Promoter Trapping of a Novel Medium-chain Acyl-CoA Oxidase, Which Is Induced Transcriptionally during Arabidopsis Seed Germination
2000; Elsevier BV; Volume: 275; Issue: 44 Linguagem: Inglês
10.1074/jbc.m004945200
ISSN1083-351X
AutoresPeter J. Eastmond, Mark A. Hooks, Dawn Williams, Peter Lange, Nichole Bechtold, Catherine Sarrobert, Laurent Nussaume, Ian A. Graham,
Tópico(s)Plant nutrient uptake and metabolism
ResumoThe first step of peroxisomal fatty acid β-oxidation is catalyzed by a family of acyl-CoA oxidase isozymes with distinct fatty acyl-CoA chain-length specificities. Here we identify a new acyl-CoA oxidase gene from Arabidopsis(AtACX3) following the isolation of a promoter-trapped mutant in which β-glucuronidase expression was initially detected in the root meristem. In acx3 mutant seedlings medium-chain acyl-CoA oxidase activity was reduced by 95%, whereas long- and short-chain activities were unchanged. Despite this reduction in activity lipid catabolism and seedling development were not perturbed.AtACX3 was cloned and expressed in Escherichia coli. The recombinant enzyme displayed medium-chain acyl-CoA substrate specificity. Analysis of β-glucuronidase activity inacx3 revealed that, in addition to constitutive expression in the root axis, AtACX3 is also up-regulated strongly in the hypocotyl and cotyledons of germinating seedlings. This suggests that β-oxidation is regulated predominantly at the level of transcription in germinating oilseeds. After the discovery ofAtACX3, the Arabidopsis acyl-CoA oxidase gene family now comprises four isozymes with substrate specificities that encompass the full range of acyl-CoA chain lengths that exist in vivo. The first step of peroxisomal fatty acid β-oxidation is catalyzed by a family of acyl-CoA oxidase isozymes with distinct fatty acyl-CoA chain-length specificities. Here we identify a new acyl-CoA oxidase gene from Arabidopsis(AtACX3) following the isolation of a promoter-trapped mutant in which β-glucuronidase expression was initially detected in the root meristem. In acx3 mutant seedlings medium-chain acyl-CoA oxidase activity was reduced by 95%, whereas long- and short-chain activities were unchanged. Despite this reduction in activity lipid catabolism and seedling development were not perturbed.AtACX3 was cloned and expressed in Escherichia coli. The recombinant enzyme displayed medium-chain acyl-CoA substrate specificity. Analysis of β-glucuronidase activity inacx3 revealed that, in addition to constitutive expression in the root axis, AtACX3 is also up-regulated strongly in the hypocotyl and cotyledons of germinating seedlings. This suggests that β-oxidation is regulated predominantly at the level of transcription in germinating oilseeds. After the discovery ofAtACX3, the Arabidopsis acyl-CoA oxidase gene family now comprises four isozymes with substrate specificities that encompass the full range of acyl-CoA chain lengths that exist in vivo. acyl-CoA oxidase transfer DNA β-glucuronidase polymerase chain reaction rapid amplification of cDNA ends untranslated region protein signature 4-DB, 2,4-dichlorophenoxybutyric acid 4-D, 2,4-dichlorophenoxyacetic acid flavin adenosine dinucleotide N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine base pair(s) Peroxisomal β-oxidation is the primary pathway of fatty acid catabolism in plants. The pathway plays a fundamental role in breaking down stored lipid reserves to provide metabolic energy and carbon skeletons during processes such as oilseed germination, leaf senescence, and starvation (1Kindl H. Stumpf P.K. The Biochemistry of Plants. 9. Academic Press, Inc., New York1987: 31-50Google Scholar, 2Hooks M.A. Bode K. Couee I. Phytochemistry. 1995; 40: 657-660Crossref Scopus (29) Google Scholar). β-Oxidation may also play a constitutive role in membrane lipid turnover and be involved in the synthesis of important fatty acid-derived signals such as jasmonic acid (3Wasternack C. Parthier B. Trends Plant Sci. 1997; 2: 302-307Abstract Full Text PDF Scopus (3) Google Scholar) and traumatin (4Farmer E.E. Plant Mol. Biol. 1994; 26: 1423-1437Crossref PubMed Scopus (179) Google Scholar). Recently an Arabidopsis mutant (aim1) has been isolated that is deficient in a multifunctional protein isoform (5Richmond T.A. Bleeker A.B. Plant Cell. 1999; 11: 1911-1923PubMed Google Scholar). The fact that aim1displays an altered inflorescencemeristem phenotype has lead to the suggestion that β-oxidation may be involved in flower development (5Richmond T.A. Bleeker A.B. Plant Cell. 1999; 11: 1911-1923PubMed Google Scholar). Peroxisomal β-oxidation consists of three components: (i) acyl-CoA oxidase, (ii) the multifunctional protein (which exhibits 2-trans-enoyl-CoA hydratase, l-3-hydroxyacyl-CoA dehydrogenase, d-3-hydroxyacyl-CoA epimerase, and Δ3,Δ2-enoyl-CoA isomerase activities), and (iii) l-3-ketoacyl-CoA thiolase. Together these enzymes are capable of the complete degradation of both saturated and unsaturated long-chain fatty acyl-CoAs to acetyl-CoA (6Kleiter A.E. Gerhardt B. Planta. 1998; 206: 125-130Crossref Scopus (12) Google Scholar). The process involves the repeated cleavage of acetate units from the thiol end of the fatty acid. Acyl-CoA oxidase (ACX,1 EC1.3.3.6) catalyzes the conversion of fatty acyl-CoAs to trans-2-enoyl-CoAs. The reaction requires FAD as a cofactor, which is subsequently re-oxidized by O2 to form H2O2. This first step is believed to be predominant in exerting control over the rate of carbon flux through the pathway (7Holtman W.L. Heistek J.C. Mattem K.A. Bakhuisen R. Douma A.C. Plant Sci. 1994; 99: 43-53Crossref Scopus (39) Google Scholar, 8Chu C. Mao L.F. Schulz H. Biochem. J. 1994; 302: 23-29Crossref PubMed Scopus (26) Google Scholar). Biochemical evidence suggests that plants contain a family of acyl-CoA oxidase isozymes with distinct but partially overlapping substrate specificities (9Gerhardt B. Phytochemistry. 1985; 24: 351-352Crossref Scopus (16) Google Scholar, 10Kirsch T. Loffler H.G. Kindl H. J. Biol. Chem. 1986; 261: 8570-8575Abstract Full Text PDF PubMed Google Scholar, 11Hooks M.A. Bode K. Couee I. Biochem. J. 1996; 320: 607-614Crossref PubMed Scopus (32) Google Scholar). cDNA clones of several acyl-CoA oxidase homologues have been identified in plants (12Grossi M. Gulli M. Stanca A.M. Cattivelli L. Plant Sci. 1995; 105: 71-80Crossref Scopus (46) Google Scholar, 13Do Y.Y. Huang P.L. Arch. Biochem. Biophys. 1997; 344: 295-300Crossref PubMed Scopus (15) Google Scholar, 14Hayashi H. De Bellis L. Hayashi M. Yamaguchi K. Kato A. Nishimura M. J. Biol. Chem. 1998; 273: 8301-8307Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). However, direct evidence of their identity has only recently been obtained by overexpression and characterization of the recombinant proteins. Hooks et al. (15Hooks M.A. Kellas F. Graham I.A. Plant J. 1999; 19: 1-13Crossref PubMed Scopus (84) Google Scholar) have identified and characterized two long-chain acyl-CoA oxidases from the oilseedArabidopsis thaliana (AtACX1 andAtACX2). The preferred substrate of AtACX1 is myristoyl-CoA (C14:0), whereas that of AtACX2 is oleoyl-CoA (C18:1). In addition Hayashi et al. (16Hayashi H. De Bellis L. Ciurli A. Kondo M. Hayashi M. Nishimura M. J. Biol. Chem. 1999; 274: 12715-12721Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) have recently described a gene (referred to here as AtACX4) that encodes an enzyme with specificity for the short-chain substrate hexanoyl-CoA (C6:0). A comparison of the substrate specificities of known acyl-CoA oxidases from Arabidopsis suggests that a gene encoding a medium-chain acyl-CoA oxidase with a preference for decanoyl-CoA (C10:0) and lauroyl-CoA (C12:0) remains to be discovered (15Hooks M.A. Kellas F. Graham I.A. Plant J. 1999; 19: 1-13Crossref PubMed Scopus (84) Google Scholar). Three acyl-CoA oxidase genes, a multifunctional protein, and a 3-keto acyl-CoA thiolase are up-regulated co-ordinately duringArabidopsis seed germination and post-germinative growth, correlating with the period of most rapid fatty acid degradation (Refs.15Hooks M.A. Kellas F. Graham I.A. Plant J. 1999; 19: 1-13Crossref PubMed Scopus (84) Google Scholar, 16Hayashi H. De Bellis L. Ciurli A. Kondo M. Hayashi M. Nishimura M. J. Biol. Chem. 1999; 274: 12715-12721Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 17Eastmond P.J. Graham I.A. Biochem. Soc. Trans. 2000; 28: 95-99Crossref PubMed Google Scholar, 18Hayashi H. Toriyama K. Kondo M. Nishimura M. Plant Cell. 1998; 10: 183-195PubMed Google Scholar, respectively). In addition genes encoding glyoxylate cycle and gluconeogenesis enzymes are also induced during oilseed germination (19Zhang J.Z. Santes C.M. Engel M.L. Gasser C.S. Harada J.J. Plant Physiol. 1996; 110: 1069-1079Crossref PubMed Scopus (12) Google Scholar, 20Kim D.J. Smith S.M. Plant Mol. Biol. 1994; 26: 423-434Crossref PubMed Scopus (56) Google Scholar). Although transcription is known to play a major role in the regulation of the glyoxylate cycle (19Zhang J.Z. Santes C.M. Engel M.L. Gasser C.S. Harada J.J. Plant Physiol. 1996; 110: 1069-1079Crossref PubMed Scopus (12) Google Scholar, 21Graham I.A. Smith L.M. Leaver C.J. Smith S.M. Plant Mol. Biol. 1990; 15: 539-549Crossref PubMed Scopus (34) Google Scholar, 22Sarah C.J. Graham I.A. Reynolds S.J. Leaver C.J. Smith S.M. Mol. Gen. Genet. 1996; 250: 153-161Crossref PubMed Scopus (34) Google Scholar), the level at which peroxisomal β-oxidation is regulated has not yet been determined. The regulation of β-oxidation in plants may be important for a variety of biotechnological applications. In the majority of cases where crops have been genetically engineered to produce novel fatty acids, the accumulation of these products is much lower than that required for commercial exploitation (23Eccleston V.S. Cranmer A.M. Voelker T.A. Ohlrogge J.B. Planta. 1996; 198: 46-53Crossref Scopus (54) Google Scholar, 24Broun P. Boddupalli S. Somerville C.R. Plant J. 1998; 13: 201-210Crossref PubMed Scopus (140) Google Scholar). It is believed that in these plants the novel fatty acids are synthesized at significant rates but are subsequently degraded by peroxisomal β-oxidation (25Eccleston V.S. Ohlrogge J.B. Plant Cell. 1998; 10: 613-621PubMed Google Scholar, 26Hooks M.A. Fleming Y. Larson T.R. Graham I.A. Planta. 1999; 207: 385-392Crossref PubMed Scopus (25) Google Scholar). Moreover, Eccleston and Ohlrogge (25Eccleston V.S. Ohlrogge J.B. Plant Cell. 1998; 10: 613-621PubMed Google Scholar) provide evidence that in the case of plants producing medium-chain fatty acids, medium-chain acyl-CoA oxidase activity is up-regulated to facilitate this degradation. The down-regulation of acyl-CoA oxidases may therefore promote the accumulation of unusual fatty acids in genetically modified crops. In this study we identify and characterize a promoter-trappedArabidopsis mutant disrupted in a gene encoding a new member of the acyl-CoA oxidase family with medium-chain substrate specificity. This gene is up-regulated at the level of transcription during seed germination. Combined with the three genes previously identified and characterized, our data reveal that Arabidopsis comprises a family of isoforms that together are capable of utilizing the full range of fatty acyl-CoA chain lengths present in vivo. A T-DNA-mutagenizedA. thaliana (ecotype Wassilewskija) population consisting of 10,000 lines (27Bechtold N. Ellis J. Pelletier G. C. R. Acad. Sci. ( Paris ). 1993; 316: 1194-1199Google Scholar) transformed with the pGKB5 vector designed for promoter trapping (28Bouchez D. Camilleri C. Caboche M. C. R. Acad. Sci. ( Paris ). 1993; 316: 1188-1193Google Scholar) was screened for transformants exhibiting GUS expression in the roots. Seeds were surface-sterilized and germinated on modified Hoagland solution (1 mm MgSO4, 2 mm Ca(NO3)2, 1.7 mmKNO3, 0.5 mmNH4H2PO4, 1.6 mmFeSO47H2O, 46.2 μmH3BO3, 9.1 μm MnCl2, 0.87 μm ZnSO4, 0.32 μmCuSO4, and 1.03 μmNa2MoO4) plus 0.8% (w/v) agar. After 7 days of growth in a 16-h photoperiod (23 °C light/18 °C dark) seedlings were stained for GUS as described below. For subsequent analysis of line Rm 328, seeds were germinated in continuous light on 0.8% (w/v) agar plates containing half-strength Murashige and Skoog media (29Murashige T. Skoog F. Physiol. Plant. 1962; 15: 473-496Crossref Scopus (53693) Google Scholar) (plus 1% (w/v) sucrose where indicated) at 20 °C after 4 days of imbibition at 4 °C in the dark. For experiments with etiolated seedlings, plates were transferred back to the dark at 20 °C after 30 min exposure to white light. The sequence flanking the right border of the T-DNA inserts in lines displaying GUS expression in the roots was obtained by inverse PCR. Genomic DNA was digested with EcoRI and ligated using T4 DNA ligase. PCR was then carried out using the primers GUS1 (5′-CCAGACTGAATGCCCACACGCCGTC) and inverse PCR 2200 (5′-GTATCACCGCGTCTTTGATCGCGTC). The conditions for amplification were 15 s at 94 °C, 30 s at 65 °C, and 30 s at 68 °C, repeated 38 times followed by a 2-min extension. The products were sequenced directly using the nested primer GUS2 (5′-TCACGGGTTGGGGTTTCTACAGGAC). A partial clone ofAtACX3 was isolated from a λZAPII cDNA library constructed from 3-day-old etiolated hypocotyls (30Kieber J.J. Rothenburg M. Roman G. Ecker J.R. Cell. 1993; 72: 427-441Abstract Full Text PDF PubMed Scopus (1487) Google Scholar) by 3′ RACE using the gene specific primer ACX3S (5′-AGGAGTATTTATCACAAACTCTTCAG) in combination with the T7 primer. The library was then screened by colony hybridization according to Sambrook et al. (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 320-328Google Scholar) using the partial clone as a probe (15Hooks M.A. Kellas F. Graham I.A. Plant J. 1999; 19: 1-13Crossref PubMed Scopus (84) Google Scholar). Four full-length clones were identified and sequenced. RNA was prepared from 2-day-old seedlings (see below), and the 5′-UTR was mapped by 5′ RACE using the 5′ RACE System Version 2 (Life Technologies, Inc.) following the manufacturer's protocols. GSP1 and GSP2 were (5′-ACTTTACAGCATTACCCCACAGGA) and (5′-GTGATCATAAATCCCGCAAACCT), respectively. Three 5′ RACE products from separate PCR reactions were cloned and sequenced. One AtACX3 cDNA clone was determined to be in-frame with the N terminus of the pBluescript β-galactosidase gene and could be expressed as a fusion protein following induction of Escherichia coli XL1-blue MRFTM cells with isopropyl-β-d-thiogalactoside. Cells were grown at 37 °C to an optical density of 0.5 at 600 nm in Luria broth media. Isopropyl-β-d-thiogalactoside (0.4 mm) was then added to the culture, and the cells grown overnight at 28 °C. The culture was centrifuged at 700 × g for 10 min, and the pellet was resuspended in extraction buffer (150 mmTris/HCl, pH 7.5, 10 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 10 mm FAD, 10% (v/v) glycerol). The cells were lysed by sonication using a Soniprep 150 ultrasonic disintegrator (Sanyo Gallenkamp PLC, Leicester, UK), and cell debris was removed by centrifugation at 21,000 × g for 10 min. Assays were performed on the supernatant. Crude tissue extracts were prepared from approximately 2000 2-day-old seedlings. The tissue was ground in 1 ml of extraction buffer using a glass homogenizer. The extract was then centrifuged at 13,000 ×g for 10 min, and the supernatant was desalted using a Sephadex G-50 spin column as described by Hooks et al.(15Hooks M.A. Kellas F. Graham I.A. Plant J. 1999; 19: 1-13Crossref PubMed Scopus (84) Google Scholar). For subcellular fractionation experiments, approximately 10,000 2-day-old seedlings were homogenized in 3 ml of a medium containing: 150 Tricine/KOH, pH 7.5, 1 mm EDTA, 0.5 msucrose using an Ultra-Turrax (Janke and Kunkel KG). The homogenate was filtered through four layers of mira cloth, and 2 ml of homogenate was layered on top of a gradient consisting of a 1-ml cushion of 60% (w/v) sucrose plus 8 ml of a 60 to 30% linear sucrose gradient. The gradient was centrifuged at 30,000 × gfor 3 h in a Sorvall OTD55B centrifuge using a TST 41.14 swing out rotor. 0.5-ml fractions were removed and assayed for various enzyme activities. The sucrose concentration of the fractions was determined using a refractometer. ACX assays were performed on plant tissue and bacterial cell extracts according to the method of Hryb and Hogg (32Hryb D.J. Hogg J.F. Biochem. Biophys. Res. Commun. 1979; 87: 1200Crossref PubMed Scopus (136) Google Scholar) using 50 μm acyl-CoAs as substrate. GUS assays and histochemical staining were carried out as described by Jefferson (33Jefferson R.A. Plant Mol. Biol. Rep. 1987; 5: 387-405Crossref Scopus (4029) Google Scholar). Catalase and cytochrome c oxidase were assayed according to Takahashi et al. (34Takahashi H. Chen Z. Du H. Liu Y. Klessig D.F. Plant J. 1997; 11: 993-1005Crossref PubMed Scopus (170) Google Scholar) and Denyer and Smith (35Denyer K. Smith A.M. Planta. 1988; 173: 172-182Crossref PubMed Scopus (58) Google Scholar), respectively. Protein content was determined as described by Bradford (36Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215608) Google Scholar) using bovine serum albumin as a standard. Fatty acids were measured using the method of Browse et al. (37Browse J. Mc Court P.J. Somerville C.R. Anal. Biochem. 1986; 152: 141-145Crossref PubMed Scopus (399) Google Scholar). Total RNA from various tissues was isolated using the Purescript RNA isolation kit (Flowgen) or the hot phenol method (38Kay R. Chan A. Daly M. McPherson J. Science. 1987; 236: 1299-1302Crossref PubMed Scopus (729) Google Scholar). Ten μg of total RNA was separated by electrophoresis using a 1.1% formaldehyde gel and alkaline-blotted onto Zeta-Probe membrane (Bio-Rad) using 50 mm NaOH. Genomic DNA was purified using the Puregene DNA isolation kit (Flowgen). Three μg of DNA were digested using EcoRI, separated on a 0.8% agarose gel, and blotted using 0.4 mNaOH. Probes were prepared from AtACX3 and gusA, and membranes were hybridized using the digoxygenin system (Roche Molecular Biochemicals) following the manufacturer protocols. Bands were detected using a ChemiImager (Alpha Innotech Corp.). Reverse transcriptase-PCR was performed using the Reverse-iT kit from Advanced Biotechnologies Ltd. The primers used were GUS1, ACX3S, and ACX3A (5′-GAAACATCAGCAACTGCATTCAAC). As part of a research program focused on root-rhizosphere interactions, Arabidopsis promoter trapped lines that displayed GUS expression in the roots of 7-day-old seedlings were isolated from the INRA-Versailles T-DNA-mutagenized population (27Bechtold N. Ellis J. Pelletier G. C. R. Acad. Sci. ( Paris ). 1993; 316: 1194-1199Google Scholar). Genomic DNA sequences that flank the right borders of the T-DNAs were obtained by inverse PCR. One of two inverse PCR products from a line with GUS expression in the root meristem (Rm 328) was found to be homologous to acyl-CoA oxidases. These enzymes catalyze the first step in the pathway of peroxisomal β-oxidation and are known to be highly active in the root tips of maize (2Hooks M.A. Bode K. Couee I. Phytochemistry. 1995; 40: 657-660Crossref Scopus (29) Google Scholar). This putative acyl-CoA oxidase was different from those previously identified inArabidopsis, and the new gene and mutant were designatedAtACX3 and acx3, respectively. A partial AtACX3 cDNA clone was isolated by performing 3′ RACE on a cDNA library constructed from 3-day-old etiolated seedling hypocotyls (30Kieber J.J. Rothenburg M. Roman G. Ecker J.R. Cell. 1993; 72: 427-441Abstract Full Text PDF PubMed Scopus (1487) Google Scholar). This clone was used as a homologous probe to screen the same library by colony hybridization. Four full-length cDNAs were isolated and sequenced. Finally the 5′-UTR was mapped using 5′ RACE. The assembled sequence was submitted to the GenBank data base. The cDNA was 2246 bp long and contained a 2028-bp putative open reading frame (Fig.1). The 5′-UTR extended 53 bp 5′ of the start of translation. The 3′-UTR contained a putative polyadenylation signal (AATAAA) 96 bp 3′ of the stop codon. The deduced protein was 675 amino acids long (Fig. 1) with a calculated molecular mass of 75676.33 Da and an isoelectric point (pI) of 8.17. Both the molecular weight and pI values are similar to those of long-chain acyl-CoA oxidases from Arabidopsis (15Hooks M.A. Kellas F. Graham I.A. Plant J. 1999; 19: 1-13Crossref PubMed Scopus (84) Google Scholar). A similarity search of available data bases revealed that expressed sequence tags homologous to AtACX3 (>70% amino acid identity) are present in a variety of plant species including Glycine maxand Gossypium hirsutum. Comparison of theArabidopsis acyl-CoA oxidase proteins shows that AtACX3 shares 28, 23, and 14% identity at the amino acid level with AtACX2, AtACX1 (15Hooks M.A. Kellas F. Graham I.A. Plant J. 1999; 19: 1-13Crossref PubMed Scopus (84) Google Scholar), and AtACX4 (16Hayashi H. De Bellis L. Ciurli A. Kondo M. Hayashi M. Nishimura M. J. Biol. Chem. 1999; 274: 12715-12721Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), respectively. All fourArabidopsis acyl-CoA oxidases have regions homologous to the mammalian acyl-CoA dehydrogenase protein signatures PS1 ((G/A/C)(L/I/V/M)(S/T)EX 2(G/S/A/N)GSDX 2(G/S/A)) and PS2 (Q/E)X 2G(G/S)XG(L/I/V/M/F/Y)X 2(D/E/N)X 4(K/R)X 3(D/E)) (39Bairoch A. Bucher P. Hofmann K. Nucleic Acids Res. 1997; 25: 217-221Crossref PubMed Scopus (756) Google Scholar). In the AtACX3 sequence, 7 of the 9 positions in PS1 and 7 of 8 positions in PS2 are conserved (Fig. 1). Consensus motifs characteristic of type 1 or 2 peroxisomal targeting signals (14Hayashi H. De Bellis L. Hayashi M. Yamaguchi K. Kato A. Nishimura M. J. Biol. Chem. 1998; 273: 8301-8307Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 40Wimmer C. Schmid M. Veenhuis M. Gietl C. Plant J. 1998; 16: 453-464Crossref PubMed Google Scholar) are not obvious in the AtACX3 amino acid sequence. Southern analysis using a probe to gusA revealed that acx3 contained three copies of the T-DNA (data not shown). As shown in Fig. 2 A, one copy was segregated out by back-crossing acx3. The remaining two copies formed a tandem inverted repeat inserted inAtACX3. In Fig. 2 B this was demonstrated by a PCR experiment on acx3 genomic DNA. Primers 5′ or 3′ of the site of insertion in AtACX3 were used in combination with a gusA primer to demonstrate that gusA is present at both borders. The PCR products were sequenced, and comparison with the AtACX3 cDNA sequence revealed that the insertion is situated in an exon, 806 bp 3′ of the putative start of transcription. In Fig. 2 B a reverse transcriptase-PCR experiment using the same primer combinations on RNA from 2-day-old acx3seedlings demonstrated also that the 5′ end of AtACX3 is expressed in vivo as a gusA transcriptional fusion. In contrast, the gusA copy bordering the 3′ end ofAtACX3 was not expressed. No product was detected when primers specific to a region of AtACX3 that is 3′ of the insertion site were used in combination (data not shown). These data showed that wild type transcripts are absent from acx3mutant seedlings. Arabidopsis mutants defective in peroxisomal β-oxidation have previously been selected by their resistance to 2,4-dichlorophenoxybutyric acid (2,4-DB) (18Hayashi H. Toriyama K. Kondo M. Nishimura M. Plant Cell. 1998; 10: 183-195PubMed Google Scholar, 41Lange P.R. Hooks M.A. Ahmed M. Rylott E.L. Eastmond P.J. Graham I.A. Sanchez J. Advances in Plant Lipid Research. Secretariado de Publicaciones, Universidad de Sevilla, Seville, Spain1998: 343-345Google Scholar). This compound is bio-activated to the herbicide and auxin analogue 2,4-dichlorophenoxyacetic acid (2,4-D) by β-oxidation (5Richmond T.A. Bleeker A.B. Plant Cell. 1999; 11: 1911-1923PubMed Google Scholar, 18Hayashi H. Toriyama K. Kondo M. Nishimura M. Plant Cell. 1998; 10: 183-195PubMed Google Scholar, 41Lange P.R. Hooks M.A. Ahmed M. Rylott E.L. Eastmond P.J. Graham I.A. Sanchez J. Advances in Plant Lipid Research. Secretariado de Publicaciones, Universidad de Sevilla, Seville, Spain1998: 343-345Google Scholar). To investigate whether acx3 is impaired in β-oxidation, seeds were germinated on media containing 1.5 μm 2,4-DB (41Lange P.R. Hooks M.A. Ahmed M. Rylott E.L. Eastmond P.J. Graham I.A. Sanchez J. Advances in Plant Lipid Research. Secretariado de Publicaciones, Universidad de Sevilla, Seville, Spain1998: 343-345Google Scholar), and root growth was used as an indicator of resistance. As shown in Fig. 3, homozygous acx3seedlings were significantly more resistant to 2,4-DB than the wild type. In contrast, heterozygous acx3 seedling were sensitive to 2,4-DB, showing that the phenotype is recessive (Fig. 3). Bothacx3 and wild type seedlings were susceptible to 2,4-D (Fig.3). It has also been demonstrated that the post-germinative growth ofArabidopsis mutants disrupted in storage lipid breakdown can be prevented if exogenous sugars are not supplied to the seedling (18Hayashi H. Toriyama K. Kondo M. Nishimura M. Plant Cell. 1998; 10: 183-195PubMed Google Scholar,42Eastmond P.J. Germain V. Lange P.R. Bryce J.H. Smith S.M. Graham I.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5669-5674Crossref PubMed Scopus (224) Google Scholar). To determine if the germination or post-germinative growth ofacx3 is impaired, the rate of hypocotyl elongation and fatty acid breakdown were measured in etiolated seedlings in the absence of exogenous sucrose. The data in Fig. 4show that the rate of hypocotyl growth and fatty acid breakdown were not significantly different from wild type over the course of 5 days following germination. Furthermore, no visible vegetative or reproductive phenotype was observed throughout the life cycle ofacx3 plants. To investigate whether the acx3mutant displayed altered acyl-CoA oxidase activity, the enzyme was measured in 2-day-old germinating seedlings (TableI). Saturated acyl-CoAs ranging from 4 to 20 carbons in length were used at a saturating concentration (50 μM). The acx3 mutant was almost deficient in medium-chain acyl-CoA oxidase activity ( 0.01). Open table in a new tab Activity was measured using saturated acyl-CoAs of varying chain length at a saturating concentration of 50 μm. Values are the mean ± S.E. of measurements made on three separate batches of seedlings. NS, not significant (p > 0.01). To confirm the function of AtACX3, the cDNA was expressed in E. coli as a β-galactosidase fusion protein transcribed from the pBluescript cloning vector upon induction with isopropyl-β-d-thiogalactoside. As previously reported (15Hooks M.A. Kellas F. Graham I.A. Plant J. 1999; 19: 1-13Crossref PubMed Scopus (84) Google Scholar), no inducible acyl-CoA oxidase activity was observed in extracts of E. coli harboring pBluescript without insert and endogenous levels of acyl-CoA oxidase activity were below the limits of detection. There was also no induction when the AtACX3cDNA is in the antisense orientation (data not shown). As shown in Fig. 5 A, AtACX3expressed in the correct orientation encoded a protein with medium-chain acyl-CoA oxidase activity. The optimum substrate was lauroyl-CoA (C12:0). No activity was detected with substrates of chain length greater than C14:0. In Fig. 5 B, kinetic analysis of recombinant AtACX3 showed that the apparent K m value of the enzyme for lauroyl-CoA was 3.7 μm. The optimum pH was between 8.5 and 9.0 and activity was dependent on the provision of the cofactor FAD (data not shown). To investigate whether AtACX3 is a peroxisomal protein, the subcellular location of medium-chain acyl-CoA oxidase activity was determined. The acx3 mutant specifically lacks this activity (Table I), making it a reliable marker for the subcellular localization of the protein in wild type. A homogenate of two-day-old wild type seedlings was fractionated on a sucrose density gradient by centrifugation to separate the subcellular compartments. Catalase and cytochrome c oxidase activities were used as peroxisomal and mitochondrial markers, respectively. As shown in Fig. 6, medium-chain acyl-CoA oxidase activity co-localized with that of catalase (fraction 16), suggesting that the majority of AtACX3 is located in the peroxisome. Both activities were present in the supernatant as well as in the peroxisomal fraction. This is likely to be due to a proportion of the organelles rupturing during the tissue homogenization step. In Fig. 7, Northern blot analysis of total RNA from imbibed seeds, germinating seedlings, and various tissues from wild type plants showed thatAtACX3 is expressed at low levels in all tissues but is up-regulated strongly during germination and leaf senescence.AtACX3 transcripts were detectable in imbibed seeds before radicle emergence. Steady-state AtACX3 mRNA levels increased during germination to a maximum between 2 and 3 days after imbibition (DAI) and subsequently decreased (Fig.7 A). The analysis of GUS expression in acx3, displayed in Fig.8 A, revealed that theAtACX::gusA tra
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