Arabidopsis mutants in short- and medium-chain acyl-CoA oxidase activities accumulate acyl-CoAs and reveal that fatty acid beta-oxidation is essential for embryo development

The short-chain acyl-CoA oxidase (ACX4) is one of a family of ACX genes that together catalyze the first step of peroxisomal fatty acid beta-oxidation during early, postgerminative growth in oilseed species. Here we have isolated and characterized an Arabidopsis thaliana mutant containing a T-DNA insert in ACX4. In acx4 seedlings, short-chain acyl-CoA oxidase activity was reduced by greater than 98%, whereas medium-chain activity was unchanged from wild type levels. Despite the almost complete loss of short-chain activity, lipid catabolism and seedling growth and establishment were unaltered in the acx4 mutant. However, the acx4 seedlings accumulated high levels (31 mol %) of short-chain acyl-CoAs and showed resistance to 2,4-dichlorophenoxybutyric acid, which is converted to the herbicide and auxin analogue 2,4-dichlorophenoxyacetic acid by beta-oxidation. A mutant in medium-chain length acyl-CoA activity (acx3) (1) shows a similar phenotype to acx4, and we show here that acx3 seedlings accumulate medium-chain length acyl-CoAs (16.4 mol %). The acx3 and acx4 mutants were crossed together, and remarkably, the acx3acx4 double mutants aborted during the first phase of embryo development. We propose that acx3acx4 double mutants are nonviable because they have a complete block in short-chain acyl-CoA oxidase activity. This is the first demonstration of the effects of eliminating (short-chain) beta-oxidation capacity in plants and shows that a functional beta-oxidation cycle is essential in the early stages of embryo development.     

Mutations in Arabidopsis acyl-CoA oxidase genes reveal distinct and overlapping roles in beta-oxidation

Indole-3-butyric acid (IBA) is an endogenous auxin used to enhance rooting during propagation. To better understand the role of IBA, we isolated Arabidopsis IBA-response (ibr) mutants that display enhanced root elongation on inhibitory IBA concentrations but maintain wild-type responses to indole-3-acetic acid, the principle active auxin. A subset of ibr mutants remains sensitive to the stimulatory effects of IBA on lateral root initiation. These mutants are not sucrose dependent during early seedling development, indicating that peroxisomal beta-oxidation of seed storage fatty acids is occurring. We used positional cloning to determine that one mutant is defective in ACX1 and two are defective in ACX3, two of the six Arabidopsis fatty acyl-CoA oxidase (ACX) genes. Characterization of T-DNA insertion mutants defective in the other ACX genes revealed reduced IBA responses in a third gene, ACX4. Activity assays demonstrated that mutants defective in ACX1, ACX3, or ACX4 have reduced fatty acyl-CoA oxidase activity on specific substrates. Moreover, acx1 acx2 double mutants display enhanced IBA resistance and are sucrose dependent during seedling development, whereas acx1 acx3 and acx1 acx5 double mutants display enhanced IBA resistance but remain sucrose independent. The inability of ACX1, ACX3, and ACX4 to fully compensate for one another in IBA-mediated root elongation inhibition and the ability of ACX2 and ACX5 to contribute to IBA response suggests that IBA-response defects in acx mutants may reflect indirect blocks in peroxisomal metabolism and IBA beta-oxidation, rather than direct enzymatic activity of ACX isozymes on IBA-CoA.     

Peroxisomal Acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana

In plants and other eukaryotes, long-chain acyl-CoAs are assumed to be imported into peroxisomes for beta-oxidation by an ATP binding cassette (ABC) transporter. However, two genes in Arabidopsis thaliana, LACS6 and LACS7, encode peroxisomal long-chain acyl-CoA synthetase (LACS) isozymes. To investigate the biochemical and biological roles of peroxisomal LACS, we identified T-DNA knockout mutants for both genes. The single-mutant lines, lacs6-1 and lacs7-1, were indistinguishable from the wild type in germination, growth, and reproductive development. By contrast, the lacs6-1 lacs7-1 double mutant was specifically defective in seed lipid mobilization and required exogenous sucrose for seedling establishment. This phenotype is similar to the A. thaliana pxa1 mutants deficient in the peroxisomal ABC transporter and other mutants deficient in beta-oxidation. Our results demonstrate that peroxisomal LACS activity and the PXA1 transporter are essential for early seedling growth. The peroxisomal LACS activity would be necessary if the PXA1 transporter delivered unesterified fatty acids into the peroxisomal matrix. Alternatively, PXA1 and LACS6/LACS7 may act in parallel pathways that are both required to ensure adequate delivery of acyl-CoA substrates for beta-oxidation and successful seedling establishment.     

Peroxisomal Acyl-CoA oxidase 4 activity differs between Arabidopsis accessions

In plants, peroxisomes are the primary site of fatty acid β-oxidation. Following substrate activation, fatty acids are oxidized by Acyl-CoA Oxidase (ACX) enzymes. Arabidopsis has six ACX genes, although ACX6 is not expressed. Biochemical characterization has revealed that each ACX enzyme acts on specific chain-length targets, but in a partially overlapping manner, indicating a degree of functional redundancy. Genetic analysis of acx single and double mutants in the Columbia (Col-0) accession revealed only minor phenotypes, but an acx3acx4 double mutant from Wassileskija (Ws) is embryo lethal. In this study, we show that acx3acx4(Col) and acx1acx3acx4(Col) mutants are viable and that enzyme activity in these mutants is significantly reduced on a range of substrates compared to wild type. However, the triple mutant displays only minor defects in seed-storage mobilization, seedling development, and adult growth. Although the triple mutant is defective in the three most active and highly-expressed ACX proteins, increases in ACX2 expression may support partial β-oxidation activity. Comparison of acx mutant alleles in the Col-0 and Ws accessions reveals independent phenotypes; the Ws acx4 mutant uniquely shows increased sensitivity to propionate, whereas the Col-0 acx4 allele has sucrose-dependent growth in the light. To dissect the issues between Col-0 and Ws, we generated mixed background mutants. Although alleles with the Col-0 acx4 mutant were viable, we were unable to isolate an acx3acx4 line using the Ws acx4 allele. Reducing ACX4 expression in several Arabidopsis backgrounds showed a split response, suggesting that the ACX4 gene and/or protein functions differently in Arabidopsis accessions.     

Role of beta-oxidation in jasmonate biosynthesis and systemic wound signaling in tomato

Jasmonic acid (JA) is a lipid-derived signal that regulates plant defense responses to biotic stress. Here, we report the characterization of a JA-deficient mutant of tomato (Lycopersicon esculentum) that lacks local and systemic expression of defensive proteinase inhibitors (PIs) in response to wounding. Map-based cloning studies demonstrated that this phenotype results from loss of function of an acyl-CoA oxidase (ACX1A) that catalyzes the first step in the peroxisomal beta-oxidation stage of JA biosynthesis. Recombinant ACX1A exhibited a preference for C12 and C14 straight-chain acyl-CoAs and also was active in the metabolism of C18 cyclopentanoid-CoA precursors of JA. The overall growth, development, and reproduction of acx1 plants were similar to wild-type plants. However, the mutant was compromised in its defense against tobacco hornworm (Manduca sexta) attack. Grafting experiments showed that loss of ACX1A function disrupts the production of the transmissible signal for wound-induced PI expression but does not affect the recognition of this signal in undamaged responding leaves. We conclude that ACX1A is essential for the beta-oxidation stage of JA biosynthesis and that JA or its derivatives is required both for antiherbivore resistance and the production of the systemic wound signal. These findings support a role for peroxisomes in the production of lipid-based signaling molecules that promote systemic defense responses.     

Functional diversification of acyl-coenzyme A oxidases in jasmonic acid biosynthesis and action

The biosynthesis of jasmonic acid (JA) in plant peroxisomes requires the action of acyl-coenzyme A oxidase (ACX). Among the five expressed members (ACX1-5) of the ACX gene family in Arabidopsis (Arabidopsis thaliana), only ACX1 is known to serve a role in JA production. Here, we used transgenic promoter-reporter lines to show that ACX1 is highly expressed in mature and germinating pollen, stem epidermal cells, and other tissues in which jasmonate-signaled processes occur. Wound-induced JA accumulation was reduced in a mutant that is defective in ACX1 and was abolished in a mutant that is impaired in both ACX1 and its closely related paralog, ACX5. The severe JA deficiency in acx1/5 double mutants was accompanied by decreased resistance to the leaf-eating insect Trichoplusia ni. The double mutant also showed reduced pollen viability and fecundity. Treatment of acx1/5 plants with JA restored both protection against T. ni larvae and normal seed set. Unexpectedly, acx1/5 plants accumulated JA in response to infection by the necrotrophic fungal pathogen Alternaria brassicicola. In contrast to mutants that are impaired in jasmonate perception or early steps of the JA biosynthetic pathway, acx1/5 plants maintained resistance to A. brassicicola infection. These results indicate that ACX1/5-mediated JA synthesis is essential for resistance to chewing insects and male reproductive function and further suggest that other ACX isozymes contribute to JA production in response to A. brassicicola challenge. Thus, different types of biotic stress may induce JA synthesis via distinct enzymatic routes.     

The Arabidopsis thaliana multifunctional protein gene (MFP2) of peroxisomal beta-oxidation is essential for seedling establishment

The multifunctional protein (MFP) of peroxisomal beta-oxidation catalyses four separate reactions, two of which (2-trans enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase) are core activities required for the catabolism of all fatty acids. We have isolated and characterized five Arabidopsis thaliana mutants in the MFP2 gene that is expressed predominantly in germinating seeds. Seedlings of mfp2 require an exogenous supply of sucrose for seedling establishment to occur. Analysis of mfp2-1 seedlings revealed that seed storage lipid was catabolized more slowly, long-chain acyl-CoA substrates accumulated and there was an increase in peroxisome size. Despite a reduction in the rate of beta-oxidation, mfp2 seedlings are not resistant to the herbicide 2,4-dichlorophenoxybutyric acid, which is catabolized to the auxin 2,4-dichlorophenoxyacetic acid by beta-oxidation. Acyl-CoA feeding experiments show that the MFP2 2-trans enoyl-CoA hydratase only exhibits activity against long chain (C18:0) substrates, whereas the MFP2 L-3-hydroxyacyl-CoA dehydrogenase is active on C6:0, C12:0 and C18:0 substrates. A mutation in the abnormal inflorescence meristem gene AIM1, the only homologue of MFP2, results in an abnormal inflorescence meristem phenotype in mature plants (Richmond and Bleecker, Plant Cell 11, 1999, 1911) demonstrating that the role of these genes is very different. The mfp2-1 aim1double mutant aborted during the early stages of embryo development showing that these two proteins share a common function that is essential for this key stage in the life cycle.     

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 beta-oxidation of long-chain fatty acids possessing different extents of unsaturation.

Rates of peroxisomal beta-oxidation were measured as fatty acyl-CoA-dependent NAD+ reduction, by using solubilized peroxisomal fractions isolated from livers of rats treated with clofibrate. Medium- to long-chain saturated fatty acyl-CoA esters as well as long-chain polyunsaturated fatty acyl-CoA esters were used. Peroxisomal beta-oxidation shows optimal specificity towards long-chain polyunsaturated acyl-CoA esters. Eicosa-8,11,14-trienoyl-CoA, eicosa-11,14,17-trienoyl-CoA and docosa-7,10,13,16-tetraenoyl-CoA all gave Vmax. values of about 150% of that obtained with palmitoyl-CoA. The Km values obtained with these fatty acyl-CoA esters were 17 +/- 6, 13 +/- 4 and 22 +/- 3 microM respectively, which are in the same range as the value for palmitoyl-CoA (13.8 +/- 1 microM). Myristoyl-CoA gave the higher Vmax. (110% of the palmitoyl-CoA value) of the saturated fatty acyl-CoAs tested. Substrate inhibition was mostly observed with acyl-CoA esters giving Vmax. values higher than 50% of that given by palmitoyl-CoA.     

Biochemical and molecular characterization of ACH2, an acyl-CoA thioesterase from Arabidopsis thaliana

By using computer-based homology searches of the Arabidopsis genome, we identified the gene for ACH2, a putative acyl-CoA thioesterase. With the exception of a unique 129-amino acid N-terminal extension, the ACH2 protein is 17-36% identical to members of a family of acyl-CoA thioesterases that are found in both prokaryotes and eukaryotes. The eukaryotic homologs of ACH2 are peroxisomal acyl-CoA thioesterases that are up-regulated during times of increased fatty acid oxidation, suggesting potential roles in peroxisomal beta-oxidation. We investigated ACH2 to determine whether it has a similar role in the plant cell. Like its eukaryotic homologs, ACH2 carries a putative type 1 peroxisomal targeting sequence (-SKL(COOH)), and maintains all the catalytic residues typical of this family of acyl-CoA thioesterases. Analytical ultracentrifugation of recombinant ACH2-6His shows that it associates as a 196-kDa homotetramer in vitro, a result that is significant in light of the cooperative kinetics demonstrated by ACH2-6His in vitro. The cooperative effects are most pronounced with medium chain acyl-CoAs, where the Hill coefficient is 3.8 for lauroyl-CoA, but decrease for long chain acyl-CoAs, where the Hill coefficient is only 1.9 for oleoyl-CoA. ACH2-6His hydrolyzes both medium and long chain fatty acyl-CoAs but has highest activity toward the long chain unsaturated fatty acyl-CoAs. Maximum rates were found with palmitoleoyl-CoA, which is hydrolyzed at 21 micromol/min/mg protein. Additionally, ACH2-6His is insensitive to feedback inhibition by free CoASH levels as high as 100 microm. ACH2 is most highly expressed in mature tissues such as young leaves and flowers rather than in germinating seedlings where beta-oxidation is rapidly proceeding. Taken together, these results suggest that ACH2 activity is not linked to fatty acid oxidation as has been suggested for its eukaryotic homologs, but rather has a unique role in the plant cell.     

Fatty acid beta-oxidation in germinating Arabidopsis seeds is supported by peroxisomal hydroxypyruvate reductase when malate dehydrogenase is absent

Peroxisomal malate dehydrogenase (PMDH) oxidises NADH produced by fatty acid beta-oxidation during seed germination and seedling growth. Arabidopsis thaliana beta-oxidation mutants exhibit seed dormancy or impaired seed germination and failure of seedlings to degrade triacylglycerol (TAG), but the pmdh1 pmdh2 null mutant germinates readily and degrades TAG slowly during seedling growth. We reasoned that in the pmdh1 pmdh2 mutant an alternative means of oxidising NADH operates to allow a slow rate of beta-oxidation, such as NADH and NAD⁺ transport across the peroxisomal membrane or activity of another peroxisomal oxido-reductase. Here we show that peroxisomal hydroxypyruvate reductase (HPR) is present in germinating seeds and although knocking out HPR has little effect on germination and early seedling growth, when knocked out in combination with PMDH it exacerbates the pmdh1 pmdh2 phenotype. It greatly increases the proportion of dormant seeds and reduces the rate of seed germination. Seedlings have increased sucrose dependence and resistance to 2,4-dichlorophenoxybutyric acid (2,4-DB), and slower rate of TAG breakdown. When PMDH is absent, malate is lower in amount in germinating seeds and when HPR is also absent, serine (the immediate precursor of hydroxypyruvate) is much higher. These results indicate that HPR can oxidise NADH at sufficient rate in the absence of PMDH to support beta-oxidation and hence seed germination. We conclude that while HPR normally plays little role in seed germination our results support the growing body of evidence that peroxisomal NADH cannot be exported to the cytosol for oxidation but is oxidised by resident oxido-reductases.     

Controlling electron transfer in Acyl-CoA oxidases and dehydrogenases: a structural view

Plants produce a unique peroxisomal short chain-specific acyl-CoA oxidase (ACX4) for beta-oxidation of lipids. The short chain-specific oxidase has little resemblance to other peroxisomal acyl-CoA oxidases but has an approximately 30% sequence identity to mitochondrial acyl-CoA dehydrogenases. Two biochemical features have been linked to structural properties by comparing the structures of short chain-specific Arabidopsis thaliana ACX4 with and without a substrate analogue bound in the active site to known acyl-CoA oxidases and dehydrogenase structures: (i) a solvent-accessible acyl binding pocket is not required for oxygen reactivity, and (ii) the oligomeric state plays a role in substrate pocket architecture but is not linked to oxygen reactivity. The structures indicate that the acyl-CoA oxidases may encapsulate the electrons for transfer to molecular oxygen by blocking the dehydrogenase substrate interaction site with structural extensions. A small binding pocket observed adjoining the flavin adenine dinucleotide N5 and C4a atoms could increase the number of productive encounters between flavin adenine dinucleotide and O2.     

IBR3, a novel peroxisomal acyl-CoA dehydrogenase-like protein required for indole-3-butyric acid response

Indole-3-butyric acid (IBA) is an endogenous auxin that acts in Arabidopsis primarily via its conversion to the principal auxin indole-3-acetic acid (IAA). Genetic and biochemical evidence indicates that this conversion is similar to peroxisomal fatty acid β-oxidation, but the specific enzymes catalyzing IBA β-oxidation have not been identified. We identified an IBA-response mutant (ibr3) with decreased responses to the inhibitory effects of IBA on root elongation or the stimulatory effects of IBA on lateral root formation. However, ibr3 mutants respond normally to other forms of auxin, including IAA. The mutant seedlings germinate and develop normally, even in the absence of sucrose, suggesting that fatty acid β-oxidation is unaffected. Additionally, double mutants between ibr3 and acx3, which is defective in an acyl-CoA oxidase acting in fatty acid β-oxidation, have enhanced IBA resistance, consistent with a distinct role for IBR3. Positional cloning revealed that IBR3 encodes a putative acyl-CoA dehydrogenase with a consensus peroxisomal targeting signal. Based on the singular defect of this mutant in responding to IBA, we propose that IBR3 may act directly in the oxidation of IBA to IAA. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

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 PTE2, a human peroxisomal long-chain acyl-CoA thioesterase

Computer-based approaches identified PTE2 as a candidate human peroxisomal acyl-CoA thioesterase gene. The PTE2 gene product is highly similar to the rat cytosolic and mitochondrial thioesterases, CTE1 and MTE1, respectively, and terminates in a tripeptide sequence, serine-lysine-valine(COOH), that resembles the consensus sequence for type-1 peroxisomal targeting signals. PTE2 was targeted to peroxisomes and recombinant PTE2 showed intrinsic acyl-CoA thioesterase activity with a pH optimum of 8.5. A comparison of PTE2 and PTE1 thioesterase activities across multiple acyl-CoA substrates indicated that while PTE1 was most active on medium-chain acyl-CoAs, with little activity on long-chain acyl-CoAs, PTE2 displayed high activity on medium- and long-chain acyl-CoAs. The identification of PTE2 therefore offers an explanation for the observed long-chain acyl-CoA thioesterase activity of mammalian peroxisomes.     

A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes

Short-chain acyl-CoA oxidases are beta-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete beta-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform beta-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C8). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C4), hexanoyl-CoA (C6), and octanoyl-CoA (C8). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acyl-CoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid beta-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid beta-oxidation in plant peroxisomes, and that by the cooperative action of long- and short-chain acyl-CoA oxidases, plant peroxisomes are capable of performing the complete beta-oxidation of acyl-CoA.     

Long-chain acyl-CoA oxidases of Arabidopsis

Full-length cDNAs coding for two distinct acyl-CoA oxidases were isolated by screening an Arabidopsis cDNA library. The genes for the two acyl-CoA oxidases have been termed AtACX1 and AtACX2. AtACX1 encodes a peptide of 664 amino acids possessing a molecular mass of 74.3 kDa. AtACX2 encodes a peptide of 691 amino acids in length with a molecular mass of 77.5 kDa. Peroxisomal targeting signals were identified in the primary sequences. AtACX1 has a putative PTS1, whereas AtACX2 has a characteristic PTS2. Expression of AtACX1 and AtACX2 in Escherichia coli gave active enzymes for enzymatic and biochemical analysis. AtACX1 was active with both medium-and long-chain saturated fatty acyl-CoAs and showed maximal activity with C14-CoA. Activity with mono-unsaturated acyl-CoAs was slightly higher than with the corresponding saturated acyl-CoA. AtACX2 was active with long-chain acyl-CoAs and showed maximal activity with C18-CoA. AtACX2 activity with mono-unsaturated acyl-CoAs was approximately twice as high as with the corresponding saturated acyl-CoA. Both enzymes have an apparent Km of approximately 5 microM with the preferred substrate. Northern analysis was conducted to determine the expression patterns of AtACX1 and AtACX2 during germination and in various tissues of a mature plant. The two genes showed generally similar expression profiles and steady-state mRNA levels in seedlings and mature tissues, but subtle differences were observed. Enzymatic analyses of plant extracts revealed that AtACX1 and AtACX2 are members of a family that includes acyl-CoA oxidases specific for shorter-chain acyl-CoAs. Through expression of antisense constructs of the individual genes, we were able to decrease long-chain oxidase activity only in antisense AtACX1 plants. Seedlings with long-chain oxidase activity reduced down to 30% of wild-type levels germinated and established normally; however, reduced root growth appeared to be a general feature of antisense AtACX1 plants.