Lipoxygenase, Hydroperoxide Lyase and Volatile C6-Aldehyde Formation from C18-Fatty Acids during Development of Phaseolus vulgaris L.

Kidney bean plants (Phaseolus vulgaris) were found to have thecapability to produce C₆-aldehydes (hexanal and hexenals) fromlinoleic and linolenic acids. The various organs tested hadlipoxygenase and hydroperoxide lyase activities responsiblefor the C₆-aldehyde formation. Young leaves showed relativelyhigh activities for C₆-aldehyde formation. However, the activitiesof the leaves decreased gradually with leaf expansion. Seedlingsand seeds containing cotyledons showed low activities for C₆-aldehydeformation, because of the occurrence of an inhibitory factorin the cotyledons. The substrate specificity of the enzymeswas essentially the same among the various developmental stagesof leaves tested. (Received February 5, 1982; Accepted March 19, 1982)     

Natural Inhibitor for Volatile C6-Aldehyde Formation from C18-Unsaturated Fatty Acids

The homogenate of tea seed cotyledons contained an inhibitor for C₆-aldehyde formation from linoleic acid and linolenic acid by isolated tea chloroplasts. Seed homogenates of other plants, such as soybean, kidney bean, cucumber, Japanese radish and rice, also contained the inhibitor for C₆-aldehyde formation. The inhibitor from tea seed and cucumber seed inhibited C₆-aldehyde formation by the homogenate of cucumber hypocotyl. Hydroperoxides of linoleic acid detected were reduced when the tea seed inhibitor was added to the reaction mixture, but the enzyme activities of lipoxygenase and hydroperoxide lyase were not inhibited. This means that the inhibitor is a decomposer of fatty acid hydroperoxides as an intermediate of C₆-aldehyde formation. The tea seed inhibitor was formed during the seed ripening and it was stable during the seed germination. These findings obtained here suggest that the inhibitor is widely present in plant seeds and inhibits C₆-aldehyde formation by a variety of plant tissues.     

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.     

Specificity of Enzyme System Producing C6-Aldehyde in Tea Chloroplasts

The substrate specificity of enzyme system producing C₆-aldehyde in Thea chloroplasts was clarified with an entire series of synthesized positional isomers, in which the position of cis-1, cis-4-pentadiene system varies from C-3 to C-13 in C₁₈ fatty acid and geometrical isomers of linoleic acid. The structural requirement for the substrate of enzyme system producing C₆-aldehyde is the presence of cis-1, cis-4-pentadiene system between ω-6 and ω-10.     

Purification and Properties of Fatty Acid Hydroperoxide Lyase from Green Bell Pepper Fruits

Fatty acid hydroperoxide lyase (HPO lyase) was purified to apparentlyhomogeneity state from immature fruits of green bell pepper(Capsicum annuum L.) by differential centrifugation, ion-exchangechromatography, hydroxylapatite chromatography and gel filtration.The enzymatic activity was separated into two fractions (HPOlyases I and II) during the chromatography on hydroxylapatite.Both the isoforms were deduced to be trimers of 55-kDa subunitsand have similar enzymatic properties. Peptide maps revealedonly slight differences between them. Furthermore, immunoblotanalysis showed that an antibody raised against HPO lyase Ireacted with HPO lyase II as strongly as with the original antigen.These results indicate that there is only limited heterogeneityin terms of amino acid sequence and/or post-translational modification.The activities of both HPO lyases were considerably inhibitedby lipophilic antioxidants, such as nordihydroguaiaretic acidand     

Enantioselectivity of Enzymatic Cleavage Reaction of 13-Hydroperoxylinolenic Acid to C6-Aldehyde and C12-Oxo Acid in Tea Chloroplasts

A protease with strict specificity to lysyl peptide bonds like that of Achromobacter protease I was purified from a crude enzyme powder obtained from a culture filtrate of Achromobacter lyticus M497-1 and characterized. The purified enzyme had the following differences from protease I. The enzyme had an isoelectric point of 5.3, lower than the value of 6.9 for protease I. The amino acid composition of the enzyme had higher proportions of His, Glu, and Gly and lower proportions of Arg and Thr than protease I. The enzyme was unstable (30% residual activity) in the presence of 7 m urea (pH 8.0, 30°C, 20 min); protease I was resistant to the same conditions (80% residual activity). The k_cat/Km values for the hydrolysis of Tos-Lys-OMe and Lys-pNA by the enzyme were lower than those of protease I.     

Promotive Effect of C18-Unsaturated Fatty Acids on the Abscission of Bean Petiole Explants

The abscission-promoting effects of C₁₈-unsaturated fatty acidswere studied in bean (Phaseolus vulgaris L. cv. Masterpiece)petiole explants with the junction between the petiole and thepulvinus in the primary leaves in the light. Linolenic, linoleicand oleic acids promoted the abscission of the explants in thelight. Linolenic acid was the most effective among the compoundstested and its promotive effect was evident without any accompanyingincrease in the production of ethylene from the explants, ascompared with non-treated explants. Linolenic acid is easilyconverted to its hydroperoxide during the incubation with explants,as indicated by the formation of the conjugated diene and thegeneration of ethane. The production of ethylene from the explantstreated with linolenic acid was completely inhibited by theaddition of aminoethoxyvi-nylglycine (AVG), but large amountsof ethane were still generated. The promotive effect of linolenicacid was almost eliminated by the addition of scavengers offree radicals. Hydrogen peroxide and tert-butyl hydroperoxidepromoted abscission in the light. From these results, we concludedthat the abscission-promoting effect of linolenic acid are notmediated by the effect of ethylene but by the effect of itshydroperoxide, while the well-established pathway for the biosynthesisof ethylene from S-adenosylmethionine to ethylene, via 1-aminocyclopropane-l-carboxylicacid (ACC), was apparently operative. (Received May 1, 1991; Accepted July 10, 1991)     

Fat Metabolism in Higher Plants: XLVII. The Effect of Nitrite and Other Anions on the Formation of Unsaturated Fatty Acids by Isolated Chloroplasts

Intact spinach and barley chloroplast normally incorporate ¹⁴C-acetate into palmitate and oleate as the major ¹⁴C fatty acids. Addition of nitrite markedly altered the relative patterns of the products with the appearance of stearate, a drop in oleate, but no marked change in palmitate. Arsenite greatly increased appearance of palmitate with a concomitant decrease in the C₁₈ fatty acids. The effect of other anions was also examined. Spinach and barley plants grown under different nitrogen nutritional conditions also served as sources of chloroplasts, and their activities suggest a correlation between nitrite reductase activity and stearate accumulation.     

气相色谱—质谱技术对艰难梭菌细胞的挥发性脂肪酸成份的鉴定

应用气相色谱技术测定艰难梭菌培养物和悉生小鼠盲肠内含物中的挥发性脂肪酸含量,并用气相色谱—质谱技术加以鉴定。结果表明2-甲基丁酸是艰难梭菌的特征性产物,以前曾被其他作者错误地鉴定为异戊酸。     

Formation of Extracellular C14-C18 2-D-Hydroxy Fatty Acids by Species of Saccharomycopsis

Eighteen of 19 strains of Saccharomycopsis fibuligera and one of two strains of S. capsularis produced mixtures of C(14)-C(18) 2-D-hydroxy acids in liquid culture medium. The mixture of these acids showed antimicrobial activity against Vibrio tyrogenus but not against the other microorganisms tested. Candida lactosa, a recently described species, was shown to be an isolates of S. fibuligera.     

Changes in Volatile C6-Aldehydes Emitted from and Accumulated in Tea Leaves

C₆-Aldehydes emitted from intact tea leaves were analyzed quantitatively.Emission of the aldehydes increased temporarily in mid-May whenenzymatic activities involved in aldehyde formation from lipidsbegan to increase. Levels of C₆-aldehydes in tea leaves alsoincreased temporarily. However, the accumulated C₆-aldehydesdid not always correspond to emitted ones. (Received December 1, 1991; Accepted March 18, 1992)     

Effects of Hydroxamic Acids Isolated from Gramineae on Adenosine 5'-triphosphate Synthesis in Chloroplasts

Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7-methoxy-1,4-(2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-beta-d-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chloroplasts. Both cyclic and noncyclic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF(1)) and of purified CF(1). On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation.     

Influence of Volatile Fatty Acids on Nitrite Accumulation by a Pseudomonas stutzeri Strain Isolated from a Denitrifying Fluidized Bed Reactor

Intermediate nitrite accumulation during denitrification by Pseudomonas stutzeri isolated from a denitrifying fluidized bed reactor was examined in the presence of different volatile fatty acids. Nitrite accumulated when acetate or propionate served as the carbon and electron source but did not accumulate in the presence of butyrate, valerate, or caproate. Nitrite accumulation in the presence of acetate was caused by differences in the rates of nitrate and nitrite reduction and, in addition, by competition between nitrate and nitrite reduction pathways for electrons. Incubation of the cells with butyrate resulted in a slower nitrate reduction rate and a faster nitrite reduction rate than incubation with acetate. Whereas nitrate inhibited the nitrite reduction rate in the presence of acetate, no such inhibition was found in butyrate-supplemented cells. Cytochromes b and c were found to mediate electron transport during nitrate reduction by the cells. Cytochrome c was reduced via a different pathway when nitrite-reducing cells were incubated with acetate than when they were incubated with butyrate. Furthermore, addition of antimycin A to nitrite-reducing cells resulted in partial inhibition of electron transport to cytochrome c in acetate-supplemented cells but not in butyrate-supplemented cells. On the basis of these findings, we propose that differences in intermediate nitrite accumulation are caused by differences in electron flow to nitrate and nitrite reductases during oxidation of either acetate or butyrate.     

Phosphate Translocator of Isolated Guard-Cell Chloroplasts from Pisum sativum L. Transports Glucose-6-Phosphate

Chloroplasts were isolated from ruptured guard-cell protoplasts of the Argenteum mutant of Pisum sativum L. and purified by centrifugation through a Percoll layer. The combined volume of the intact plastids and the uptake of phosphate were determined by silicone oil-filtering centrifugation, using tritiated water and [14C]sorbitol as membrane-permeating and nonpermeating markers and [32P]phosphate as tracer for phosphate. The affinities of the phosphate translocator for organic phosphates were assessed by competition with inorganic phosphate. The affinities for dihydroxyacetone phosphate, 3-phosphoglycerate (PGA), and phosphoenolpyruvate were in the same order as those reported for mesophyll chloroplasts of several species. However, the guard-cell phosphate translocator had an affinity for glucose-6-phosphate that was as high as that for PGA. Guard-cell chloroplasts share this property with amyloplasts from the root of pea (H.W. Heldt, U.I. Flugge, S. Borchert [1991] Plant Physiol 95: 341-343). An ability to import glucose-6-phosphate enables guard-cell chloroplasts to synthesize starch despite the reported absence of a fructose-1,6-bisphosphatase activity in the plastids, which would be required if only C3 phosphates could enter through the translocator.     

Effects of Cyanide on Rates and Products of Fatty Acid Synthesis by Chloroplasts Isolated from Spinacia oleracea

Cyanide inhibited unesterified fatty acid synthesis but stimulated glyceride synthesis from [1-¹⁴C]acetate when Spinacia oleracea chloroplasts were incubated in basal media. Both unesterified fatty acid and glyceride accumulation were inhibited when chloroplasts were incubated in a diacylglycerol mode. Stimulation of chloroplast fatty acid synthesis by either exogenous coenzyme A or Triton X-100 was almost completely abolished in the presence of cyanide. Stearoyl-ACP desaturation is considered to be inhibited to a greater extent than is fatty acid synthesis de novo.     

红树植物木榄胚轴中的挥发性成分和脂肪酸成分分析

应用毛细管气相色谱-质谱联用技术对红树植物木榄胚轴中挥发性成分和脂肪酸成分进行了分析检测,检测到挥发性成分30个,鉴定了其中的27种成分,占所检测挥发性成分总量的97.01%,以烷烃类为主,占鉴定成分的67.43%,还有酸、酯及萜类化合物等;检测到脂肪酸组分23种,确定了其中的19种,脂肪酸检出量为97.81%,其中饱和脂肪酸占64.87%,不饱和脂肪酸占35.13%。     

Cytokinins in tRNA Obtained from Spinacia oleracea L. Leaves and Isolated Chloroplasts

Cytokinin-active ribonucleosides have been isolated from tRNA of whole spinach (Spinacia oleracea L.) leaves and isolated spinach chloroplasts. The tRNA from spinach leaf blades contained: 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine (cis and trans isomers), 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine, and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine (cis and trans isomers). A method for isolation of large amounts of intact chloroplasts was developed and subsequently used for the isolation of chloroplast tRNA. The chloroplast tRNA contained 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine (the cis isomer only). The structures of these compounds were assigned on the basis of their chromatographic properties and mass spectra of trimethylsilyl derivatives which were identical with those of the corresponding synthetic compounds. The results of this study indicate that ribosylzeatin was present in spinach leaf tRNA, but absent from the purified chloroplast tRNA preparation.     

Polypeptide Composition of Envelope Membranes Isolated from Chloroplasts of C3, C4, and CAM Plants

Chloroplast envelopes were isolated from chloroplasts purifiedfrom Spinacea oleracea L. (C₃), Panicum miliaceum L. (NAD-malicenzyme-type C₁), Digitaria sanguinalis (L.) Scop. (NADP-malicenzyme-type C₄), Kalanchoe daigremontiana Hamet et Perrier (constitutiveCAM), and from Mesembryanthemum crystallinum L. (inducible CAM)performing either C₃ photosynthesis or Crassulacean acid metabolism(CAM). For each species, methods were developed to isolate chloroplastenvelopes free of thylakoid contamination. The polypeptidesof ribulose bisphosphate (RuBP) carboxylase which has been consistentlyreported in envelope preparations of spinach were not foundin envelope preparations of C₄ mesophyll chloroplasts. Silverstaining of envelope polypeptides resolved electrophoreticallyon sodium dodecylsulfate polyacrylamide gradient slab gels produceda more complex profile than did Coomassie staining which haspreviously been used with C₃ envelope preparations, even thoughsilver reacted poorly with polypeptides corresponding to thesubunits of RuBP carboxylase. All of the plants examined possesseda major polypeptide of 27 to 29 kilodaltons (kD) which was previouslysuggested to be the phosphate translocator in spinach. WithC₃ M. crystallinum, the 29 kD polypeptide stained most intensely.After induction of CAM, a 32 kD polypeptide also stained intensely,giving a profile similar to that obtained with the constitutiveCAM species. A 32 kD polypeptide was also prominent in C₄ envelopepreparations, suggesting that a 32 kD polypeptide may be a translocatorprotein which is required in Crassulacean acid metabolism andC4 photosynthesis, but not in C₃ photosynthesis. (Received April 25, 1983; Accepted July 9, 1983)     

Fat Metabolism in Higher Plants: LVII. A Comparison of Fatty Acid-Synthesizing Enzymes in Chloroplasts Isolated from Mature and Immature Leaves of Spinach

Chloroplasts isolated from immature leaves of spinach (Spinacia oleracea) differ in enzyme levels from those isolated from mature leaves. On a chlorophyll basis, immature chloroplast preparations had 5- to 6-fold higher capacity to synthesize fatty acids from 2-¹⁴C-acetate compared to plastids isolated from mature leaves. This difference was correlated with higher activities for the enzymes, acetyl coenzyme A synthetase, malonyl coenzyme A synthetase, acetyl coenzyme A carboxylase, and oleyl coenzyme A transferase in plastid pressates obtained from immature leaves. Disrupted chloroplast preparations from both mature and immature leaves retained the ability to incorporate 2-¹⁴C-acetate into fatty acids in a pattern similar to that by isolated chloroplasts. 2-¹⁴C-Acetate, 2-¹⁴C-acetyl coenzyme A, 2-¹⁴C-malonate, and 1,3-¹⁴C malonyl coenzyme A were readily incorporated into a number of fatty acids. Moreover, the synthesis of oleate by chloroplast pressates from these substrates was strongly inhibited by KCN, flavin adenine mononucleotides and dinucleotides, and anaerobic conditions, while linolenic acid synthesis was unaffected by these compounds.