Formation of (-⊃-1′,2′-epi-2-cis-xanthoxin acid from a precursor of abscisic acid

Tomato shoots and avocado mesocarp supplied with (±)-[2-¹⁴C]-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl)-3-methylpenta-cis-2-trans-4-dienoic acid metabolize it into (+)-abscisic acid and a more polar material that was isolated and identified as (?)-epi-1′(R),2′(R)-4′(S)-2-cis-xanthoxin acid. The (+)-1′(S),2′(S)-4′(S)-2-cis-xanthoxin acid recently synthesized from natural violaxanthin, has the 1′,2′-epoxy group on the opposite side of the ring to that of the 4′(S)-hydroxyl group and the compound is rapidly converted into (+)-abscisic acid. The 1′,2′-epoxy group of (?)-1′,2′-epi-2-cis-xanthoxin acid is on the same side of the ring as the 4′(S) hydroxyl group: the compound is not metabolized into abscisic acid. The configuration of the 1′,2′-epoxy group probably controls whether or not the 4′(S) hydroxyl group can be oxidized. (+)-2-cis-Xanthoxin acid is probably not a naturally occurring intermediate because a ‘cold trap’, added to avocado fruit forming [¹⁴C]-labelled abscisic acid from [2-¹⁴C]mevalonate, failed to retain [¹⁴C] label.     

Carotenogenic and abscisic acid biosynthesizing activity in a cell-free system

Abscisic acid is considered an apocarotenoid formed by cleavage of a C-40 precursor and subsequent oxidation of xanthoxin and abscisic aldehyde. Confirmation of this reaction sequence is still awaited, and might best be achieved using a cell-free system capable of both carotenoid and abscisic acid biosynthesis. An abscisic acid biosynthesizing cell-free system, prepared from flavedo of mature orange fruits, was used to demonstrate conversion of farnesyl pyrophosphate, geranylgeranyl pyrophosphate and all-trans-β-carotene into a range of β,β-xanthophylls, xanthoxin, xanthoxin acid, 1′,4′-trans-abscisic acid diol and abscisic acid. Identification of product carotenoids was achieved by high-performance liquid chromatography and on-line spectral analysis of individual components together with co-chromatography. Putative C-15 intermediates and product abscisic acid were identified by combined capillary gas chroma-tography-mass spectrometry. Kinetic studies revealed that β-carotene, formed from either famesyl pyrophosphate or geranylgeranyl pyrophosphate, reached a maximum within 30 min of initiation of the reaction. Thereafter, β-carotene levels declined exponentially. Catabolism of substrate β-carotene into xanthophylls, putative abscisic acid precursors and product abscisic acid was restricted to the all-trans-isomer. However, when a combination of all-trans- and 9-cis-β-carotene in the ratio 1:1 was used as substrate, formation of abscisic acid and related metabolites was enhanced. Biosyn-thetically prepared [¹⁴C]-all-trans-violaxanthin, [¹⁴C]-all-trans-neoxanthin and [¹⁴C]-9′-cis-neoxanthin were used as substrates to confirm the metabolic interrelationship between carotenoids and abscisic acid. The results are consistent with 9′-cis-neoxan-thin being the immediate carotenoid precursor to ABA, which is oxidatively cleaved to produce xanthoxin. Formation of abscisic aldehyde was not observed. Rather, xanthoxin appeared to be converted to abscisic acid via xanthoxin acid and 1′,4′-trans-abscisic acid diol. An alternative pathway for abscisic acid biosynthesis is therefore proposed.     

Epoxyoctadecadienoic acids from Crepis conyzaefolia seed oil

The seed oil of Crepis conyzaefolia (Gouan) Dalle Torre contains previously unidentified (±)-cis-12,13-epoxyoctadeca-trans-6-cis-9-dienoic (14%) and cis-12,13-epoxyoctadeca-cis-6-cis-9-dienoic (2%) acids and the more common vernolic [(±)-12,13-epoxyoctadec-cis-9-enoic] (32%) acid.     

Activity and metabolism of C-(+/-)-abscisic Acid derivatives

Several radioactive analogues of abscisic acid have been tested for their growth-inhibitory effects and their metabolism in excised embryonic axes of Phaseolus vulgaris. The compounds tested were the methyl and ethyl esters of 2-¹⁴C-abscisic acid and the cis- and trans-1′,4′-diols of 2-¹⁴C-abscisic acid. All four compounds cause less growth inhibition than abscisic acid, and all four compounds are converted to abscisic acid in the axes at rates which are sufficient to account for most, if not all, of the observed growth-inhibitory activity. None of the four compounds is metabolized to the extent that abscisic acid is metabolized in the axes, suggesting that the structural requirements for growth-inhibitory activity and metabolism may be similar.     

5-(1,2-Epoxy-2,6,6-trimethyl-1-cyclohexyl)-3-methyl-cis,trans-2,4-pentadienoic acid and its esters: New plant growth inhibitors structurally related to abscisic acid

Summary 5-(1,2-Epoxy-2,6,6-trimethyl-1-cyclohexyl)-3-methyl-cis,trans-2,4-pentadienoic acid and its esters were found to be potent plant growth inhibitors. Their activities were comparable to that of abscisic acid.     

Formation of 12-oxo-trans-10-dodecenoic acid in chloroplasts from Thea sinensis leaves

Linolenic acid-[1-¹⁴C] was converted to 12-oxo-trans-10-dodecenoic acid, via 12-oxo-cis-9-dodecenoic acid by incubation with chloroplasts of Thea sinensis leaves. Thus, it was confirmed that linolenic acid is split into a C₁₂-oxo-acid, 12-oxo-trans-10-dodecenoic acid, and a C₆-aldehyde, trans-2-hexenal, leaf aldehyde, by an enzyme system in chloroplasts of tea leaves.     

The biohydrogenation of α-linoleic acid and oleic acid by rumen micro-organisms

1. α-[U-¹⁴C]Linolenic acid was incubated with the rumen contents of sheep and the metabolic products were characterized by thin-layer chromatography, gas–liquid chromatography and absorption spectroscopy in the ultraviolet and infrared. 2. A tentative scheme for the biohydrogenation route to stearic acid is presented. The main pathway is through diconjugated cis–cis–cis-octadecatrienoic acid, non-conjugated trans–cis (cis–trans)-octadecadienoic acid and trans-octadecenoic acid, but other pathways are apparent. 3. Washed rumen micro-organisms possessed only a limited capacity to hydrogenate α-linolenic acid and oleic acid but the rate was greatly stimulated by a factor(s) present in the supernatant rumen liquor. 4. Pure cultures of Clostridium perfringens, Streptococcus faecalis, Escherichia coli and a coliform organism isolated from sheep faeces possessed negligible ability to hydrogenate unsaturated fatty acids compared with a mixed population of rumen micro-organisms. Butyrivibrio fibrisolvens slowly converted linoleic acid into octadecenoic acid.     

Synthesis of Some Analogs of Abscisin II

(±)-trans, cis-Abscisin II has been synthesized starting from 2,6,6-trimethyl-4-ethylenedioxy-2-cyclohexene-1-one. Similarly the synthesis of (±)-cis, cis-abscisin II has been attempted, but the product has turned out to be 1-oxa-8-oxo-2,6,10,10-tetramethyl-spiro-[4,5]deca-3,6-diene-2-acetic acid.     

Effects of Dichloroethene Isomers on the Induction and Activity of Butane Monooxygenase in the Alkane-Oxidizing Bacterium “Pseudomonas butanovora”

We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium “Pseudomonas butanovora.” Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of ~20 nmol mg protein⁻¹ of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein⁻¹. Oxidation of similar amounts of each DCE isomer (~20 nmol mg protein⁻¹) produced different negative effects on lactic acid-dependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown “P. butanovora” did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.     

Compartmentation and equilibration of abscisic Acid in isolated xanthium cells

The compartmentation of endogenous abscisic acid (ABA), applied (±)-[³H]ABA, and (±)-trans-ABA was measured in isolated mesophyll cells of the Chicago strain of Xanthium strumarium L. The release of ABA to the medium in the presence or absence of DMSO was used to determine the equilibration of ABA in the cells. It was found that a greater percentage of the (±)-[³H]ABA and the (±)-trans-ABA was released into the medium than of the endogenous ABA, indicating that applied ABA did not equilibrate with the endogenous material.     

The Resolution by HPLC of RS-[2-14C]Me 1', 4'-cis-Diol of Abscisic acid and the Metabolism of (-)-R- and (+)-S-Abscisic Acid

The R- and S-enantiomers of racemic [2-¹⁴C]Me 1', 4'-cis-diolof abscisic acid have been separated by high performance liquidchromatography on an optically-active Pirkle column. R-[2-¹⁴C]-and S-[2-¹⁴C]abscisic acids, formed from the Me 1', 4'-cis-diolby oxidation and alkyline hydrolysis were fed to tomato shootsand the extracts analysed by reversed phase high performanceliquid chromatography. R-[2-¹⁴C]abscisic acid formed mainlythe abscisic acid glucose ester (ABAGE), abscisic acid l'-glucoside(ABAGS) and an uncharacterized conjugate. Dihydrophaseic acid4'-B-D-glucoside, the major metabolite of RS-abscisic acid intomato shoots, was found to be derived virtually exclusivelyfrom the natural, S-abscisic acid. Phaseic acid and conjugatesof abscisic acid were also found as products of the naturallyoccurring enantiomer. The resolution method was used to measurethe relative proportions of R and S enantiomers in the freeacid liberated from conjugates formed from RS-[2-¹⁴C]ABA fedto shoots. The ratios show an excess of the R-enantiomer: 5.8:1, ABAGE; 29.4: 1, ABAGE; 8.3: 1 for an uncharacterized conjugateand 6.1: 1 for the residual free [2-¹⁴C]ABA. Key words: ABA, HPLC, Tomato     

A criterion to determine whether cis-4-hydroxyproline is produced in animal tissues

Hydrolyzates of tissues that had been labeled with [¹⁴C]proline often contain significant amounts of cis-4-hydroxy[¹⁴C]proline. Since animal cells do not contain an enzyme which can effect formation of cis-4-hydroxyproline, there are only two possible explanations for its presence. Either it is formed during acid hydrolysis of trans-4-hydroxyproline (which is synthesized by cells and is a common constituent of connective tissues), or it is produced by a nonenzymatic mechanism such as attack by oxygen radicals. It is important to resolve this issue because if a nonenzymatic mechanism is active in connective tissues, then it will be necessary to reevaluate currently accepted ideas about production of hydroxyproline. This communication describes a method for distinguishing between the two alternate explanations. Tissues or cells are labeled with [¹⁴C]proline, and then a known amount of trans-4-hydroxy[³H]proline is added to each sample before hydrolysis; the relative amounts of [¹⁴C]- and [³H]-cis-4-hydroxyproline are compared after hydrolysis. It is known from a separate series of measurements with mixtures of [¹⁴C]- and [³H]-trans-4-hydroxyproline standards that there is a very high correlation (r = 0.998) between acid-induced formation of the [¹⁴C]- and [³H]-cis epimers. One can thus compare the amount of cis-4-hydroxy[¹⁴C]proline in a hydrolyzate from a biological system with the amount that would be expected if it were all formed during acid hydrolysis. This method was used to show that fibroblasts cultured under conditions commonly used to study collagen metabolism do not produce cis-4-hydroxyproline. This result strongly suggests that nonenzymatic hydroxylation does not normally occur in cell culture systems.     

Chloroethene Biodegradation in Sediments at 4°C

Microbial reductive dechlorination of [1,2-¹⁴C]trichloroethene to [¹⁴C]cis-dichloroethene and [¹⁴C]vinyl chloride was observed at 4°C in anoxic microcosms prepared with cold temperature-adapted aquifer and river sediments from Alaska. Microbial anaerobic oxidation of [1,2-¹⁴C]cis-dichloroethene and [1,2-¹⁴C]vinyl chloride to ¹⁴CO₂ also was observed under these conditions.     

2-(7,13-Dihydroxy-2-trans-octadecenoylamino) ethanesulfonic acid (lipotaurine) as an intermediate of taurolipids biosynthesis

A hydroxy fatty-acid-combined taurine (lipotaurine) was found in the taurolipids fraction of Tetrahymena thermophila. Lipotaurine accounted for about 1.4% of the total taurolipids of the cells, and was composed of taurine and 7,13-dihydroxy-2-trans-octadecenoic acid. By nuclear magnetic resonance, mass and infrared spectrometries, the chemical structure of lipotaurine was identified as 2-(7,13-dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid. When cells of T. thermophila were incubated with the double-labeled lipotaurine which was biosynthesized from [2(n)-³H]taurine and [1-¹⁴C]stearic acid, both the radioactivities were detected in taurolipid A, B and C. Furthermore, the ratio of the radioactivities of ³H and ¹⁴C in the lysotaurolipids were the same as that of the lipotaurine. From these results, it is suggested that lipotaurine is an intermediate of taurolipid biosynthesis.     

The action of alkali on di-O-(2-bromoethylidene)-d-mannitols: Formation of an unusually acid-stable acetal linkage

When cis,trans-1,2:5,6-di-O-(2-bromoethylidene)-d-mannitol and cis,cis-1,2:5,6-di-O-(2-bromoethylidene)-d-mannitol were treated with dilute, boiling sodium hydroxide, 5,6-O-(S)-(2-bromoethylidene)-3:1,2-O-[(R)-1-ethanyl-2-ylidene]-d-mannitol (3) and 3:1,2;4:5,6-di-O-[(R)-1-ethanyl-2-ylidene]-d-mannitol (10) were produced; the structures were established by a combination of chemical transformations, ¹H-n.m.r. spectroscopy, and mass spectrometry. The bicyclo ether-acetal linkage in 3 and 10 proved unusually resistant to hydrolysis by acid.     

The unexpected reactions of [RuCl3(2mqn)NO] (H2mqn=2-methyl-8-quinolinol) with 2-chloro-8-quinolinol (H2cqn) and of [RuCl(2cqn)(2mqn)NO] on photoirradiation

The reaction of [RuCl₃(2mqn)NO]⁻ (H2mqn=2-methyl-8-quinolinol) with 2-chloro-8-quinolinol (H2cqn) afforded cis-1 [RuCl(2cqn)(2mqn)NO] (the oxygen of 2cqn is trans to the NO) (complex 1), cis-1 [RuCl(2cqn)(2mqn)NO] (the oxygen of 2mqn is trans to the NO) (complex 2) and a 1:1 mixture of cis-2 [RuCl(2cqn)(2mqn)NO] (the oxygen of 2mqn is trans to the NO) and cis-2 [RuCl(2cqn)(2mqn)NO] (the oxygen of 2cqn is trans to the NO) (complex 3). The reaction was compared with that of [RuCl₃(2mqn)NO]⁻ with 8-quinolinol (Hqn) or 5-chloro-8-quinolinol (H5cqn). Photoirradiation reaction of complex 1 at room temperature in deaerated CH₂Cl₂ in the presence of NO gave trans-[RuCl(2cqn)(2mqn)NO] (the Cl is trans to the NO) and complex 2 with recovery of complex 1. The reaction was contrasted with that of cis-1 [RuCl(qn)(2mqn)NO] or cis-1 [RuCl(5cqn)(2mqn)NO]. The crystal structure of complex 1 was determined by X-ray diffraction. The reactions were examined under consideration of atomic charge of the phenolato oxygen in 8-quinolinol and its derivatives calculated at the restricted Hartree-Fock/6-311G** level.     

Stereochemical aspects of the metabolism of the isomeric methylcyclohexanols and methylcyclohexanones

1. The seven isomeric optically inactive forms of methylcyclohexanol (i.e. 1-, and cis- and trans-2-, -3- and -4-) are excreted by rabbits mainly as glucuronides of the thermodynamically more stable forms of the alcohols. The eighth isomer, cyclohexylmethanol, however, undergoes aromatization in vivo, giving rise to benzoic acid and hippuric acid. The (±)-2-, (±-3- and 4-methylcyclohexanones are reduced in the rabbit and excreted mainly as the glucuronides of the thermodynamically more stable forms of the corresponding methylcyclohexanols. 2. Racemic cis- and trans-2-methylcyclohexanol and 2-methylcyclohexanone are all excreted as conjugated trans-2-methylcyclohexanol. However, when the (±)-cis-alcohol or the (±)-ketone is fed, (+)-trans-2-methylcyclohexanol is excreted, whereas when the (±)-trans-alcohol is fed it is excreted as the (±)-trans-alcohol. 3. Racemic cis- and trans-3-methylcyclohexanol and 3-methylcyclohexanone are all excreted as conjugated racemic cis-3-methylcyclohexanol. cis- and trans-4-Methylcyclohexanol and 4-methylcyclohexanone are all excreted as conjugated trans-4-methylcyclohexanol. 4. The metabolic differences between the various methylcyclohexanols are explicable in terms of their conformations and of Vennesland's (1958) hypothesis of the role of NADH in dehydrogenation reactions.     

Abscisic Acid Metabolism in Salt-Stressed Cells of Dunaliella salina: Possible Interrelationship with beta-Carotene Accumulation

The interrelationship between abscisic acid (ABA) production and β-carotene accumulation was investigated in salt-stressed cells of the halotolerant green alga Dunaliella salina var bardawil. Cells were supplied with either R-[2-¹⁴C]mevalonolactone or [¹⁴C] sodium bicarbonate for 20 hours and then exposed to increased salinity (1.5 to 3.0 molar NaCl) for various lengths of time. Incorporation of label into abscisic acid and phaseic acid and the distribution of [¹⁴C]ABA between the cells and incubation media were monitored. [¹⁴C]ABA and [¹⁴C]phaseic acid were identified as products of both R-[2-¹⁴C]mevalonolactone and [¹⁴C]sodium bicarbonate metabolism. ABA metabolism was enhanced by hypersalinity stress. Actinomycin D, chloramphenicol, and cycloheximide abolished the stress-induced production of ABA, suggesting a role for gene activation in the process. Kinetic analysis of both ABA and β-carotene production demonstrated two stages of accelerated β-carotene production. In addition, ABA levels increased rapidly, and this increase occurred coincident with the early period of accelerated β-carotene production. A possible role for ABA as a regulator of carotenogenesis in cells of D. salina is therefore discussed.     

Cytokinins: synthesis and biological activity of geometric and position isomers of zeatin

Geometric and position isomers of zeatin and of ribosylzeatin and other compounds closely related to zeatin have been tested in the tobacco (Nicotiana tabacum var. Wisconsin No. 38) bioassay. None was more active than zeatin itself. There was a much greater difference in activity (> 50-fold) between trans- and cis-zeatin than between trans-isozeatin [6-(4-hydroxy-2-methyl-trans-2-butenylamino) purine] and cis-isozeatin [6-(4-hydroxy-2-methyl-cis-2-butenylamino) purine], the latter being less active than cis-zeatin and trans-isozeatin. Higher concentrations were required for equivalent callus growth stimulated by the 9-ribosyl derivatives, which followed an order of decreasing activity: ribosyl-trans-zeatin > ribosyl-cis-zeatin > ribosyl-trans-isozeatin > ribosyl-cis-isozeatin, corresponding roughly to that of the bases. The effect of side chain, double bond saturation was to diminish the activity, and in the dihydro series the shift of the methyl group from the 3- to the 2-position in going from dihydrozeatin to dihydroisozeatin [6-(4-hydroxy-2-methylbutylamino) purine] resulted in a 70-fold decrease in activity. cis-Norzeatin [6-(4-hydroxy-cis-2-butenylamino) purine], which was less than one-fifth as active as cis-zeatin, showed the effect of complete removal of the side chain methyl group, and cyclic-norzeatin [6-(3,6-dihydro-1,2-oxazin-2-yl) purine] was about 1/100 as active as cis-norzeatin. These findings delineate completely the effect on the cytokinin activity of zeatin of variation in side chain geometry, presence and position of the methyl substituent, presence and geometry of hydroxyl substitution, presence of the double bond, and of side chain cyclization.     

Biosynthesis of p-hydroxybenzoic acid in poplar lignin

Biosynthetic pathways to p-hydroxybenzoic acid in polar lignin were examined by tracer experiments. High incorporation of radioactivity to the acid was observed when shikimic acid-[1-¹⁴C], phenylalanine-[3-¹⁴C], trans-cinnamic acid-[3-¹⁴C], p-coumaric acid-[3-¹⁴C] and p-hydroxybenzoic acid-[COOH-¹⁴C] were administered, while incorporation was low from shikimic acid-[COOH-¹⁴C], phenylalanine-[1-¹⁴C], phenylalanine-[2-¹⁴C], tyrosine-[3-¹⁴C], benzoic acid-[COOH-¹⁴C], sodium acetate-[1-¹⁴C] and d-glucose-[U-¹⁴C]. Thus p-hydroxybenzoic acid in poplar lignin is formed mainly via the pathway: shikimic acid → phenylalanine → trans-cinnamic acid → p-coumaric acid → p-hydroxybenzoic acid.