Enzyme system catalyzing formation of cis-3-hexenal and n-hexanal from linolenic and linoleic acids in Japanese silver (Farfugium iaponicum Kitamura) leaves

Leaf alcohol (cis-3-hexenol) and leaf aldehyde (trans-2-hexenal)are responsible for the green odor in leaves and fruits. cis-3-Hexenal,a precursor of cis-3-hexenol and trans-2-hexenal, was producedfrom linolenic acid by a homogenate of Farfugium japonicum (Japanesesilver) leaves. n-Hexanal was produced from linoleic acid bya homogenate of the leaves. The enzyme system catalyzing formationof C₆-aldehydes from linolenic and linoleic acids was localizedin chloroplast lamellae, and required oxygen for reaction. C₁₈-unsaturatedfatty acids such as linolenic acid, linoleic acid and -linolenicacid, which have carboxyl groups and cis-1, cis-4-pentadienesystems including a double bond at C-12, acted as substrates,and C₆-aldehydes (cis-3-hexenal or n-hexanal), but not C₉-aldehydes,were produced from them. The properties of the enzyme systemin chloroplasts were as follows: optimal pH 7.0; stable at pH5 to 7; thermolabile and no activity at 50?C. These propertieswere very similar to those of tea chloroplasts. The enzyme systemcould be solubilized from chloroplasts by 2% Triton X-100, butwas very unstable in solubilized form. (Received July 9, 1976; )     

Seasonal changes in activity of the enzyme system producing cis-3-hexenal and n-hexanal from linolenic and linoleic acids in tea leaves

The enzyme system producing cis-3-hexenal, a precursor of cis-3-hexenol(leaf alcohol) and trans-2-hexenal (leaf aldehyde), from linolenicacid showed high activity in summer and no activity in winterin tea (Thea sinensis) leaves and isolated chloroplasts. Theenzyme system producing n-hexanal from linoleic acid also showedsimilar seasonal changes in activity. These changes were closelyrelated to temperature and solar radiation. Enzyme activitycould not be induced after the leaves had been cut and was notaccompanied by de novo protein synthesis. (Received July 9, 1976; )     

Seasonal Changes in Activities of Enzymes Responsible for the Formation of C6-aldehydes and C6-alcohols in Tea Leaves, and the Effects of Environmental Temperatures on the Enzyme Activities

When tea leaves were homogenized and incubated, the volatileC₆-compounds hexanal, cis-3-hexenal, cis-3-hexenol and trans-2-hexenalwere formed much more by summer leaves than by winter leavesof tea plants (Camellia sinensis). The enzymes lipolytic acylhydrolase (LAH), lipoxygenase, fatty acid hydroperoxide lyase(HPO lyase) and alcohol dehydrogenase (ADH) and an isomerizationfactor were responsible for the sequential reactions of C₆-compoundformation from linoleic and linolenic acids in tea leaf lipids,and there were seasonal changes in their activities. The tealeaf enzymes were of 3 types: LAH and lipoxygenase, which hadhigh activities in summer leaves and low activities in winterleaves; ADH, which had low activity in summer leaves and highactivity in winter ones; and HPO lyase and the isomerizationfactor, which did not seem to have any effect on the rate ofC₆-compound formation throughout the year. Changes in enzymeactivities were induced by shifts in the environmental air temperaturerather than by the age of the leaves. The combined activitiesof these enzymes determined the amounts and compositions ofthe volatile C₆-compounds formed, which are the factors thatcontrol the quality of the raw leaves processed for green tea. (Received October 6, 1983; Accepted December 20, 1983)     

Production of volatiles by degradation of lipids during manufacture of black tea

During manufacture of black tea, lipids are degraded to volatile constituents. Cis-3-hexenal was present in appreciable amounts in the various parts of fresh shoots and decreased in the second leaves during manufacture. There was a simultaneous increase in trans-2-hexenal. Linalol and methyl salicylate also increased appreciably during rolling and fermentation. Most of the volatiles were lost during the firing process. The above trend was borne out by the ‘potential’ of the leaves for the production of volatiles as indicated by the increased amounts of volatiles produced by homogenizing the tissue in water against controls homogenized in 0.1 N acid. The C₆-aldehydes present in the headspace of withered shoots increased significantly following mechanical damage. The major fatty acids of the lipids in the various parts of the shoots were linolenic, linoleic, palmitic, oleic and stearic acids. The ratio of linoleic to linolenic acid in the stems was much higher than that of the leaves or buds and this was reflected in its higher 'potential for formation of hexanal. During withering and rolling of the second leaves, the unsaturated fatty acids showed substantial losses compared with the saturated acids. It is suggested that the enzymic breakdown of membrane lipids initiate the formation of volatile carbonyl compounds which are partly responsible for the flavour of black tea.     

Enzymic formation of carbonyls from linoleic acid in leaves of Phaseolus vulgaris

Homogenization of Phaseolus vulgaris leaves at acid pH results in the evolution of hexanal, cis-3- and trans-2-hexenal. With cell-free extracts of leaves, linoleic and linolenic acids are enzymically converted to their hydroperoxides (predominantly the 13-hydroperoxide isomers) and to hexanal or hexenal respectively. Activity was highest in young, dark-green leaves and was stimulated by Triton X-100. Oleic acid is not a substrate for these reactions. Both 9- and 13-hydroperoxides were cleaved to carbonyl fragments and are proposed as intermediates in the formation of volatile aldehydes and non-volatile ω-oxoacids in P. vulgaris leaves. Properties of the enzyme systems are described.     

Lipoxygenase-mediated cleavage of fatty acids to carbonyl fragments in tomato fruits

Homogenates of tomato fruits catalysed the enzymic conversion of linoleic and linolenic acids (but not oleic acid) to C₆ aldehydes in low (3–5%) molar yield. Hexanal was formed from linoleic acid; cis-3-hexenal and smaller amounts of trans-2-hexenal were formed from linolenic acid. With the fatty acids as substrates, the major products were fatty acid hydroperoxides (50–80% yield) and the ratio of 9- to 13-hydroperoxides as isolated from an incubation with linoleic acid was at least 95:5 in favour of the 9-hydroperoxide isomer. When the 9- and 13-hydroperoxides of linoleic acid were used as substrates with tomato homogenates, the 13-hydroperoxide was readily cleaved to hexanal in high molar yield (60%) but the 9-hydroperoxide isomer was not converted to cleavage products. Properties of the hydroperoxide cleavage system are described. The results indicate that the C₆ aldehydes are formed from C₁₈ polyunsaturated fatty acids in a sequential enzyme system involving lipoxygenase (which preferentially oxygenates at the 9-position) followed by a hydroperoxide cleavage system which is, however, specific for the 13-hydroperoxy isomers.     

Formation of cis-3-hexenal,trans-2-hexenal and cis-3-hexenol in macerated Thea sinensis leaves

Thea sinensis; Theaceae; tea; cis-3-hexenal: leaf aldehyde; leaf alcohol; linolenic acid; biosynthesis of leaf alcohol.Linolenic acid and cis-3-hexenal were found in macerated leaves of Thea sinensis and this aldehyde may be produced from linolenic acid by an enzyme contained in macerated leaves in the presence of oxygen. This aldehyde was easily isomerized to trans-2-hexenal, and was converted to cis-3-hexenol by alcohol dehydrogenase. During maceration of freshly picked tea leaves, the amounts of trans-2-hexenal quickly increased and were influenced by maceration time, heating, oxygen and the pH. But in unpicked tea leaves the occurrence of trans-2-hexenal is extremely doubtful.     

Flavour biogenesis. Partial purification and properties of a fatty acid hydroperoxide cleaving enzyme from fruits of cucumber

A membrane-bound enzyme, which catalyses the cleavage of fatty acid hydroperoxides to carbonyl fragments, has been partially purified from cucumber fruit. The isomeric 9- and 13-hydroperoxydienes (but not the hydroxydienes) derived from both linoleic and linolenic acids are cleaved by the enzyme but a mixture of 9- and 10-hydroperoxymonoenoic derivatives of oleic acid was not attacked. No evidence was obtained for free intermediates between fatty acid hydroperoxides and the cleavage products. Major volatile products were: cis-3-nonenal and hexanal (from 9- and 13-hydroperoxides of linoleic acid respectively) or cis-3,cis-6-nonadienal and cis-3-hexenal (from 9- and 13-hydroperoxides of linolenic acid). The increase in the ratio of cis-3- to trans-2-enal products with enzyme purification indicated that cis-3-enals are the immediate cleavage products and that the trans-2- forms are produced by subsequent isomerization.     

Seasonal variations in trans-2-hexenal and linolenic acid in homogenates of Thea sinensis leaves

The seasonal variations in the amounts of C₆-volatile components cis-3-hexenal trans-2-hexenal n-hexanal) and their precursors (linoleic and linolenic acid) in homogenates of Thea sinensis leaves were quantitatively analyzed throughout the year. Formation of trans-2-hexenal began in the middle of April and reached a maximum during July. Towards autumn the aldehyde gradually decreased and, in winter (December to March), was virtually absent. The levels of cis-3-hexenol remained constant during May–December. cis-3-Hexenal showed a similar variation pattern to that of trans-2-hexenal. The major fatty acids in the leaves were palmitic, palmitoleic, oleic, linoleic and linolenic acid, and occurred in non-ionic lipids and phospholipid fractions. The amounts of linoleic and linolenic acid did not show any marked variation except for a big peak in October.     

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.     

Studies on chlorogenic acid biosynthesis in sweet potato root tissue using trans-cinnamic acid-2-14C and quinic acid-G-3H

When either trans-cinnamic acid-2-¹⁴C or quinic acid-G-³H wasadministered to sweet potato root discs, each compound was incorporatedinto chlorogenic acid. Hydrolysis analysis revealed that trans-cinnamicacid-2-¹⁴C and quinic acid-G-³H were selectively incorporatedinto the aromatic and non-aromatic moieties of chlorogenic acid,respectively. Quinic acid-G-³H was considered a more efficient precursor thantrans-cinnamic acid-2-¹⁴C, based on data of dilution values,incorporation percents and pool sizes in the tissue. No conjugatesof trans-cinnamic acid and quinic acid were detected in discsadministered trans-cinnamic acid-2-¹⁴C or quinic acid-G-³H.From these experimental results, a possible biosynthetic pathwayfor chlorogenic acid has been proposed. ¹ This paper constitutes Part 98 of the Phytopathological Chemistryof Sweet Potato with Black Rot or Injury. (Received November 2, 1971; )     

Metabolism of unsaturated fatty acids in tomato fruit: linoleic and linolenic acid as precursors of hexanal

Incubation of linoleic or linolenic acid with tissue slicesas well as cell-free extracts of tomato fruits produced hexanal.Biogenesis of hexanal from these fatty acids was further substantiatedby the use of uniformally labelled ¹⁴C substrates. Based onthe fact that hydrogen peroxide inhibited oxygen uptake andalso production of carbonyls, it is apparent that lipoxidaseis involved in these reactions. The activity of the crude solubleextract was increased by dialysis and ammonium sulphate fractionation. In general, ripe fruits contained greater enzymatic activitiesbut smaller amounts of linoleic and linolenic acid than greenfruits. The enzymatic activity was enhanced by metal ions andcompounds containing free -SH groups. (Received December 27, 1971; )     

The Incorporation of Ciliatine (2-Aminoethylphosphonic Acid) into Lipids of the Rat Liver

Four alcohols, 1-penten-3-ol, n-amylalcohol, trans-2-hexenol and one of the linalool oxides, were newly identified as the components of carbonyl-free neutral fraction of the essential oil of black tea.

On the gas chromatogram of carbonyl fraction three unknown peaks were identified with those of n-valeraldehyde, n-heptanal and trans-2-octenal.

From these results almost all main components of carbonyl and carbonyl free fractions were clarified.

Flavor change during the manufacture of black tea was investigated by gas chromatography. During withering, hexylalcohol, nerol, trans-2-hexenoic acid, trans-2-heхenol, linalool oxide (cis, furanoid), n-valeraldehyde, capronaldehyde, n-heptanal, trans-2-hexenal, trans-2-octenal, benzaldehyde, phenylacetaldehyde, n-butyric, isovaleric, n-caproic, cis-3-hexenoic and salicylic acids and o-cresol were increased, especially the former three greatly increased, while cis-2-pentenol, linalool, geraniol, benzylalcohol, phenylethanol and acetic acid diminished markedly. In the process of fermentation almost all constituents increased, especially, 1-penten-3-ol, cis-2-pentenol, benzylalcohol, trans-2-hexenal, benzaldehyde, n-caproic, cis-3-hexenoic and salicylic acids were remarkable.

On firing, most alcohols, carbonyl and phenolic compounds decreased remarkably whereas acetic, propionic and isobutyric acids greatly increased.     

The photosynthetic production of oxalic acid in Oxalis corniculata

The biogenesis of oxalic acid in Oxalis corniculata has beeninvestigated. In O. corniculata the bulk of the oxalic acidis produced by CO₂ fixation both in light and in darkness butthe rate of its photosynthetic formation is much higher thanin darkness. Several other plants some of which are known toaccumulate oxalic acid e.g., Biophytum sensitivum, Averrhoacarambola, Impatiens balsamina, Amorphophallus campanulatusand Colocassia antiquorum also fix ¹⁴CO₂ into oxalic acid photosyntheticallywithin 1 min of exposure to the gas. In O. corniculata ¹⁴C canbe detected in oxalic acid within 5 sec and about half of thetotal ¹⁴C fixed in the 70% ethanol soluble fraction can be locatedin this compound after 5 min. This is accompanied by a declineof radioactivity in two compounds, the chromatographic behaviourand melting points of one of which and its DNP hydrazone aresimilar to those of an authentic sample of glyoxylic acid. Whenglyoxylate 1, 2-¹⁴C is incubated with Oxalis leaf homogenateit is converted to oxalate-¹⁴C. Glycolate is also metabolizedto oxalate. The conversion of both glycolate and glyoxylateare favoured by light. The C₂ compounds acetate and glycinehowever are utilized rather poorly. Sucrose-¹⁴C is also notmetabolized markedly for this purpose. (Received August 20, 1969; )     

Volatile C6-Aldehyde Formation via Hydroperoxides from C18-Unsaturated Fatty Acids in Etiolated Alfalfa and Cucumber Seedlings

1. Etiolated seedlings of alfalfa and cucumber evolved n-hexanal from linoleic acid and cis-3-hexenal and trans-2-hexenal from linolenic acid when they were homogenized.

2. The activities for n-hexanal formation from linoleic acid, lipoxygenase and hydro-peroxide lyase were maximum in dry seeds and 1~2 day-old etiolated seedlings of alfalfa, and in 6~7 day-old etiolated seedlings of cucumber.

3. n-Hexanal was produced from linoleic acid and 13-hydroperoxylinoleic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. cis-3-Hexenal and trans-2-hexenal were produced from linolenic acid and 13-hydroperoxylinolenic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. But these extracts, particulariy cucumber one, showed a high isomerizing activity from cis-3-hexenal to trans-2-hexenal.

4. When the C₈-aldehydes were produced from linoleic acid and linolenic acid by the crude extracts, formation of hydroperoxides of these C₁₈-fatty acids was observed.

5. When 9-hydroperoxylinoleic acid was used as a substrate, trans-2-nonenal was produced by the cucumber homogenate but not by the alfalfa homogenate.

6. As the enzymes concerned with C₆-aldehyde formation, lipoxygenase was partially purified from alfalfa and cucumber seedlings and hydroperoxide lyase, from cucumber seedlings. Lipoxygenase was found in a soluble fraction, but hydroperoxide lyase was in a membrane bound form. Alfalfa lipoxygenase catalyzed formation of 9- and 13-hydroperoxylinoleic acid (35: 65) from linoleic acid and cucumber one, mainly 13-hydroperoxylinoleic acid formation. Alfalfa hydroperoxide lyase catalyzed n-hexanal formation from 13-hydroperoxylinoleic acid, but cucumber one catalyzed formation of n-hexanal and trans-2-nonenal from 13- and 9-hydroperoxylinoleic acid, respectively.

7. From the above results, the biosynthetic pathway for C₆-aldehyde formation in etiolated alfalfa and cucumber seedlings is established that C₆-aldehydes (n-hexanal, cis-3-hexenal and trans-2-hexenal) are produced from linoleic acid and linolenic acid via their 13-hydroperoxides by lipoxygenase and hydroperoxide lyase.     

Biosynthesis of trans-2-hexenal in chloroplasts from Thea sinensis

The biosynthetic pathway of trans-2-hexenal, leaf aldehyde, in isolated chloroplasts of Thea sinensis leaves. was examined using a tracer experiment. A high and specific incorporation of radioactivity into cis-3-hexenal and trans-2-hexenal, was observed when linolenic acid-[U-¹⁴C] was incubated with the isolated chloroplasts. Thus, trans-2-hexenal was biosynthesized via cis-3-hexenal from linolenic acid in the chloroplasts.     

Compositions and Positional Distributions of Fatty Acids in Phospholipids from Leaves of Chilling-Sensitive and Chilling-Resistant Plants

The compositions and positional distributions of fatty acidsin the major leaf phospholipids of phosphatidylglycerol, phosphatidylcholineand phosphatidylethanolamine were analyzed by gas-liquid chromatographyand enzymic hydrolysis, and chilling-sensitive and chilling-resistantplants were comparcd with respect to the relative contents ofpalmitic and trans-³-hexadecenoic acids in the separated phospholipids.A distinct difference between these plants was found in thefatty acid compositions of phosphatidylglycerol, in which thesum of palmitic and trans-³-hexadecenoic acids ranged from 60to 78% of the total fatty acids in 8 species of chilling-sensitiveplants, and from 50 to 57% in 11 species of chilling-resistantplants. The only exception among the chilling sensitive plantsin this respect was the tomato, in which the sum of palmiticand trans-³-hexadecenic acids in phosphatidylglycerol amountedto 54%. The fatty acid compositions and the positional distributionsof fatty acids in phosphatidylglycerol suggest that the occurrenceof high proportions of dipalmitoyl and 1-palmitoyl-2-(trans-³-hexadecenoyl)species in this lipid is correlated with the susceptibilityto chilling of the leaves of higher plants. In the compositionsand positional distributions of fatty acids in phosphatidylcholineand phosphatidylethanolamine, no difference was found betweenthe chilling-sensitive and chilling-resistant plants. ¹ Present address: Department of Biology, Faculty of Science,Universityof Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. (Received May 21, 1982; Accepted June 25, 1982)     

The Metabolism of Abscisic Acid

The light-catalysed isomerization of (+)-abscisic acid (ABA)to its trans isomer during isolation from leaves was monitoredby the addition of (±)-[2-¹⁴C]ABA to the extraction medium.(+)Trans-abscisic acid (t-ABA) was found to occur naturallyin rose (Rosa arvensis) leaves at 20µg/kg fresh weight;(+)-ABA was present at 594µg/kg. (±)-[2-¹⁴D]Trans-abscisicacid was not isomerized enzymically to ABA in tomato shoots. (±)-Abscisic acid was converted by tomato shoots to awater-soluble neutral product, ‘Metabolite B’, whichwas identified as abscisyl-ß-D-glucopyranoside. When(±)-[2-¹⁴C]trans-abscisic acid in an equimolar mixturewith (±)-[2-¹⁴C}ABA was fed to tomato shoots it was convertedto its glucose ester 10 times faster than was ABA. Trans-abscisyl-ß-D-glucopyrano8ide only was formedfrom (±)-[2-¹⁴C]t-ABA in experiments lasting up to 30h. Glucosyl abscisate was formed slowly from ABA and the freeacid fraction contained an excess of the unnatural (–).ABAas did the ABA released from abscisyl-ß-D-glucopyranosideby alkaline hydrolysis. The (+).ABA appeared to be the solesource of the acidic ‘Metabolite C" previously noted. The concentrations of endogenous (+)-, (+)-[2-¹⁴C]-, and (–)-[2-¹⁴C]ABAremaining as free acid, and also in the hydrolysate of abscisyl-ß-D-glucopyranoside,were measured by the ORD, UV absorption, and scintillation spectrometryof highly purified extracts of ABA from tomato shoots whichhad been supplied with racemic [2-^l4C]ABA.     

Isolation of 1-O-trans-p-Coumaroyl-r{beta}-D-glucopyranose from Sweet Potato Roots and Examination of Its Role in Chlorogenic Acid Biosynthesis

1-O-trans-p-Coumaroyl-rß-D-glucopyranose (p-coumaroyl-D-glucose)was isolated from slices of sweet potato root which had beenincubated with trans-cinnamic acid. Pre-loaded trans-cinnamicacid efficiently trapped the radioactivity from L-[U-¹⁴C]-phenylalanineand reduced its incorporation into chlorogenic acid by 75% ofcontrol values in disks of sweet potato root. In the root diskssupplied with trans-[3-¹⁴C]-cinnamic acid, the radioactivitywas transferred first to trans-cinnamoyl- D-glucose, then top-coumaroyl-D-glucose, and subsequently to chlorogenic acidand isochlorogenic acid. These results support our earlier propositionthat p-coumaroyl-D-glucose is involved in the biosynthesis ofchlorogenic acid as an intermediate adjacent in the pathwayto trans-cinnamoyl-D-glucose in sweet potato roots. (Received April 11, 1988; Accepted August 9, 1988)     

Volatile Flavor of Black Tea

The volatile components extracted from fresh tea leaf, fermented leaf and black tea were analysed by gas chromatography.

Quantitative difference in the composition of essential oils was observed between fresh leaf and manufactured black tea; the former was rich in alcohols, whereas the latter in aldehydes and acids.

During fermentation process the following components mainly brought about changes: n-capronaldehyde (4.1 times after fermentation for 3hrs.), trans-2-hexen-l-al (13.2 times) and cis-3-hexenoic acid (1.2 times) increased, but n-hexylalcohol (0.7 time), cis-3-hexen-l-ol (0.7 time) and methylsalicylate (0.8 time) decreased.

These changes during fermentation were scarcely carried out in nitrogen atmosphere.