrocarbons of V. faba flowers. The radioactivity distribution among these three alkanes (C₂₇, 15%; C₂₉, 48%; C₃₁, 38%) was similar to the per cent composition of the alkanes (C₂₇, 12%; C₂₉, 43%; C₃₁, 44%). [1-¹⁴C]Stearic acid was also incorporated into C₂₇, C₂₉, and C₃₁n-alkanes in good yield (3%). Trichloroacetate, which has been postulated to be an inhibitor of fatty acid elongation, inhibited the conversion of [1-¹⁴C]stearic acid to alkanes, and the inhibition was greatest for the longer alkanes. Developing flower petals also incorporated exogenous C₂₈, C₃₀, and C₃₂ acids into alkanes in 0.5% to 5% yields. [G-³H]n-octacosanoic acid (C₂₈) was incorporated into C₂₇, C₂₉, and C₃₁n-alkanes. [G-³H]n-triacontanoic acid (C₃₀) was incorporated mainly into C₂₉ and C₃₁ alkanes, whereas [9, 10, 11-³H]n-dotriacontanoic acid (C₃₂) was converted mainly to C₃₁ alkane. Trichloroacetate inhibited the conversion of the exogenous acids into alkanes with carbon chains longer than the exogenous acid, and at the same time increased the amount of the direct decarboxylation product formed. These results clearly demonstrate direct decarboxylation as well as elongation and decarboxylation of exogenous fatty acids, and thus constitute the most direct evidence thus far obtained for an elongation-decarboxylation mechanism for the biosynthesis of alkanes.     

Labelling of lipids by D-[1-14C]glucose, D-[6-14C]glucose and D-[3-3H]glucose in pancreatic islets from normal and GK rats

In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-¹⁴C]glucose than D-[6-¹⁴C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired ¹⁴C/³H ratio found in islets exposed to both D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose and D-[3-³H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-¹⁴C]glucose. The labelling of islet lipids by D-[6-¹⁴C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphate generated from the C₁-C₂-C₃ moiety of D-glucose than D-glyceraldehyde-3-phosphate produced from the C₄-C₅-C₆ moiety of the hexose, (iii) that only a limited amount of [3-³H]glycerone 3-phosphate generated from D-[3-³H]glucose is detritiated at the triose phosphate isomerase level before being converted to L-glycerol-3-phosphate, and (iv) that a rise in D-glucose concentration results in an increased labelling of islet lipids, this phenomenon being somewhat more pronounced in the case of D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose rather than D-[3-³H]glucose.