Microbody defective mutants of arabidopsis

In germinating fatty seedlings, microbodies are differentiated to leaf peroxisomes from glyoxysomes during greening, and then transformed to glyoxysomes from leaf peroxisomes during senescence. These transformations of microbodies are regulated at various level, such as gene expression, splicing of the mRNA and degradation of microbody proteins. In order to clarify the regulatory mechanisms underlying these transformations of microbodies, we tried to obtain glyoxysome-deficient mutants of Arabidopsis. We screened 2,4-dichlorophenoxybutyric acid (2,4-DB) mutants of Arabidopsis which have defects in glyoxysomal fatty acid β-oxidation. Four mutants can be classified as carrying alleles at three independent loci, which we designatedped1, ped2, andped3, respectively (whereped stands for peroxisome defective). The characteristics of theseped mutants are described. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Acyl-CoA oxidase 1 from Arabidopsis thaliana. Structure of a key enzyme in plant lipid metabolism

The peroxisomal acyl-CoA oxidase family plays an essential role in lipid metabolism by catalyzing the conversion of acyl-CoA into trans-2-enoyl-CoA during fatty acid beta-oxidation. Here, we report the X-ray structure of the FAD-containing Arabidopsis thaliana acyl-CoA oxidase 1 (ACX1), the first three-dimensional structure of a plant acyl-CoA oxidase. Like other acyl-CoA oxidases, the enzyme is a dimer and it has a fold resembling that of mammalian acyl-CoA oxidase. A comparative analysis including mammalian acyl-CoA oxidase and the related tetrameric mitochondrial acyl-CoA dehydrogenases reveals a substrate-binding architecture that explains the observed preference for long-chained, mono-unsaturated substrates in ACX1. Two anions are found at the ACX1 dimer interface and for the first time the presence of a disulfide bridge in a peroxisomal protein has been observed. The functional differences between the peroxisomal acyl-CoA oxidases and the mitochondrial acyl-CoA dehydrogenases are attributed to structural differences in the FAD environments.     

Peroxisomal lipid degradation via β-and α-oxidation in Mammals

Peroxisomal β-oxidation is involved in the degradation of long chain and very long chain fatty acyl-(coenzyme A)CoAs, long chain dicarboxylyl-CoAs, the CoA esters of eicosanoids, 2-methyl-branched fatty acyl-CoAs (e.g. pristanoyl-CoA), and the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids (side chain of cholesterol). In the rat, straight chain acyl-CoAs (including the CoA esters of dicarboxylic fatty acids and eicosanoids) are β-oxidized via palmitoyl-CoA oxidase, multifunctional protein-1 (which displays 2-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA, dehydrogenase activities) and peroxisomal thiolase. 2-Methyl-branched acyl-CoAs are degraded via pristanoyl-CoA oxidase, multifunctional protein-2 (MFP-2) (which displays 2-enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase activities) and sterol carrier protein-X (SCPX; displaying 2-methyl-3-oxoacyl-CoA thiolase activity). The side chain of the bile acid intermediates is shortened via one cycle of β-oxidation catalyzed by trihydroxycoprostanoyl-CoA oxidase, MFP-2 and SCPX. In the human, straight chain acyl-CoAs are oxidized via palmitoyl-CoA oxidase, multifunctional protein-1, and peroxisomal thiolase, as is the case in the rat. The CoA esters of 2-methyl-branched acyl-CoAs and the bile acid intermediates, which also possess a 2-methyl substitution in their side chain, are shortened, via branched chain acyl-CoA oxidase (which is the human homolog of trihydroxycoprostanoyl-CoA oxidase), multifunctional protein-2, and SCPX. The rat and the human enzymes have been purified, cloned, and kinetically and stereochemically characterized. 3-Methyl-branched fatty acids such as phytanic acid are not directly β-oxidizable because of the position of the methyl-branch. They are first shortened by one carbon atom through the a-oxidation process to a 2-methyl-branched fatty acid (pristanic acid in the case of phytanic acid), which is then degraded via peroxisomal β-oxidation. In the human and the rat, α-oxidation is catalyzed by an acyl-CoA synthetase (producing a 3-methylacyl-CoA), a 3-methylacyl-CoA 2-hydroxylase (resulting in a 2-hydroxy-3-methylacyl-CoA), and a 2-hydroxy-3-methylacyl-CoA lyase that cleaves the 2-hydroxy-3-methylacyl-CoA into a 2-methyl-branched fatty aldehyde and formyl-CoA. The fatty aldehyde is dehydrogenated by an aldehyde dehydrogenase to a 2-methyl-branched fatty acid while formyl-CoA is hydrolyzed to formate, which is then converted to CO₂. The activation, hydroxylation and cleavage reactions and the hydrolysis of formyl-CoA are performed by peroxisomal enzymes; the aldehyde dehydrogenation remains to be localized whereas the conversion of formate to CO₂ occurs mainly in the cytosol.     

Arabidopsis peroxisomal malate dehydrogenase functions in beta-oxidation but not in the glyoxylate cycle

The aim was to determine the function of peroxisomal NAD⁺-malate dehydrogenase (PMDH) in fatty acid β-oxidation and the glyoxylate cycle in Arabidopsis. Seeds in which both PMDH genes are disrupted by T-DNA insertions germinate, but seedling establishment is dependent on exogenous sugar. Mutant seedlings mobilize their triacylglycerol very slowly and growth is insensitive to 2,4-dichlorophenoxybutyric acid. Thus mutant seedlings are severely impaired in β-oxidation, even though microarray analysis shows that β-oxidation genes are expressed normally. The mutant phenotype was complemented by expression of a cDNA encoding PMDH with either its native peroxisome targeting signal-2 (PTS2) targeting sequence or a heterologous PTS1 sequence. In contrast to the block in β-oxidation in mutant seedlings, [¹⁴C]acetate is readily metabolized into sugars and organic acids, thereby demonstrating normal activity of the glyoxylate cycle. We conclude that PMDH serves to reoxidize NADH produced from fatty acid β-oxidation and does not participate directly in the glyoxylate cycle.     

Localisation of β-oxidation enzymes in peroxisomes of rice coleoptiles

Enzymes of the β-oxidation pathway in rice ( Oryza sativa L., cv. Arborio) coleoptiles were investigated. The coleoptiles contain acyl-CoA oxidase (EC 1.3.99.3), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), enoyl-CoA hydratase (EC 4.2.1.17) and thiolase (EC 2.3.1.9). Analysis of coleoptile homogenates by sucrose density fractionation showed a preferential distribution of these enzymes in the unspecialized peroxisomes. The enzymatic activity found in the mitochondrial fraction was due to peroxisomal contamination since electron micrographs show the peroxisomes to be intact and pure whereas the mitochondrial fraction was contaminated by other organelles. It appears that the β-oxidation pathway is localized in the unspecialized peroxisomes of rice coleoptiles, extending the number of plant species in which such a localization has been observed.     

Localization of Some Enzymes of β-oxidation of Fatty Acids in the Peroxisomes of Tetrahymena*

Mitochondria and peroxisomes were prepared from homogenates of Tetrahymena pyriformis by sedimentation through sucrose gradients. Catalase and isocitrate lyase served as peroxisomal markers; lactic dehydrogenase and glutamic dehydrogenase as mitochondrial markers. Acetyl-CoA synthetase, octanoyl-CoA synthetase, palmitoyl-CoA synthetase, 3-β hydroxyacyl CoA dehydrogenase, and thiolase activities were found in both the peroxisomes and the mitochondria. It is suggested that β-oxidation of fatty acids accurs in both organelles in Tetrahymena.     

Non-destructive transformation ofArabidopsis

Culture conditions are reported in which roots ofArabidopsis thaliana grow abundantly along the surface of, rather than down into, a solid matrix. Such root segments can be explanted without killing of the plant, which can then complete its life cycle. Non-destructive explanting permits transformation of heterozygous plants without preventing subsequent segregation and of plants that can only be obtained from a population segregating for a marker expressed late in growth. Moreover, the speed and abundance of surface root growth provides a rapid non-destructive phenotypic assay of response to selective conditions.     

Localization of Glyoxylate Cycle Enzymes in Glyoxysomes in Euglena*

SYNOPSIS. We demonstrated previously microbodies in Euglena gracilis grown in the dark on 2-carbon substrates. We have now established in Euglena the particulate nature of enzymes known in other organisms to be localized in microbodies (glyoxysomes and leaf peroxisomes). On a linear sucrose gradient the glyoxylate cycle enzymes band together at a nigner equilibrium density (1.20 g/cm3) than mitochondrial marker enzymes (1.17 g/cm3), establishing the existence in Euglena of glyoxysomes similar to those of higher plants. Glyoxylate (hydroxypyruvate) reductase and, under certain conditions, also glycolate dehydrogenase co-band with the glyoxylate cycle enzymes, suggesting that Euglena glyoxysomes, like those of higher plants, may contain peroxisomal-type enzymes. Catalase, an enzyme characteristic of microbodies from a variety of sources, was not detected in Euglena.     

The use of norbornene derivatives in the synthesis of conformationally constrained peptides and pseudo-peptides

Summary The desymmetrisation ofendo-norborn-5-ene-2,3-dicarboxylic anhydride by proline esters has been used to prepare conformationally constrained pseudo-peptides with two peptide chains parallel to one another. A Curtius rearrangement on the desymmetrication adduct produced the corresponding isocyanate which was used to prepare both a peptide incorporating anendo-2-amino-3-carboxy-norborn-5-ene unit, and a pseudo-peptide with two peptide chains parallel to one another but offset by the presence of a urea unit. The conformational analysis of the resulting peptides was carried out, and the norbornene unit was found to induce the formation of β-turns and parallel β-sheets.     

Metabolism and decarboxylation of glycollate and serine in leaf peroxisomes

The linked utilization of glycollate and L-serine has been studied in peroxisomal preparations from leaves of spinach beet (Beta vulgaris L.). The generation of glycine from glycollate was found to be balanced by the production of hydroxypyruvate from serine and similarly by 2-oxoglutarate when L-glutamate was substituted for L-serine. In the presence of L-malate and catalytic quantities of NAD⁺, about 40% of the hydroxypyruvate was converted further to glycerate, whereas with substrate quantities of NADH, this conversion was almost quantitative. CO₂ was released from the carboxyl groups of both glycollate and serine. Since the decarboxylation of both substrates was greatly in creased by the catalase inhibitor, 3-amino-1,2,4-triazole, and abolished by bovine liver catalase, it was attributed to the nonenzymic attack of H₂O₂, generated in glycollate oxidation, upon glyoxylate and hydroxypyruvate respectively. At 25–30° C, about 10% of the glyoxylate and hydroxypyruvate accumulated was decarboxylated, and the release of CO₂ from each keto-acid was related to the amounts present. It is suggested that hydroxypyruvate decarboxylation might contribute significantly to photorespiration and provide a metabolic route for the complete oxidation of glycollate, the magnitude of this contribution depending upon the concentrations of glyoxylate and hydroxypyruvate in the peroxisomes.     

In vitro regeneration and Agrobacterium-mediated genetic transformation of Euonymus alatus

An in vitro plant regeneration method and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Euonymus alatus. More than 60% of cotyledon and 70% of hypocotyl sections from 10-day-old seedlings of E. alatus produced 2–4 shoots on woody plant medium (WPM) supplemented with 5.0 mg/l 6-benzylaminopurine (BA) plus 0.2 mg/l α-naphthalene acetic acid (NAA), and 77% of shoots produced roots on WPM medium with 0.3 mg/l NAA and 0.5 mg/l Indole-3-butyricacid (IBA). On infection with Agrobacterium tumefaciens strain EHA105 harboring a gusplus gene that contained a plant recognizable intron from the castor bean catalase gene to ensure plant-specific β-glucuronidase (GUS) expression, 16% of cotyledon and 15% of hypocotyl explants produced transgenic shoots using kanamycin as a selection agent, and 67% of these shoots rooted. Stable insertion of T-DNA into the host genome was determined with organ- and tissue-specific expression of the gusplus gene and further confirmed with a PCR-based molecular analysis.     

Biochemistry of peroxisomes in health and disease

The ubiquitous distribution of peroxisomes and the identification of a number of inherited diseases associated with peroxisomal dysfunction indicate that peroxisomes play an essential part in cellular metabolism. Some of the most important metabolic functions of peroxisomes include the synthesis of plasmalogens, bile acids, cholesterol and dolichol, and the oxidation of fatty acids (very long chain fatty acids > C22, branched chain fatty acids (e.g. phytanic acid), dicarboxylic acids, unsaturated fatty acids, prostaglandins, pipecolic acid and glutaric acid). Peroxisomes are also responsible for the metabolism of purines, polyamines, amino acids, glyoxylate and reactive oxygen species (e.g. O-2 and H2O2). Peroxisomal diseases result from the dysfunction of one or more peroxisomal metabolic functions, the majority of which manifest as neurological abnormalities. The quantitation of peroxisomal metabolic functions (e.g. levels of specific metabolites and/or enzyme activity) has bec ome the basis of clinical diagnosis of diseases associated with the organelle. The study of peroxisomal diseases has also contributed towards the further elucidation of a number of metabolic functions of peroxisomes. (Mol Cell Biochem 167:1-29, 1997)     

Nine 3-ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoA thiolases (ACATs) encoded by five genes in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> are targeted either to peroxisomes or cytosol but not to mitochondria

The sub-cellular location of enzymes of fatty acid β-oxidation in plants is controversial. In the current debate the role and location of particular thiolases in fatty acid degradation, fatty acid synthesis and isoleucine degradation are important. The aim of this research was to determine the sub-cellular location and hence provide information about possible functions of all the putative 3-ketoacyl-CoA thiolases (KAT) and acetoacetyl-CoA thiolases (ACAT) in Arabidopsis. Arabidopsis has three genes predicted to encode KATs, one of which encodes two polypeptides that differ at the N-terminal end. Expression in Arabidopsis cells of cDNAs encoding each of these KATs fused to green fluorescent protein (GFP) at their C-termini showed that three are targeted to peroxisomes while the fourth is apparently cytosolic. The four KATs are also predicted to have mitochondrial targeting sequences, but purified mitochondria were unable to import any of the proteins in vitro. Arabidopsis also has two genes encoding a total of five different putative ACATs. One isoform is targeted to peroxisomes as a fusion with GFP, while the others display no targeting in vivo as GFP fusions, or import into isolated mitochondria. Analysis of gene co-expression clusters in Arabidopsis suggests a role for peroxisomal KAT2 in β-oxidation, while KAT5 co-expresses with genes of the flavonoid biosynthesis pathway and cytosolic ACAT2 clearly co-expresses with genes of the cytosolic mevalonate biosynthesis pathway. We conclude that KATs and ACATs are present in the cytosol and peroxisome, but are not found in mitochondria. The implications for fatty acid β-oxidation and for isoleucine degradation in mitochondria are discussed.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Overexpression of proline oxidase induces proline-dependent and mitochondria-mediated apoptosis

The neuropathogenesis of influenza-associated encephalopathy in children and Reye's syndrome remains unclear. A surveillance effort conducted during 2000-2003 in South-West Japan reveals that almost all fatal and handicapped influenza-associated encephalopathy patients exhibit a disorder of mitochondrial β-oxidation with elevated serum acylcarnitine ratios (C_16:0+C_18:1)/C₂. Here we show invasion by a non-neurotropic epidemic influenza A H3N2 virus in cerebral capillaries with progressive brain edema after intranasal infection of mice having impaired mitochondrial β-oxidation congenitally or posteriorly in the newborn/ suckling periods. Mice genetically lacking of carnitine transporter OCTN2, resulting in carnitine deficiency and impaired β-oxidation, exhibited significant higher virus-genome numbers in the brain, accumulation of virus antigen exclusively in the cerebral capillaries and increased brain vascular permeability compared to in wild type mice. Mini-plasmin, which proteolytically potentiates influenza virus multiplication in vivo and destroys the blood-brain barrier, accumulated with virus antigen in the brain capillaries of OCTN2-deficient mice but only a little in wild-type mice. These results suggest that the impaired mitochondrial β-oxidation changes the susceptibility to a non-neurotropic influenza A virus as to multiplication in the brain capillaries and to cause brain edema. These pathological findings in the brain of mice having impaired mitochondrial β-oxidation after influenza virus infection may have implications for human influenza-associated encephalopathy.     

1β,7β-Dihydroxydehydroabietic acid,a new biotransformation product of dehydroabietic acid by Aspergillus niger

Microbial transformation of dehydroabietic acid by Aspergillus niger afforded the new derivative 1β,7β-dihydroxydehydroabietic acid and the known 1β-hydroxy and 7β-hydroxy derivatives. The structures were elucidated by spectroscopic methods. The compounds were assessed towards Gram (+) and Gram (−) bacteria and showed a weak antimicrobial effect. This revised version was published online in August 2006 with corrections to the Cover Date.     

Termini and telomeres in T-DNA transformation

A T-DNA vector for plant transformation has been constructed in which the cloning site is located 9 bp from the right-border (RB) end and 27 bp from the left-border (LB) end. In this vector cloned DNA homologous to plant chromosomal sequences is located at the T-DNA termini, and will thus be exposed by even limited exonucleolysis in planta. The arabidopsis ADH (alcohol dehydrogenase) locus was mobilized from Agrobacterium, and integration into the recipient genome was studied. Despite the terminal location of ADH homology in this vector, the T-DNA integrated essentially at random in the Arabidopsis genome rather than at the endogenous ADH locus. T-DNA integration was blocked, however, when Arabidopsis telomeric sequences were added to the construct at each end of the ADH homology. Thus the predominant mode by which incoming T-DNA is integrated into the continuity of chromosomal DNA involves free DNA ends, but, in contrast to modes of recombination such as gap repair, does not involve extensive terminal DNA sequence homology.     

Metabolic significance and expression of Caenorhabditis elegans type II 3-oxoacyl-CoA thiolase

The authors cloned the cDNA of the nematode Caenorhabditis elegans encoding a 44-kDa protein (P-44), which is similar to sterol carrier protein x (SCPx). Genomic DNA data and Northern blot analysis excluded the possibility of P-44 forming SCPx-like fusion protein. P-44 is required in the formation of bile acid in vitro from CoA esters of their enoyl-form intermediate in the presence of d-3-hydroxyacyl-CoA dehydratase/d-3-dehydrogenase bifunctional protein. Also, rat SCPx converts 24-hydroxy-form intermediate to bile acid under similar conditions. From this and other evidence, P-44 and SCPx were categorized as type II thiolase. The mRNA encoding P-44 was detected in every developmental stage of C. elegans: egg, larval stages, and adult. P-44, therefore, seems essential for the normal functioning of this organism.     

Plant Peroxisomes: Molecular Basis of the Regulation of their Functions

β -oxidation, the glyoxylate cycle and the glycolate pathway for photorespiration. Recent molecular biological studies have revealed that most of these enzymes possess either one of two peroxisomal targeting signals (PTS) within their amino acid sequence. One of the signals, PTS1, is found at the carboxy-terminus, while the other, PTS2, is found within the amino-terminal presequence. Subsequent to the synthesis and folding of these enzymes in the cytosol, the targeting signal in the folded proteins may bind to the corresponding receptors. At present, only a receptor that recognizes PTS1 has been identified in higher plants. After the binding of the protein and the receptor, the protein complex may be recognized by docking proteins that exist in the peroxisomal membrane. The mechanisms responsible for the recognition of peroxisomal proteins are now under investigation. Genetic analyses of Arabidopsis mutants with defective peroxisomes may give us some clues to understanding the mechanisms of peroxisomal protein import. Received 18 November 1999/ Accepted in revised form 13 January 2000     

Decalactone Production by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> under increased O<Subscript>2</Subscript> Transfer Rates

Yarrowia lipolytica converts methyl ricinoleate to γ-decalactone, a high-value fruity aroma compound. The highest amount of 3-hydroxy-γ-decalactone produced by the yeast (263 mg l^-1) occurred by increasing the k_La up to 120 h⁻¹ at atmospheric pressure; above it, its concentration decreased, suggesting a predominance of the activity of 3-hydroxyacyl-CoA dehydrogenase. Cultures were grown under high-pressure, i.e., under increased O₂ solubility, but, although growth was accelerated, γ-decalactone production decreased. However, by applying 0.5 MPa during growth and biotransformation gave increased concentrations of dec−2-en-4-olide and dec-3-en-4-olide (70 mg l⁻¹).