Oxidation of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids by Acetobacter and Serratia strains

Conversion of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids was carried out using Acetobacter rancens IFO3297, A. pasteurianus IFO13753 and Serratia liquefaciens LF14. IFO3297 produced 110g 2-furoic acid l^-1 from furfural with a 95% molar yield. 5-Hydroxymethyl-2-furancarboxylic acid was produced from the corresponding aldehyde by using whole cells LF14. IFO13753 and LF14 both converted isophthalaldehyde, 2,5-furandicarbaldehyde, 2,5-thiophenedicarbaldehyde and 2,2 biphenyldicarbaldehyde to the corresponding formylcarboxylic acid with 86--91% molar yields.Revisions requested 21 July 2004; Revisions received 7 September 2004     

Oxidation and reduction of lignin-related aromatic compounds by Aureobasidium pullulans

Summary A yeast-like fungus, identified as Aureobasidium pullulans, was isolated from a kraft mill settling pond by enrichment culture on 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-propane-1,3-diol (I). The fungus was also able to use the following aromatic acids as sole carbon source: Benzoic, p-hydroxybenzoic, vanillic, syringic, ferulic and protocatechuic acids. Various aromatic alcohols were oxidised to their corresponding aldehydes and acids during aerobic culture, while aromatic aldehydes were both oxidised and reduced. However, the aromatic acids were not reduced, but were slowly metabolised. Dimer I was cleaved at the alkyl-phenyl linkage to give glycerol-2-guaiacyl ether in high yield. The identity of the latter was determined by mass spectrometry and proton nmr. The dimers 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-propane-1,3-diol (II), 3,4-dimethoxy--(2,6-dimethoxy-4-carboxyphenoxy)-acetophenone (III) and 5-carboxy-7-methoxy-2-(4-hydroxy-3-methoxyphenyl)-3-methyl-2,3-dihydrobenzo-[b]-furan (IV) were not metabolised. It is concluded that the fungus resembles Fusarium in many of its metabolic properties, and could be considered as a potential lignin degrader.     

On a reversible molybdenum-containing aldehyde oxidoreductase from Clostridium formicoaceticum

Clostridium formicoaceticum grown in the presence of 1 mM molybdate and about 1.5×10^-5 mM tungsten (present in the 5 g yeast extract/l of the growth medium) forms two reversible aldehyde oxidoreductases in an activity ratio of about 45:55. The fraction of 45% does not bind to the octyl-Sepharose column, whereas the 55% aldehyde oxidoreductase binds to this column. From cells grown on a synthetic medium without the addition of tungstate only about 2% of the aldehyde oxidoreductase of the crude extract binds to octyl-Sepharose. The enzyme not binding to octyl-Sepharose has been purified as judged by electrophoresis. It is pure after about 50 fold enrichment. According to SDS gel electrophoresis the enzyme consists of identical 100 kD subunits. Based on gel chromatography it seems to be a trimer. Per subunit 0.6 molybdenum, 7 iron, 6.6 acid labile sulphur, about 0.1 pterin-6-carboxylic and <0.05 tungsten have been found. The first 13 amino acids from the amino end show no similarity with the W-containing aldehyde oxidoreductase from the same bacterium. With reduced tetramethylviologen (E₀=–550 mV) the new molybdenum containing enzyme can reduce various aliphatic and aromatic acids to aldehydes. The pH optimum is at 6.0. For the dehydrogenation of butyraldehyde a rather broad pH region from pH 6 to 10 shows almost no variation of rate. From 15 different aldehydes acetaldehyde exhibits the highest rate. The K_m value for butanal is 0.002 and for propionate 7.0 mM. Compared with the tungsten enzyme the molybdenum enzyme is only moderately oxygen-sensitive.Abbreviations AOR aldehyde oxidoreductase - BV benzylviologen - MV methylviologen - NH₂CO-MV 1,1-carbamoylmethylviologen - TMV 1,1,2,2-tetramethylviologen     

Utilization of aromatic amino acids as nitrogen sources in Brevibacterium linens: an inducible aromatic amino acid aminotransferase

The first step of the utilization of the aromatic amino acids as sole nitrogen sources by Brevibacterium linens strain 47 was found to be a transamination. The deaminated metabolites of the amino acids were detected in culture supernatants, and the enzyme activity was identified in cell free extracts. The cells contained increased aromatic amino acid aminotransferase activities on growth on the aromatic amino acids as sole nitrogen sources. Two aromatic aminotransferases (AT-I and AT-II) were separated upon diethylaminoethyl-Trisacryl M column chromatography of cell free extracts. Only AT-I was responsible for the increased level of aromatic amino acid aminotransferase activity of induced cells. The results suggested a catabolic role of AT-I in vivo.Abbreviations DNP dinitrophenyl - HPLC high performance liquid chromatography - PLP pyridoxal-5-phosphate     

Overexpression of a cyanobacterial phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase with diminished sensitivity to feedback inhibition in<Emphasis Type="Italic"> Arabidopsis</Emphasis> changes amino acid metabolism

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Synechococcus vulcanus (SvPEPC) is a unique enzyme, being almost insensitive to feedback inhibition at neutral pH. In order to assess its usefulness in metabolic engineering of plants, SvPEPC was expressed in Arabidopsis thaliana (L.) Heynh. under the control of the cauliflower mosaic virus 35S promoter. About one-third of the transformants of the T1 generation showed severe visible phenotypes such as leaf bleaching and were infertile when grown on soil. However, no such phenotype was observed with Arabidopsis transformed with Zea mays L. PEPC (ZmPEPC) for C4 photosynthesis, which is normally sensitive to a feedback inhibitor, l-malate. For the SvPEPC transformants of the T2 generation, which had been derived from fertile T1 transformants, three kinds of phenotype were observed when plants were grown on an agar medium containing sucrose: Type-I plants showed poor growth and a block in true leaf development; Type-II plants produced a few true leaves, which were partially bleached; Type-III plants were apparently normal. In Type-I plants, total PEPC activity was increased about 2-fold over the control plant but there was no such increase in Type-III plants. The phenotypes of Type-I plants were rescued when the sucrose-containing agar medium was supplemented with aromatic amino acids. Measurement of the free amino acid content in whole seedlings of Type-I transformants revealed that the levels of the aromatic amino acids Phe and Tyr were lowered significantly as compared with the control plants. In contrast, the levels of several amino acids of the aspartic and glutamic families, such as Asn, Gln and Arg, were markedly enhanced (4- to 8-fold per plant fresh weight). However, when the medium was supplemented with aromatic amino acids, the levels of Asn, Gln, and Arg decreased to levels slightly higher than those of control plants, accompanied by growth recovery. Taken together, it can be envisaged that SvPEPC is capable of efficiently exerting its activity in the plant cell environment so as to cause imbalance between aromatic and non-aromatic amino acid syntheses. The growth inhibition of Type-I plants was presumed to be primarily due to a decreased availability of phosphoenolpyruvate, one of the precursors for the shikimate pathway for the synthesis of aromatic amino acids and phenylpropanoids. The possible usefulness of SvPEPC as one of the key components for installing the C₄-like pathway is proposed.Abbreviations CaMV Cauliflower mosaic virus - GUS -Glucuronidase - Kan Kanamycin - 2-ME 2-Mercaptoethanol - MS/G medium 1/2 Murashige–Skoog and 1/2 Gamborg mixed medium - PEP Phosphoenolpyruvate - PEPC Phosphoenolpyruvate carboxylase - Sv Synechococcus vulcanus - ZmPEPC Maize PEPC involved in C₄ photosynthesis     

Anaerobic degradation of phenylacetate and 4-hydroxyphenylacetate by denitrifying bacteria

From various oxic or anoxic habitats anaerobic enrichment cultures were set up which completely oxidized aromatic amino acids to CO₂ with nitrate as electron acceptor. Tyrosine and tryptophan at first were degraded to phenol and indole, respectively, prior to utilization of the aromatic ring; with phenylalanine no intermediates were detected. Attempts to isolate denitrifying bacteria able to completely degrade aromatic amino acids were unsuccessful. Starting with these enrichments several strains of denitrifying bacteria were anaerobically enriched and isolated with known fermentation products of amino acids (phenylacetate, 4-OH-phenylacetate, 2-OH-benzoate) plus nitrate as sole sources of carbon and energy.Three strains were characterized further. They grew well in defined mineral salts medium, were gram-negative and facultatively anaerobic with strictly oxidative metabolism; molecular oxygen, nitrate or nitrite served as electron acceptors. The isolates were tentatively identified as pseudomonads, but could not be aligned to known species. They oxidized a variety of aromatic compounds completely to CO₂ anaerobically and, with some exceptions, also aerobically. The substrates included among others: (4-OH)-phenylacetate, (4-OH)-phenylglyoxylate, benzoate, 2-aminobenzoate, phenol, OH-benzoates, indole and notably toluene. Reduced alicyclic compounds were not utilized. During anaerobic degradation of (4-OH)-phenylacetate transient accumulation of (4-OH)-phenylglyoxylate was observed.It is proposed that anaerobic -oxidation of the-CH₂–COOH side chain to -CO–COOH initiates anaerobic degradation of (4-OH)-phenylacetate. This implies a novel type of anaerobic -hydroxylation with water as the oxygen donor. Abbreviation. Hydroxyl groups were abbreviated as OH     

Cyclodextrin-aided microbial transformation of aromatic aldehydes by Saccharomyces cerevisiae

Summary Biotransformation of benzaldehyde and vanillin by growing cells of Saccharomyces cerevisiae was performed in an aqueous medium containing either - or -cyclodextrin at the same molar concentration as the substrate. The yeast fermentative activity, as reflected by CO₂ evolution, and bioconversion to the corresponding alcohols were both faster and greater in the presence of the cyclic dextrins. Clearly, cyclodextrins were shown to significantly alleviate the inhibitory effects of the aromatic aldehydes.     

Expression of secondary alcohol dehydrogenase in methanogenic bacteria and purification of the F420-specific enzyme from Methanogenium thermophilum strain TCI

In four species of methanogens able to grow with secondary alcohols as hydrogen donors the expression and properties of secondary alcohol dehydrogenase (sec-ADH) were investigated. Cells grown with 2-propanol and CO₂ immediately started to oxidize secondary alcohols to ketones if transferred to new media. In the presence of H₂, such cells reduced ketones or aldehydes to alcohols. In the absence of H₂, aldehydes were dismutated (without growth) to primary alcohols and fatty acids. None of these reactions was catalyzed by cells grown with only H₂ and CO₂ at non-limiting concentration. This indicated an induction or derepression of sec-ADH by its substrate. Apparently, sec-ADH in all strains enabled not only the reduction of ketones or aldehydes, but also the dismutation of the latter. Sec-ADH was also expressed if strains were grown on H₂ and CO₂ in the presence of non-oxidizable, tertiary alcohols. Methanogenium thermophilum expressed sec-ADH even without added alcohol when H₂ became limiting. From this species, an F₄₂₀-specific sec-ADH was purified; the final gel filtration chromatography yielded a single protein peak that coincided with the activity. The enrichment was 12-fold, the activity recovery 26%. SDS polyacrylamide gel electrophoresis indicated that the enzyme was a homodimer with an apparent M _r of 79,000. At the pH optimum around 4.2, the specific activity for oxidation of 2-propanol (130 mM) and reduction of acetone (20 mM) was 176 and 110 mol/ min·mg, respectively (40°C). The apparent K _m for 2-propanol and acetone (with 15 M F₄₂₀) was 2.5 and 0.25 mM, respectively. Aldehydes also were reduced.Non-standard abbreviations ADH alcohol dehydrogenase - Bis-Tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - F₄₂₀ N-(N-L-lactyl--L-glutamyl)-L-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - Mb. Methanobacterium - Mg. Methanogenium - Ms. Methanospirillum - OD₅₇₈ optical density at 578 nm - SDS sodium dodecyl sulfate     

Oxidation of Lignin-Related Aromatic Alcohols by Cell Suspensions of Methylosinus trichosporium

Cell suspensions of Methylosinus trichosporium oxidized the aromatic alcohols benzyl alcohol, vanillyl alcohol, and veratryl alcohol to the corresponding aldehydes, and with the exception of vanillyl alcohol, the aldehydes were further oxidized to the corresponding aromatic acids. No other transformation was observed, and the methoxyl moieties attached to the aromatic nucleus remained intact. More than 70% of the alcohol oxidized could be accounted for by aldehyde and/or acid. Investigation of the inhibitor kinetics of EDTA or p-nitrophenylhydrazine (specific for NAD⁺-independent methanol dehydrogenase in methylotrophs) on aromatic alcohol oxidation revealed noncompetitive inhibition in which the V_max was decreased but the K_m remained unchanged. The pattern of inhibition of aromatic alcohol oxidation matched that of methanol oxidation, and the K_m values for all of the substrates were similar (12 to 16 mM). The results indicate that the initial step in the oxidation of aromatic alcohols was similar to that for methanol, and because oxidation was incomplete (i.e., only the corresponding aldehyde or acid was produced), there may be some biotechnological advantages in using whole cells of methylotrophs to facilitate aromatic biotransformations.     

Effect of leucine on aroma volatiles production from Ceratocystis fimbriata grown in liquid culture

Aroma volatiles produced by Ceratocystis fimbriata on a defined liquid synthetic medium with and without the addition of leucine were identified by gas chromatography–mass spectrometry and quantified by gas chromatography-flame ionisation detection in the liquid medium as well as in the headspace. Volatiles were extracted from the liquid by simultaneous steam distillation–solvent extraction. Ceratocystis fimbriata produced a complex set of volatile intermediary metabolites, of which ethanol was the dominant compound (92–95% of total volatiles). Low molecular weight esters, alcohols, aldehydes, ketones, alkanes, and carboxylic acids were identified in the liquid broth. Alcohols and esters were the most abundant aroma volatiles. Leucine addition effected further growth and higher volatiles production. In the headspace, ethanol and ethyl acetate accounted for 92% of total volatiles over the synthetic medium and 89% when leucine was added. Aroma perception (fruity and banana) correlated closely with liquid and headspace total volatiles.     

Cooxidation of naphthalene and other polycyclic aromatic hydrocarbons by the nitrifying bacterium,Nitrosomonas europaea

The soil nitrifying bacterium Nitrosomonas europaea has shown the ability to transform cometabolically naphthalene as well as other 2- and 3-ringed polycyclic aromatic hydrocarbons (PAHs) to more oxidized products. All of the observed enzymatic reactions were inhibited by acetylene, a selective inhibitor of ammonia monooxygenase (AMO). A strong inhibitory effect of naphthalene on ammonia oxidation by N. europaea was observed. Naphthalene was readily oxidized by N. europaea and 2-naphthol was detected as a major product (85%) of naphthalene oxidation. The maximum naphthol production rate was 1.65 nmole/mg protein-min in the presence of 240 M naphthalene and 10 mM NH₄ ⁺. Our results demonstrate that the oxidation between ammonia and naphthalene showed a partial competitive inhibition. The relative ratio of naphthalene and ammonia oxidation, depending on naphthalene concentrations, demonstrated that the naphthalene was oxidized 2200-fold slower than ammonia at lower concentration of naphthalene (15 M) whereas naphthalene was oxidized only 100-fold slower than ammonia oxidation. NH₄ ⁺- and N₂H₄-dependent O₂ uptake measurement demonstrated irreversible inhibitory effects of the naphthalene and subsequent oxidation products on AMO and HAO activity.     

Production of carbon monoxide from aromatic amino acids by Morganella morganii

Morganella morganii produced CO when cultured in a medium containing casamino acids or peptone as the sole carbon source. Although the production of CO was distinctly enhanced by the addition of hemin to the medium, the amounts of CO produced in the absence of hemin were nearly proportional to the amounts of peptone added to the culture media. Examination of 20 amino acids for their ability to produce CO by resting cells revealed that phenylalanine, tyrosine, histidine and tryptophan were the sources of CO. Oxygen and hemin were necessary for CO production from the amino acids except tryptophan which produced CO in the absence of hemin. When cells were incubated for 4 h at 30° C in the mixture containing 40 mol tyrosine and 1 mol hemin, about 15 mol CO was produced; the activity of CO production was about 1.2 mol CO/h · mg cell nitrogen. Phenylpyruvic acid, p-hydroxyphenylpyruvic acid and imidazolepyruvic acid also produced CO in the presence of hemin, while indolepyruvic acid produced CO regardless of the presence or absence of hemin. The production of CO by the 2-oxo acids proceeded spontaneously and did not require the presence of M. morganii cells.     

Acetogenesis coupled to the oxidation of aromatic aldehyde groups

Vanillin cultures of Clostridium formicoaceticum produced higher cell densities than did vanillate cultures. During growth at the expense of vanillin, vanillate was the predominat intermediate formed; 3,4-dihydroxybenzaldehyde was not a significantly detectable intermediate. Acetate and protocatechuate were both produced in equimolar ratio relative to vanillin consumption. 4-Hydroxybenzaldehyde was a growth-supportive aromatic compound for both C. formicoaceticum and Clostridium aceticum (doubling times approximated 5 h), was oxidized stoichiometrically to 4-hydroxybenzoate, and was not appreciably toxic at concentrations up to 15 mM. Acetate was (i) the major reduced end product detected concomitant to growth and to benzaldehyde oxidation and (ii) formed in close approximation to the following stoichiometry: 4 4-hydroxybenzaldehyde + 2CO₂+2H₂O4 4-hydroxybenzoate + CH₃COOH. We conclude that these two acetogens are capable of benzaldehyde-coupled acetogenesis and growth.     

Studies on lysine analogs,aspartate-derived amino acids,and attempted mutant selection on oat seedlings

The lysine analogs S-2-aminoethyl-L-cysteine (AEC) and DL--hydroxylysine (DHL) caused severe growth inhibition in dark-grown oat seedlings (Avena sativa L. and A. nuda L.) at similar concentrations while L-lysine methyl ester (LME) had little effect. Lysine, arginine, and ornithine reversed the inhibition caused by AEC and DHL, the order of effectiveness being lysine>arginine>ornithine. Of aspartate-pathway amino acids, tested individually and in combinations for inhibitory effects on seedling growth, lysine and combinations containing lysine were the most inhibitory, but the inhibition was much less than that produced by AEC. Only slight synergistic effects occurred when oat seedlings were grown in the presence of paired combinations of aspartatepathway endproduct amino acids.Ca. 54,000 seeds obtained from 3,463 plants grown from ethyl-methanesulfonate (EMS) treated seed were screened for resistance to AEC. Three resistant variants were identified but the resistance was not recovered among their self-pollinated progeny.Abbreviations AEC S-2-aminoethyl-L-cysteine - DHL DL--hydroxylysine - EMS ethyl methanesulfonate - LME L-lysine methyl ester Paper No. 10351, Scientific Journal Series, Minnesota Agricultural Experiment Station     

Oxidation of polycyclic aromatic hydrocarbons by laccase of Coriolus hirsutus

The oxidation of five polycyclic aromatic hydrocarbons; anthracene, benzo()pyrene, fluoranthene, phenanthrene and pyrene was catalyzed by laccase from Coriolus hirsutus in the presence of the redox mediators, 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT). In the ABTS-mediated system, benzo()pyrene was the most rapidly oxidized substrate, with anthracene being the most rapidly oxidized in the HBT-mediated system. There was no clear relationship between the ionization potential and the oxidation of the substrates. ABTS increased the oxidation of benzo()pyrene more than HBT but the oxidation of the other PAHs tested were the opposite. The mediators used in conjunction increased the oxidation of benzo()pyrene compared to using the mediators alone.     

Hybrid Mn-Peroxidase from the Ligninolytic Fungus Panus tigrinus 8/18. Isolation, Substrate Specificity, and Catalytic Cycle

Increased manganese concentration during submerged cultivation of the ligninolytic white rot fungus Panus tigrinus 8/18 on N-limited mineral medium resulted in the induction of Mn-peroxidase and laccase. The Mn-peroxidase was purified with the purity factor RZ (A ₄₀₆/A ₂₈₀) = 4.3. The purified enzyme catalyzed H₂O₂-dependent oxidation of phenol oxidase substrates (aromatic amines, 2,2"-azinobis-(3-ethylbenzthiazolinesulfonic acid), hydroquinone, 2,6-dimethoxyphenol) without Mn²⁺, which is not typical for the usual Mn-peroxidases. Guaiacol and 2,4,6-trichlorophenol were not oxidized in the absence of Mn²⁺. Study of absorption spectra of the intermediates of the catalytic cycle revealed that this peroxidase is able to complete the redox cycle, reducing one-electron oxidized intermediate (Compound II) by Mn²⁺, as well as by an organic substrate (hydroquinone). This means that the enzyme is a hybrid Mn-peroxidase, different from the common Mn-peroxidases from ligninolytic fungi.     

Aspects of nitrogen fixation in Chlorobium

Four strains of the green sulfur bacterium Chlorobium were studied in respect to nitrogen nutrition and nitrogen fixation. All strains grew on ammonia, N₂, or glutamine as sole nitrogen sources; certain strains also grew on other amino acids. Acetylene-reducing activity was detectable in all strains grown on N₂ or on amino acids (except for glutamine). In N₂ grown Chlorobium thiosulfatophilum strain 8327 1 mM ammonia served to switch-off nitrogenase activity, but the effect of ammonia was much less dramatic in glutamate or limiting ammonia grown cells. The glutamine synthetase inhibitor methionine sulfoximine inhibited ammonia switch-off in all but one strain. Cell extracts of glutamate grown strain 8327 reduced acetylene and required Mg²⁺ and dithionite, but not Mn²⁺, for activity. Partially purified preparations of Rhodospirillum rubrum nitrogenase reductase (iron protein) activating enzyme slightly stimulated acetylene reduction in extracts of strain 8327, but no evidence for an indigenous Chlorobium activating enzyme was obtained. The results suggest that certain Chlorobium strains are fairly versatile in their nitrogen nutrition and that at least in vivo, nitrogenase activity in green bacteria is controlled by ammonia in a fashion similar to that described in nonsulfur purple bacteria and in Chromatium.Non-common abbreviations MSX Methionine sulfoximine - MOPS 3-(N-morpholino) propane sulfonic acid This paper is dedicated to Professor Norbert Pfennig on the occasion of his 60th birthday     

Pathway of anaerobic poly-β-hydroxybutyrate degradation byIlyobacter delafieldii

Ilyobacter delafieldii produced an extracellular poly--hydroxybutyrate (PHB) depolymerase when grown on PHB; activity was not detected in cultures grown on 3-hydroxybutyrate, crotonate, pyruvate or lactate. PHB depolymerase activity was largely associated with the PHB granules (supplied as growth substrate), and only 16% was detected free in the culture supernatant. Monomeric 3-hydroxybutyrate was detectable as a product of depolymerase activity. The monomer was fermented to acetate, butyrate and H₂. After activation by coenzyme A transfer from acetyl-CoA or butyryl-CoA, the resultant 3-hydroxybutyryl-CoA was oxidized to acetoacetyl-CoA (producing NADH), followed by thiolytic cleavage to yield acetyl-CoA which was further metabolized to acetyl-phosphate, then to acetate with concomitant ATP production. The reducing equivalents (NADH) could be disposed of by the evolution of H₂, or by a reductive pathway in which 3-hydroxybutyryl-CoA was dehydrated to crotonyl-CoA and reduced to butyryl-CoA. In cocultures ofI. delafieldii withDesulfovibrio vulgaris on PHB, the H₂ partial pressure was much lower than in the pure cultures, and sulfide was produced. Thus interspecies hydrogen transfer caused a shift to increased acetate and H₂ production at the expense of butyrate.     

Role of ω-3 fatty acid desaturases in the regulation of the level of trienoic fatty acids during leaf cell maturation

Trienoic fatty acids, namely -linolenic acid and hexadecatrienoic acid, present in leaf lipids are produced by -3 fatty acid desaturases located in the endoplasmic reticulum and plastid membranes. The changes in the level of trienoic fatty acids during leaf maturation were investigated in wild-type plants of Arabidopsis thaliana (L.) Heynh. and in the fad7 mutant deficient in the activity of a plastid -3 desaturase. The levels of trienoic fatty acids increased in 26 °C- and 15 °C-grown wild-type plants with maturation of leaves. The increase in trienoic fatty acids was mainly due to galactolipids enriched in plastid membranes. In addition, the relative levels of trienoic fatty acids in major glycerolipids, including phospholipids enriched in the endoplasmic reticulum membranes, also increased with leaf maturation. By contrast, when the fad7 mutant was grown at 26 °C, the relative levels of trienoic fatty acids in individual lipids decreased with leaf maturation. The decreases in the levels of trienoic fatty acids, however, were alleviated when the fad7 mutant was grown at 15 °C. These results suggest that the plastid -3 desaturase plays a major role in increasing the levels of trienoic fatty acids with leaf maturation.Abbreviations 163 hexadecatrienoic acid - 183 -linolenic acid - DGD digalactosyldiacylglycerol - MGD monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - TA trienoic fatty acid - WT wild type - -3 refers to the position of the double bond from the methyl end of a fatty acid This research was supported in part by Grants-in-Aid for Scientific research (#07251214 and #06804050 to K.I.) from the Ministry of Education, Science and Culture, Japan, and by the research grant from Shorai Foundation.