Gene-specific involvement of beta-oxidation in wound-activated responses in Arabidopsis

The coordinated induced expression of beta-oxidation genes is essential to provide the energy supply for germination and postgerminative development. However, very little is known about other functions of beta-oxidation in nonreserve organs. We have identified a gene-specific pattern of induced beta-oxidation gene expression in wounded leaves of Arabidopsis. Mechanical damage triggered the local and systemic induction of only ACX1 among acyl-coenzyme A oxidase (ACX) genes, and KAT2/PED1 among 3-ketoacyl-coenzyme A thiolase (KAT) genes in Arabidopsis. In turn, wounding induced KAT5/PKT2 only systemically. Although most of the beta-oxidation genes were activated by wound-related factors such as dehydration and abscisic acid, jasmonic acid (JA) induced only ACX1 and KAT5. Reduced expression of ACX1 or KAT2 genes, in transgenic plants expressing their corresponding mRNAs in antisense orientation, correlated with defective wound-activated synthesis of JA and with reduced expression of JA-responsive genes. Induced expression of JA-responsive genes by exogenous application of JA was unaffected in those transgenic plants, suggesting that ACX1 and KAT2 play a major role in driving wound-activated responses by participating in the biosynthesis of JA in wounded Arabidopsis plants.     

Identification of a peroxisomal acyl-activating enzyme involved in the biosynthesis of jasmonic acid in Arabidopsis

Jasmonic acid (JA) is a lipid-derived signal that regulates a wide variety of developmental and defense-related processes in higher plants. JA is synthesized from linolenic acid via an enzymatic pathway that initiates in the plastid and terminates in peroxisomes. The C18 JA precursor 12-oxo-phytodienoic acid (OPDA) is converted in the peroxisome to 3-oxo-2-(2'-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8:0), which subsequently undergoes three rounds of beta-oxidation to yield JA. Although most JA biosynthetic enzymes have been identified, several key steps in the pathway remain to be elucidated. To address this knowledge gap, we employed co-expression analysis to identify genes that are coordinately regulated with known JA biosynthetic components in Arabidopsis. Among the candidate genes uncovered by this approach was a 4-coumarate-CoA ligase-like member of the acyl-activating enzyme (AAE) gene family, which we have named OPC-8:0 CoA Ligase1 (OPCL1). In response to wounding, opcl1 null mutants exhibited reduced levels of JA and hyperaccumulation of OPC-8:0. Recombinant OPCL1 was active against both OPDA and OPC-8:0, as well as medium-to-long straight-chain fatty acids. Subcellular localization studies with green fluorescent protein-tagged OPCL1 showed that the protein is targeted to peroxisomes. These findings establish a physiological role for OPCL1 in the activation of JA biosynthetic precursors in leaf peroxisomes, and further indicate that OPC-8:0 is a physiological substrate for the activation step. The results also demonstrate the utility of co-expression analysis for identification of factors that contribute to jasmonate homeostasis.     

The Arabidopsis 3-ketoacyl-CoA thiolase-2 (kat2-1) mutant exhibits increased flowering but reduced reproductive success

The enzyme 3-ketoacyl-CoA thiolase (KAT) (EC 2.3.1.16) catalyses a key step in fatty acid beta-oxidation. In Arabidopsis thaliana, expression of the KAT2 gene is known to be required for the efficient mobilization of triacylglycerol during germination and seedling establishment. Here, data from the Arabidopsis kat2-1 mutant are presented, showing that perturbation of beta-oxidation also affects vegetative growth and reproductive success. In the wild type, the KAT2 protein was detected in all organs tested. In the kat2-1 mutant, rosette leaf area and dry weight, but not leaf number, were greatly increased relative to wild type. Global proliferative arrest of flowering was delayed, resulting in increased silique production in kat2-1 plants. However, total silique dry weight was not increased. kat2-1 siliques were smaller and had a reduced seed number caused by increased ovule abortion. In kat2-1 ovules, carbon flow into sugars via gluconeogeneis and respiration were both reduced in comparison to the wild type. In conclusion, these data indicate that a functional beta-oxidation pathway is required to maintain the balance between silique development and the continued initiation of floral meristems.     

The crystal structure of a plant 3-ketoacyl-CoA thiolase reveals the potential for redox control of peroxisomal fatty acid beta-oxidation

Crystal structures of peroxisomal Arabidopsis thaliana 3-ketoacyl-CoA thiolase (AtKAT), an enzyme of fatty acid beta-oxidation, are reported. The subunit, a typical thiolase, is a combination of two similar alpha/beta domains capped with a loop domain. The comparison of AtKAT with the Saccharomyces cerevisiae homologue (ScKAT) structure reveals a different placement of subunits within the functional dimers and that a polypeptide segment forming an extended loop around the open catalytic pocket of ScKAT converts to alpha-helix in AtKAT, and occludes the active site. A disulfide is formed between Cys192, on this helix, and Cys138, a catalytic residue. Access to Cys138 is determined by the structure of this polypeptide segment. AtKAT represents an oxidized, previously unknown inactive form, whilst ScKAT is the reduced and active enzyme. A high level of sequence conservation is observed, including Cys192, in eukaryotic peroxisomal, but not mitochondrial or prokaryotic KAT sequences, for this labile loop/helix segment. This indicates that KAT activity in peroxisomes is influenced by a disulfide/dithiol change linking fatty acid beta-oxidation with redox regulation.     

Expression of the beta-oxidation gene 3-ketoacyl-CoA thiolase 2 (KAT2) is required for the timely onset of natural and dark-induced leaf senescence in Arabidopsis

The onset of leaf senescence is regulated by a complex mechanism involving positive and negative regulators. Among positive regulators, jasmonic acid (JA) accumulates in senescing leaves and the JA-insensitive coi1-1 mutant displays delayed leaf senescence in Arabidopsis. A strong activated expression of the gene coding for the JA-biosynthetic beta-oxidation enzyme 3-ketoacyl-CoA thiolase 2 (KAT2) in natural and dark-induced senescing leaves of Arabidopsis thaliana is reported here. By using KAT2::GUS and KAT2::LUC transgenic plants, it was observed that dark-induced KAT2 activation occurred both in excised leaves as well as in whole darkened plants. The KAT2 activation associated with dark-induced senescence occurred soon after a move to darkness, and it preceded the detection of symptoms and the expression of senescence-associated gene (SAG) markers. Transgenic plants with reduced expression of the KAT2 gene showed a significant delayed senescence both in natural and dark-induced processes. The rapid induction of the KAT2 gene in senescence-promoting conditions as well as the delayed senescence phenotype and the reduced SAG expression in KAT2 antisense transgenic plants, point to KAT2 as an essential component for the timely onset of leaf senescence in Arabidopsis.     

Jasmonate biosynthesis in Arabidopsis thaliana requires peroxisomal beta-oxidation enzymes--additional proof by properties of pex6 and aim1

Jasmonic acid (JA) is an important regulator of plant development and stress responses. Several enzymes involved in the biosynthesis of JA from alpha-linolenic acid have been characterized. The final biosynthesis steps are the beta-oxidation of 12-oxo-phytoenoic acid. We analyzed JA biosynthesis in the Arabidopsis mutants pex6, affected in peroxisome biogenesis, and aim1, disrupted in fatty acid beta-oxidation. Upon wounding, these mutants exhibit reduced JA levels compared to wild type. pex6 accumulated the precursor OPDA. Feeding experiments with deuterated OPDA substantiate this accumulation pattern, suggesting the mutants are impaired in the beta-oxidation of JA biosynthesis at different steps. Decreased expression of JA-responsive genes, such as VSP1, VSP2, AtJRG21 and LOX2, following wounding in the mutants compared to the wild type reflects the reduced JA levels of the mutants. By use of these additional mutants in combination with feeding experiments, the necessity of functional peroxisomes for JA-biosynthesis is confirmed. Furthermore an essential function of one of the two multifunctional proteins of fatty acid beta-oxidation (AIM1) for wound-induced JA formation is demonstrated for the first time. These data confirm that JA biosynthesis occurs via peroxisomal fatty acid beta-oxidation machinery.     

The multifunctional protein AtMFP2 is co-ordinately expressed with other genes of fatty acid beta-oxidation during seed germination in Arabidopsis thaliana (L.) Heynh

In germinating oilseeds peroxisomal fatty acid beta-oxidation is responsible for the mobilization of storage lipids. This pathway also occurs in other tissues where it has a variety of additional physiological functions. The central enzymatic steps of peroxisomal beta-oxidation are performed by acyl-CoA oxidase (ACOX), the multifunctional protein (MFP) and 3-ketoacyl-CoA thiolase (thiolase). In order to investigate the function and regulation of beta-oxidation in plants it is first necessary to identify and characterize genes encoding the relevant enzymes in a single model species. Recently we and others have reported on the cloning and characterization of genes encoding four ACOXs and a thiolase from the oilseed Arabidopsis thaliana. Here we identify a gene encoding an Arabidopsis MFP (AtMFP2) that is induced transiently during germination. The pattern of AtMFP2 expression closely reflects changes in the activities of 2-trans-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase. Similar patterns of expression have previously been reported for ACOX and thiolase genes. We conclude that genes encoding the three main proteins responsible for beta-oxidation are co-ordinately expressed during oilseed germination and may share a common mechanism of regulation.     

Metabolism of 4-pentenoic acid and inhibition of thiolase by metabolites of 4-pentenoic acid

The metabolism of 4-pentenoic acid, a hypoglycemic agent and inhibitor of fatty acid oxidation, has been studied in rat heart mitochondria. Confirmed was the conversion of 4-pentenoic acid to 2,4-pentadienoyl coenzyme A (CoA), which either is directly degraded via beta-oxidation or is first reduced in a NADPH-dependent reaction before it is further degraded by beta-oxidation. At pH 6.9, the NADPH-dependent reduction of 2,4-pentadienoyl-CoA proceeds 10 times faster than its degradation by beta-oxidation. At pH 7.8, this ratio is only 2 to 1. The direct beta-oxidation of 2,4-pentadienoyl-CoA leads to the formation of 3-keto-4-pentenoyl-CoA, which is highly reactive and spontaneously converts to another 3-ketoacyl-CoA derivative (compound X). 3-Keto-4-pentenoyl-CoA is a poor substrate of 3-ketoacyl-CoA thiolase (EC 2.3..1.16) whereas compound X is not measurably acted upon by this enzyme. The effects of several metabolites of 4-pentenoic acid on the activity of 3-ketoacyl-CoA thiolase were studied. 3,4-Pentadienoyl-CoA is a weak inhibitor of this enzyme that is protected against the inhibition by acetoacetyl-CoA. The most effective inhibitor of 3-ketoacyl-CoA thiolase was found to be 3-keto-4-pentenoyl-CoA, which inhibits the enzyme in both a reversible and irreversible manner. The reversible inhibition is possibly a consequence of the inhibitor being a poor substrate of 3-ketoacyl-CoA thiolase. It is concluded that 4-pentenoic acid is metabolized in mitochondria by two pathways. The minor yields 3-keto-4-pentenoyl-CoA, which acts both as a reversible and as a irreversible inhibitor of 3-ketoacyl-CoA thiolase and consequently of fatty acid oxidation.     

Fatty acid oxidation in rat brain is limited by the low activity of 3-ketoacyl-coenzyme A thiolase

In an attempt to clarify why the brain oxidizes fatty acids poorly or not at all, the activities of beta-oxidation enzymes present in rat brain and rat heart mitochondria were measured and compared with each other. Although the apparent Km values and chain-length specificities of the brain and heart enzymes are similar, the specific activities of all but one brain enzyme are between 4 and 50% of those observed in heart mitochondria. The exception is 3-ketoacyl-CoA thiolase (EC 2.3.1.16) whose specific activity in brain mitochondria is 125 times lower than in heart mitochondria. The partially purified brain 3-ketoacyl-CoA thiolase was shown to be catalytically and immunologically identical with the heart enzyme. The low rate of fatty acid oxidation in brain mitochondria, estimated on the basis of palmitoylcarnitine-supported respiration and [1-14C]palmitoylcarnitine degradation to be less than 0.5 nmol/min/mg of protein, may be the consequence of the low activity of 3-ketoacyl-CoA thiolase. Inhibition of [1-14C]palmitoylcarnitine oxidation by 4-bromocrotonic acid proves the observed oxidation of fatty acids in brain to be dependent on 3-ketoacyl-CoA thiolase and thus to occur via beta-oxidation. Since the reactions catalyzed by carnitine palmitoyltransferase (EC 2.3.1.21) and acyl-CoA synthetase (EC 6.2.1.3) do not seem to restrict fatty acid oxidation in brain, it is concluded that the oxidation of fatty acids in rat brain is limited by the activity of the mitochondrial 3-keto-acyl-CoA thiolase.     

4-Bromo-2-octenoic acid specifically inactivates 3-ketoacyl-CoA thiolase and thereby fatty acid oxidation in rat liver mitochondria

In an attempt to develop a compound which would specifically inhibit 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in whole mitochondria, 4-bromo-2-octenoic acid was synthesized and studied. After rat liver mitochondria were preincubated with 4-bromo-2-octenoic acid for 3 min, respiration supported by either palmitoylcarnitine or pyruvate was completely abolished, whereas no inhibition was observed with rat heart mitochondria. Addition of carnitine stimulated respiration supported by pyruvate without relieving inhibition of palmitoylcarnitine-dependent respiration. Hence, this compound seems to be a specific inhibitor of beta-oxidation. When the enzymes of beta-oxidation were assayed in a soluble extract prepared from mitochondria preincubated with 4-bromo-2-octenoic acid, only 3-ketoacyl-CoA thiolase was found to be inactivated. 4-Bromo-2-octenoic acid is metabolized by mitochondrial beta-oxidation enzymes to 3-keto-4-bromooctanoyl-CoA which effectively and irreversibly inhibits 3-ketoacyl-CoA thiolase but not acetoacetyl-CoA thiolase (EC 2.3.1.9). Even though 3-keto-4-bromooctanoyl-CoA inhibits the latter enzyme reversibly, 4-bromo-2-octenoic acid does not inhibit ketogenesis in rat liver mitochondria with acetylcarnitine as a substrate. It is concluded that 4-bromo-2-octenoic acid specifically inhibits mitochondrial fatty acid oxidation by inactivating 3-ketoacyl-CoA thiolase in rat liver mitochondria.     

Up-regulation of the peroxisomal beta-oxidation system occurs in butyrate-grown Candida tropicalis following disruption of the gene encoding peroxisomal 3-ketoacyl-CoA thiolase

In the yeast Candida tropicalis, two thiolase isozymes, peroxisomal acetoacetyl-CoA thiolase and peroxisomal 3-ketoacyl-CoA thiolase, participate in the peroxisomal fatty acid beta-oxidation system. Their individual contributions have been demonstrated in cells grown on butyrate, with C. tropicalis able to grow in the absence of either one. In the present study, a lack of peroxisomal 3-ketoacyl-CoA thiolase protein resulted in increased expression (up-regulation) of acetoacetyl-CoA thiolase and other peroxisomal proteins, whereas a lack of peroxisomal acetoacetyl-CoA thiolase produced no corresponding effect. Overexpression of the acetoacetyl-CoA thiolase gene did not suppress the up-regulation or the growth retardation on butyrate in cells without peroxisomal 3-ketoacyl-CoA thiolase, even though large amounts of the overexpressed acetoacetyl-CoA thiolase were detected in most of the peroxisomes of butyrate-grown cells. These results provide important evidence of the greater contribution of 3-ketoacyl-CoA thiolase to the peroxisomal beta-oxidation system than acetoacetyl-CoA thiolase in C. tropicalis and a novel insight into the regulation of the peroxisomal beta-oxidation system.     

Hydroxylated jasmonates are commonly occurring metabolites of jasmonic acid and contribute to a partial switch-off in jasmonate signaling

In potato 12-hydroxyjasmonic acid (12-OH-JA) is a tuber-inducing compound. Here, it is demonstrated that 12-OH-JA, as well as its sulfated and glucosylated derivatives, are constituents of various organs of many plant species. All accumulate differentially and usually to much higher concentrations than jasmonic acid (JA). In wounded tomato leaves, 12-OH-JA and its sulfated, as well as glucosylated, derivative accumulate after JA, and their diminished accumulation in wounded leaves of the JA-deficient mutants spr2 and acx1 and also a JA-deficient 35S::AOCantisense line suggest their JA-dependent formation. To elucidate how signaling properties of JA/JAME (jasmonic acid methyl ester) are affected by hydroxylation and sulfation, germination and root growth were recorded in the presence of the different jasmonates, indicating that 12-OH-JA and 12-hydroxyjasmonic acid sulfate (12-HSO(4)-JA) were not bioactive. Expression analyses for 29 genes showed that expression of wound-inducible genes such as those coding for PROTEINASE INHIBITOR2, POLYPHENOL OXIDASE, THREONINE DEAMINASE or ARGINASE was induced by JAME and less induced or even down-regulated by 12-OH-JA and 12-HSO(4)-JA. Almost all genes coding for enzymes in JA biosynthesis were up-regulated by JAME but down-regulated by 12-OH-JA and 12-HSO(4)-JA. The data suggest that wound-induced metabolic conversion of JA/JAME into 12-OH-JA alters expression pattern of genes including a switch off in JA signaling for a subset of genes.     

Formation and degradation of dicarboxylic acids in relation to alterations in fatty acid oxidation in rats.

Dicarboxylic acids are excreted in urine when fatty acid oxidation is increased (ketosis) or inhibited (defects in beta-oxidation) and in Reye's syndrome. omega-Hydroxylation and omega-oxidation of C6-C12 fatty acids were measured by mass spectrometry in rat liver microsomes and homogenates, and beta-oxidation of the dicarboxylic acids in liver homogenates and isolated mitochondria and peroxisomes. Medium-chain fatty acids formed large amounts of medium-chain dicarboxylic acids, which were easily beta-oxidized both in vitro and in vivo, in contrast to the long-chain C16-dicarboxylic acid, which was toxic to starved rats. Increment of fatty acid oxidation in rats by starvation or diabetes increased C6:C10 dicarboxylic acid ratio in rats fed medium-chain triacylglycerols, and increased short-chain dicarboxylic acid excretion in urine in rats fed medium-chain dicarboxylic acids. Valproate, which inhibits fatty acid oxidation and may induce Reye like syndromes, caused the pattern of C6-C10-dicarboxylic aciduria seen in beta-oxidation defects, but only in starved rats. It is suggested, that the origin of urinary short-chain dicarboxylic acids is omega-oxidized medium-chain fatty acids, which after peroxisomal beta-oxidation accumulate as C6-C8-dicarboxylic acids. C10-C12-dicarboxylic acids were also metabolized in the mitochondria, but did not accumulate as C6-C8-dicarboxylic acids, indicating that beta-oxidation was completed beyond the level of adipyl CoA.     

Identification of the peroxisomal beta-oxidation enzymes involved in the biosynthesis of docosahexaenoic acid.

DHA (C22:6n-3) is an important PUFA implicated in a number of (patho)physiological processes. For a long time, the exact mechanism of DHA formation has remained unclear, but now it is known that it involves the production of tetracosahexaenoic acid (C24:6n-3) from dietary linolenic acid (C18:3n-3) via a series of elongation and desaturation reactions, followed by beta-oxidation of C24:6n-3 to C22:6n-3. Although DHA is deficient in patients lacking peroxisomes, the intracellular site of retroconversion of C24:6n-3 has remained controversial. By making use of fibroblasts from patients with defined mitochondrial and peroxisomal fatty acid oxidation defects, we show in this article that peroxisomes, and not mitochondria, are involved in DHA formation by catalyzing the beta-oxidation of C24:6n-3 to C22:6n-3. Additional studies of fibroblasts from patients with X-linked adrenoleukodystrophy, straight-chain acyl-CoA oxidase (SCOX) deficiency, d-bifunctional protein (DBP) deficiency, and rhizomelic chondrodysplasia punctata type 1, and of fibroblasts from l-bifunctional protein and sterol carrier protein X (SCPx) knockout mice, show that the main enzymes involved in beta-oxidation of C24:6n-3 to C22:6n-3 are SCOX, DBP, and both 3-ketoacyl-CoA thiolase and SCPx. These findings are of importance for the treatment of patients with a defect in peroxisomal beta-oxidation.     

Fatty acid degradation in Caulobacter crescentus.

Fatty acid degradation was investigated in Caulobacter crescentus, a bacterium that exhibits membrane-mediated differentiation events. Two strains of C. crescentus were shown to utilize oleic acid as sole carbon source. Five enzymes of the fatty acid beta-oxidation pathway, acyl-coenzyme A (CoA) synthase, crotonase, thiolase, beta-hydroxyacyl-CoA dehydrogenase, and acyl-CoA dehydrogenase, were identified. The activities of these enzymes were significantly higher in C. crescentus than the fully induced levels observed in Escherichia coli. Growth in glucose or glucose plus oleic acid decreased fatty acid uptake and lowered the specific activity of the enzymes involved in beta-oxidation by 2- to 3-fold, in contrast to the 50-fold glucose repression found in E. coli. The mild glucose repression of the acyl-CoA synthase was reversed by exogenous dibutyryl cyclic AMP. Acyl-CoA synthase activity was shown to be the same in oleic acid-grown cells and in cells grown in the presence of succinate, a carbon source not affected by catabolite repression. Thus, fatty acid degradation by the beta-oxidation pathway is constitutive in C. crescentus and is only mildly affected by growth in the presence of glucose. Tn5 insertion mutants unable to form colonies when oleic acid was the sole carbon source were isolated. However, these mutants efficiently transported fatty acids and had beta-oxidation enzyme levels comparable with that of the wild type. Our inability to obtain fatty acid degradation mutants after a wide search, coupled with the high constitutive levels of the beta-oxidation enzymes, suggest that fatty acid turnover, as has proven to be the case fatty acid biosynthesis, might play an essential role in membrane biogenesis and cell cycle events in C. crescentus.     

Peroxisomal Plant 3-Ketoacyl-CoA Thiolase Structure and Activity Are Regulated by a Sensitive Redox Switch

The breakdown of fatty acids, performed by the β-oxidation cycle, is crucial for plant germination and sustainability. β-Oxidation involves four enzymatic reactions. The final step, in which a two-carbon unit is cleaved from the fatty acid, is performed by a 3-ketoacyl-CoA thiolase (KAT). The shortened fatty acid may then pass through the cycle again (until reaching acetoacetyl-CoA) or be directed to a different cellular function. Crystal structures of KAT from Arabidopsis thaliana and Helianthus annuus have been solved to 1.5 and 1.8 Å resolution, respectively. Their dimeric structures are very similar and exhibit a typical thiolase-like fold; dimer formation and active site conformation appear in an open, active, reduced state. Using an interdisciplinary approach, we confirmed the potential of plant KATs to be regulated by the redox environment in the peroxisome within a physiological range. In addition, co-immunoprecipitation studies suggest an interaction between KAT and the multifunctional protein that is responsible for the preceding two steps in β-oxidation, which would allow a route for substrate channeling. We suggest a model for this complex based on the bacterial system.     

Shifting of the distribution of aromatic monomer-units in polyhydroxyalkanoic acid to longer units by salicylic acid in Pseudomonas fluorescens BM07 grown with mixtures of fructose and 11-phenoxyundecanoic acid

Medium-chain-length-polyhydroxyalkanoic acids (MCL-PHAs) formed in Pseudomonas spp. have a rather broad distribution of monomer-units whose precursors are supplied via beta-oxidation degradation of MCL fatty acids fed as the carbon source and/or via PhaG enzyme catalyzing the acyl-group transfer from 3-hydroxyacyl-ACPs derived from acetyl-CoA to coenzyme A. It was found that salicylic acid (SA), in a concentration dependent manner, suppressed the accumulation of PHA in Pseudomonas fluorescens BM07 from fructose as well as shifted the distribution of monomer-units derived from a MCL fatty acid co-added as carbon source (e.g., 11-phenoxyundecanoic acid (11-POU)) to longer monomer-units. Both SA and acrylic acid were found to induce high accumulations of 3-ketohexanoic acid in BM07 wild-type cells grown with n-hexanoic acid as well as to inhibit the formation of acetyl-CoA from acetoacetyl-CoA by BM07 cell extract, suggesting that 3-ketoacyl-CoA thiolase is their common beta-oxidation target. The structural motif of acrylic acid present in the molecular structure of SA may self-explain the similar actions of the two inhibitors. A comparison of monomer modulation between BM07 wild-type and DeltaphaG mutant cells grown on the mixtures of fructose and 11-POU revealed that both PhaG and beta-oxidation inhibitor may play a critical role in the synthesis of PHA with longer side-chain omega-functional substitutions.